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  • Life and Medical Sciences  (8,388)
  • Inorganic Chemistry  (3,613)
  • 1990-1994  (12,001)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 1-12 
    ISSN: 0886-1544
    Keywords: pseudopod extension ; amoebae ; uropod retraction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Employing a newly developed computer-assisted system for visualizing and quantitating cell motility in three dimensions, we have examined the 3-dimensional changes in cell shape and the dynamics of pseuodopod extension during translocation of Dictyostelium amoebae. Amoebae exhibit a 3-dimensional behavior cycle with an average period of 1.5 min. The cycle includes a transient pseudopod extension phase in the x, y axis followed by a z-axis expansion phase. Anterior pseudopod extension in the x, y axis is accompanied by a decrease in height, not by uropod retraction. The increase in height is accompanied by uropod retraction. In the pseudopod extension phase in the x, y axes, pseudopods form either anteriorly or laterally, and either on or above the substratum. Pseudopods which initially form on the substratum in almost all cases continue to expand as the anterior end of the cell. In the case of lateral pseuodopods, anteriorization leads to a turn. Approximately half of anterior pseudopod and two-thirds of lateral pseudopods which initially form above the substratum are retracted. These results suggest that pseudopod-substratum interaction plays a fundamental role in the regulation of directionality and turning in the translocation phase of the 3-dimensional behavior cycle. © 1994 Wiley-Liss, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 219-233 
    ISSN: 0886-1544
    Keywords: microtubules ; mitosis ; cytoskeleton reorganization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plant Haemanthus. In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either “free” or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus.Elevated temperature (35°-37°) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase-interphase transition and in some interphase cells, indicating temperature and stage dependence.Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle in Haemanthus endosperm. © 1994 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 180-191 
    ISSN: 0886-1544
    Keywords: sliding movement ; 22S dynein ; Tetrahymena cilia ; dynein-track ; singlet microtubule ; ATP ; polarity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chlamydomonas and Tetrahymena axonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule-dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule-dynein complexes (α-and β-complexes) were prepared from Tetrahymena ciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)-induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP-induced extrusion of single microtubules. The α-complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β-complex, prepared by adding dynein bound to DEAE-tubulin to pre-assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β-complex extruded at 12 μm/second following the addition of ATP. Dark-field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein-track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark-field microscope, the dynein-track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β-complex under electron microscope. The higher sliding velocity of the microtubules on these dynein-tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polarity on the tracks. © 1994 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 206-218 
    ISSN: 0886-1544
    Keywords: sperm motility ; sperm maturation ; flagella ; protein kinases ; protein kinase inhibitor ; cGMP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was ∼100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained 〉90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase. © 1994 Wiley-Liss, Inc.
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  • 5
    ISSN: 0886-1544
    Keywords: cytoskeleton ; cell culture ; gene expression ; Northern blot ; serum-induction ; rat ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytochalasin D and dBcAMP cause cultured astrocytes to change from flat cells to retrated process-bearing cells. F-actin was present throughout cells stimulated with dBcAMP for 16 h, whereas cytochalasin D caused F-actin to form massive aggregates at the tips of the cell processes. The two drugs differently regulated the expression of both β-actin and tropomyosin genes in astrocytes cultured in the presence or absence of serum: dBcAMP caused down-regulation and cytochalasin D caused up-regulation. Northern blot analyses indicated that: (1) serum deprivation halved the concentration of all tropomyosin transcripts (TM-1, TM-2, TM-4, TMBr-1, TMBr-2). Serum induced TM-4 via transcriptional activation, independent of protein synthesis, (2) dBcAMP induced down-regulation of β-actin (-50%) and tropomyosin transcripts (-35 to 52%) even in the presence of serum. The concentration of profilin mRNA decreased in dBcAMP-reactive astrocytes (-46%). The decrease in β-actin mRNA concentration was not blocked by cycloheximide, whereas down-regulation of tropomyosin transcripts was completely reversed when protein synthesis was inhibited, and (3) cytochalasin D induced an increase in the concentration of tropomyosin transcripts (+ 69 to 185%) which was cumulative with serum stimulation. Cytochalasin D induction of both β-actin and TM-4 operated through transcriptional activation, independent of protein synthesis.The production of all tropomyosin transcripts examined here were strictly coordinated with β-actin expression in serum-, dBcAMP- and cytochalasin D-treated astrocytes. This indicates that the differential expression of tropomyosin isoforms occurring during astrocyte maturation is due to more complex regulation than that involved in serum- or cAMP-stimulated astrocytes. © 1994 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 360-360 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 193-205 
    ISSN: 0886-1544
    Keywords: amoeboid motility ; fluorescence ratio imaging ; BCECF ; nematodes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction. © 1994 Wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 234-247 
    ISSN: 0886-1544
    Keywords: microtubule-associated protein 2 ; neurons ; microtubule-associated proteins ; cytoskeleton ; dendrites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated protein 2 (MAP-2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP-2, two MAP-2 antisense (AS) oligonucleotides complementary to the 5′ region of the rat MAP-2 cDNA were added to rat primary embryonic day 17-18 (E17-18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP-2 AS oligonucleotides resulted in decreased MAP-2 and reduction in the number of neuritic processes relative to the control or MAP-2 sense-treated cultures. By immunostaining and light microscopy the AS-treated neurons appeared smaller, more rounded, and less intensely stained for MAP-2 than the untreated or the MAP-2 sense-treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS-treated cultures were observed. We conclude that MAP-2 continues to be required for microtubule spacing and stability within neurites once they have formed. © 1994 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 262-271 
    ISSN: 0886-1544
    Keywords: 3T3 cells ; cell motility ; infrared ; phototaxis ; centrosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous experiments have suggested that 3T3 cells were able to extend pseudopodia toward latex particles up to 60 μm away from the cell body if the particles were irradiated by an infrared beam in the range of 700-900 nm [Albrecht-Buehler, 1991: J. Cell Biol. 114:493-502]. The present article reports that this response of cells to infrared light can be inhibited if the cell center is simultaneously irradiated with a beam of the same light. In marked contrast, the cells responded normally to the presence of infrared light scattering particles if the second beam irradiated other parts of the cell body. The results imply that the cellular mechanism of infrared detection is located at the cell center. The infrared sensing mechanism remains intact in enucleated cells and in cells which were incubated in monensin to vesiculate their Golgi apparatus and inhibit their Golgi functions. Accordingly, it is proposed that the centrosome which contains the centrioles is the only remaining candidate in the cell center for a cellular detection device for the direction of infrared signal sources. The results support an earlier suggestion that centrioles may be such detection devices [Albrecht-Buehler, 1981: Cell Motil. Cytoskeleton 1:237-245]. © 1994 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 272-283 
    ISSN: 0886-1544
    Keywords: cell cycle ; transcription ; mRNA decay ; autoregulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates. © 1994 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 287-298 
    ISSN: 0886-1544
    Keywords: cilium ; flagellum ; motility ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A physical model developed to explain microtubule sliding patterns in the trypsintreated ciliary axoneme has been extended to investigate the generation of bending moments by microtubules sliding in an axoneme in which the dublets are anchored at one end. With sliding restricted, a bending moment is developed by the polarized shearing interaction between neighbouring doublets, effected by the activity of dynein arms on doublet N pushing N + 1 in a tipward ( + ) direction. In arrested axonemes in which arms on several contiguous doublets are active, the bending moment causes splitting of the 9 + 2 microtubule array into two or more sets of doublets. In the absence of special constraints, splitting depends only on breaking the circumferential interdoublet links most distorted by the bending moment. The analysis, which permits assignment of arm activity to specific microtubules in each of the observed patterns of splitting, indicates that the axoneme will split between doublet N and N + 1 if arms on doublet N are inactive and arms on either N + 1 or N-1 are active. To produce the observed major splits, dynein arms on the microtubules of roughly one-half of the axoneme are predicted to be active, in a manner consistent with the switch-point hypothesis of ciliary motion. Electron microscopic examination indicates that virtually every set of doublets in the split axonemes retains its cylindrical form. Maintenance of cylindrical symmetry can be ascribed to the mechanical properties of the unbroken links, which may resist both tensile and compressive stress, and to active dynein arms. © 1994 Wiley-Liss, Inc.
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  • 12
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 313-326 
    ISSN: 0886-1544
    Keywords: myosin II ; cardiac ; sarcomeric ; cytoplasmic myosin ; LMM ; Dd ; heterologous expression ; ConA ; receptor capping ; aggregation ; filament assembly region ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cyto-kinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1-11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677-688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat β cardiac LMM was transfected into both wild-type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild-type and Dd mhc null cells. Although null cells expressing chimeric mhc at ∼10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. © 1994 Wiley-Liss, Inc.
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  • 13
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 361-372 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 14
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    Cell Motility and the Cytoskeleton 28 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 15
    ISSN: 0886-1544
    Keywords: intermediate filaments ; phosphorylation ; sea urchin embryos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 μM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase.To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 μM 6-DMAP and with 200 μCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly. © 1994 Wiley-Liss, Inc.
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  • 16
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    Cell Motility and the Cytoskeleton 29 (1994), S. 155-166 
    ISSN: 0886-1544
    Keywords: Golgi vesicles ; pollen ; pollen tube ; microtubules ; kinesin-related protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A 100-kDa polypeptide with microtubule-interacting properties was identified in a Golgi vesicle-enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132-137] localized the polypeptide on the external surface of membrane-bounded organelles. Some 100-kDa-containing vesicles co-pelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule-interacting protein, salt-extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2-fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100-kDa polypeptide is part of a molecular complex showing properties of the kinesin class. © 1994 Wiley-Liss, Inc.
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  • 17
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    Cell Motility and the Cytoskeleton 29 (1994), S. 57-71 
    ISSN: 0886-1544
    Keywords: microtubule bundling ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 μm in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. © 1994 Wiley-Liss, Inc.
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  • 18
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    Cell Motility and the Cytoskeleton 29 (1994), S. 72-81 
    ISSN: 0886-1544
    Keywords: spectrin ; intrinsic fluorescence ; spectrin elasticity ; fluorescence quenching ; spectrin α chain ; spectrin β chain ; membrane skeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, α-β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994 Wiley-Liss, Inc.
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  • 19
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    Cell Motility and the Cytoskeleton 29 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 20
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    Cell Motility and the Cytoskeleton 29 (1994), S. 177-185 
    ISSN: 0886-1544
    Keywords: flagella ; Chalamydomonas ; mutant ; high-frequency vibration ; nanometer scale measurement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Flagellar axonemes of sea urchin sperm display high frequency (200-400 Hz) vibration with nanometer scale amplitudes in the presence of ATP [Kamimura and Kamiya, 1992: J. Cell Biol. 116:1443-1454]. To investigate how various axonemal components affect the vibration, we examined vibration in wild-type and mutant axonemes of Chlamydomonas. At 1 mM ATP, wild-type axonemes underwent vibration at 100-650 Hz with amplitudes of 4-40 nm. This vibration was similar to, but less regular than, that in sea urchin sperm. Axonemes of the mutants ida1 and ida4 lacking part of the inner arm dynein underwent vibrations indistinguishable from that of wild-type. The mutant oda1 lacking the entire outer arm underwent vibration at about half the wild-type frequency. Unexpectedly, the paralyzed mutants pf18 lacking the central pair and pf14 lacking the radial spokes displayed vibration with significantly higher frequencies and smaller amplitudes than those in the wild-type vibration. These results indicate that the high-frequency vibration is common to many kinds of mutant axonemes that lack various axonemal substructures, but that its manner is sensitive to the presence of outer arm dynein and the central pair/radial spoke system. Simultaneous measurements of amplitude and frequency in wild-type and mutant axonemes suggest that the velocity of microtubule sliding in vibrating axonemes is lower than the velocity of sliding under load-free conditions. The velocity is particularly low in pf18. A possible mechanism is proposed to explain the lower sliding velocity and vibration amplitude in the pf18 axoneme, based on an assumption that central pair/radial spoke system may work to regulate the switching of two antagonizing forces within the axoneme. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 366-374 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microfilaments ; cytochalasin ; gravity ; amiprophos-methyl ; tip-growth ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 375-382 
    ISSN: 0886-1544
    Keywords: collagen ; actin ; α-actinin ; cAMP-dependent protein kinase ; NBT-II cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cyclic AMP (cAMP) has been implicated in the regulation of movement of certain cultured cell types. We have studied the effects of cAMP on epithelial cell motility using serum-free NBT-II cells, derived from a rat bladder carcinoma. The random movement of these cells on type I collagen was reduced upon elevation of intracellular cAMP by several means and this effect was reversible. Alterations in the organization of the cytoskeletal proteins F-actin and α-actinin occurred concurrently with the reduction in motility, and the arrangement of these proteins resembled that seen in non-motile cells on glass. In addition, pretreatment of cells with KT5720, a cAMP-dependent protein kinase (PKA)-specific inhibitor, prevented the dibutyryl cAMP-induced reduction in cell movement as well as the associated cytoskeletal changes. These results suggest that elevation of PKA is responsible for the observed effects on cell motility and cytoskeletal reorganization and demonstrate a role for PKA in the regulation of cell motility in this system. © 1994 Wiley-Liss, Inc.
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    Keywords: intermediate filaments ; cytoskeleton ; filament attachment sites ; immunogold labeling ; Life and Medical Sciences ; Cell & Developmental Biology
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    Notes: The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and α-actinin during development of smooth muscle were studied in gizzard samples from 10- and 16-day embryos and from 1- and 7-day post-hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side-by-side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti-desmin labeling in embryonic 10- and 16-day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti-desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti-α-actinin labeling was directly on CDBs and was often associated with the cross-connecting filaments (CCFs) (average diameter of 2-3nm) inside CDBs. We propose, based on these data, that desmin IFs, α-actinin-containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 271-279 
    ISSN: 0886-1544
    Keywords: peptide antibodies ; protein processing ; axonemes ; microtubule associated proteins ; UV photocleavage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dyneins are multi-subunit enzymes that transduce chemical energy into the mechanical energy that makes cilia and flagella beat and moves organelles towards the minus end of microtubules. The ATPase activity is borne by heavy chains, and recent molecular analysis indicates that dynein heavy chain genes form an ancient multigene family: the similarity between the same isoform of two distantly related species is greater than that between different isoforms of the same species. We have exploited sequence identities between a Paramecium axonemal dynein heavy chain gene cloned in our laboratory and sequences of dynein heavy chains from other species to prepare antibodies against active-site peptides capable of recognizing dynein heavy chains regardless of species or isoform. One of the antibodies is perfectly specific for the larger product of V1 photolysis (HUV1) and thus incorporates a unique property of the hydrolytic ATP binding site of all known dynein heavy chains, the capacity for photocleavage in the presence of micromolar vanadate. Our characterization of these reagents suggests that they will be useful for biochemical and in situ studies of known dyneins as well as identification of potential new members of the family. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 29 (1994), S. 291-300 
    ISSN: 0886-1544
    Keywords: endoplasmic reticulum ; DiOC6(3) ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Notes: Relationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double-labeling with DiOC6(3) and anti-tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at ∼0.3 μm/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol-stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. © 1994 Wiley-Liss, Inc.This Article is a US Government work and, as such, is in the public domain in the United States of America.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 301-311 
    ISSN: 0886-1544
    Keywords: ctenophore ; egg ; nucleus ; microtubule ; endoplasmic reticulum (ER) ; sperm aster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the large eggs (∼1 mm) of the ctenophore Beroe ovata, female pronuclei migrate long distances to join stationary male pronuclei in the peripheral cytoplasm that surrounds the yolky interior. We have investigated the mechanism of nuclear migration using time lapse video recording, automated image analysis, visualization of microtubules by immunofluorescence and rhodamine-tubulin injection, and electron microscopy. Female pronuclei migrated at average speeds of 0.2 μm/sec, and were found to show periodic oscillations in velocity. Alternating phases of acceleration and deceleration occurred with an average periodicity of 235 seconds covering distances of 47 μm (about 3 times the nuclear diameter). Migration velocities and velocity oscillations were similar in fertilized and unfertilized eggs; however, changes in migration direction were much more frequent in unfertilized eggs. Characteristic deformations of the pronuclear membrane and occasional rotation of the nuclear contents were observed during migration. Inhibitor studies indicated that microtubules are required for nuclear migration. In fertilized eggs the top of the nucleus was found to move through the dense layer of aligned sperm aster microtubules. The frequent changes in direction of pronuclear migration in unfertilized eggs reflect the random organization of the microtubule layer in the absence of sperm derived centrosomes. Densely packed endoplasmic reticulum was found intermeshed with sperm aster microtubules and connected extensively with the nuclear membrane during migration. Most nuclear pores were grouped in an infolding of the nuclear membrane. We suggest that in fertilized eggs the female pronucleus is transported to the minus ends of sperm aster microtubules using motor molecules attached either to the outer nuclear membrane and/or to the network of connecting ER. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 312-320 
    ISSN: 0886-1544
    Keywords: contractile ring ; cleavage furrow ; mitosis ; unconventional myosin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: During cytokinesis, daughter cells are cleaved in two by the constriction of an actin-rich contractile ring which encircles the equator of the dividing cell. Filamentous myosin II is present in the contractile ring and necessary for constriction of the furrow, as shown in several cell types [Satterwhite and Pollard, 1992: Curr. Opin. Cell Biol. 4:43-52]. However, no functional role nor distinctive localization has been previously identified for non-filamentous “unconventional” myosins, such as myosin I, during cytokinesis. Using antibodies to adrenal medullary myosin I, we report that myosin I is localized in 3T3 fibroblasts to the mid-equatorial plane during late-cytokinesis, as well as to the polar edges as previously described in ameboid cells [Fukui et al., 1989: Nature 341:328-331]. Confocal microscopy revealed that myosin I is concentrated at the midbody region in a nearly continuous transverse disk, extending from the cortical region of the furrow through the midbody itself. These findings suggest that, in addition to the accepted role of filamentous myosin II in constriction of the contractile ring, non-filamentous myosin I might contribute to motile events occurring late in cytokinesis. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 45-58 
    ISSN: 0886-1544
    Keywords: MTOC ; cytoplasmic microtubule complex ; antitubulin ; Immunofluorescence ; ultrastructure ; immunogold ; Life and Medical Sciences ; Cell & Developmental Biology
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    Notes: We investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron-opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus-associated and “remote” MTCs are structurally identical. They consist of a ring-shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 79-87 
    ISSN: 0886-1544
    Keywords: microtubule transport ; microtubules ; 2,5-hexanedione ; glutaraldehyde ; kinesin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubules treated with the γ-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 108-116 
    ISSN: 0886-1544
    Keywords: smooth muscle ; fibroblasts ; lamellipodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using a synthetic peptide mimicking the NH2-terminus of β-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the β isoform, by its exclusive recognition of the synthetic β-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the β-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 133-149 
    ISSN: 0886-1544
    Keywords: MAP4 depletion ; antibody blocking ; detyrosination ; midbody ; asymmetric cell processes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of Mr ∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994), S. 41-48 
    ISSN: 0886-1544
    Keywords: myosin-1 ; motility ; F-actin ; liver ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have recently purified and characterized from rat liver, polypeptides of 110-kDa and 130-kDa which possess several characteristics of myosin-1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189-199, 1993]. What roles these myosin-1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellular transport or locomotion. As a first step in establishing their function, we have investigated whether these molecules are capable of supporting motility in vitro. Our results clearly demonstrate that the isolated 130-kDa-calmodulin complex will translocate filaments at a rate of 0.03-0.05 μ/sec; motility is inhibited in free calcium ion concentrations above 0.1 μM. This inhibition is reversed with the addition of exogenous calmodulin. These results provide supporting evidence of a motile role for the 130-kDa-calmodulin complex in vivo. This is the first demonstration that in higher eukaryotes, myosin-1 from a tissue other than intestine will support motility. Partial peptide sequence analysis indicates that the 130-kDa polypeptide resembles the recently described myr 1 [Ruppert et al.: J. Cell Biol. 120:1393-1403, 1993] or MM1α [Sherr et al.: J. Cell Biol. 1405-1416, 1993] gene product. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 13-25 
    ISSN: 0886-1544
    Keywords: Tβ4 ; Tβ10 ; β-thymosins ; Life and Medical Sciences ; Cell & Developmental Biology
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    Topics: Biology , Medicine
    Notes: The β-thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β-thymosins, we have used isoform-specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β-thymosins are uniformly distributed within the cytoplasm. cDNA-mediated overexpression of β-thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β-thymosin were observed to divide. In these cells, β-thymosin was excluded from the midbody which contains an actin filament-rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in living cells. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 97-97 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994), S. 101-107 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994), S. 299-312 
    ISSN: 0886-1544
    Keywords: microtubule motors ; dynein ; cilia ; axoneme ; computer modeling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the “toadstool”) in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 × 24 × 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compct arms and the dynein heavy (H)-chains disappear, while isolated bouquets and dynein H-chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 × 39 × 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard-diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H-chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 327-336 
    ISSN: 0886-1544
    Keywords: HEL cells ; cell spreading ; fibronectin ; diacyl glycerol ; phorbol myristate acetate ; protein kinase C ; staurosporine ; thymosin beta four ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238-246]. We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread. Spreading was ∼85% inhibited by 100 nM staurosporine. PKC-β was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin β4 decreased significantly.To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels. Staurosporine inhibited F-actin increases under all the conditions studied.Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 μM).We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F-actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 337-349 
    ISSN: 0886-1544
    Keywords: microtubules ; glutamylation ; Paramecium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubular networks are extensively developped in many ciliate species. In several of them, we investigate the occurrence of the post-translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82-85; Eddé et al., 1991: J. Cell. Biochem. 46:134-142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425-432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy-terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold-resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules.Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 350-360 
    ISSN: 0886-1544
    Keywords: microtubules ; MAPs ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mr range. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α- and β-tubulin with pI ∼5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 179-193 
    ISSN: 0886-1544
    Keywords: acetylcholine receptor ; deep-etch replication ; sarcolemma ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent α-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 199-204 
    ISSN: 0886-1544
    Keywords: axoneme ; cilia ; flagella ; microtubule ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Observations that were interpreted to provide evidence for equivalent functions of all axonemal dyneins should be reinterpreted, and models based on this assumption should be abandoned. In the future, attempts to understand the mechanisms for flagellar bending, oscillation, and bend propagation should start from the assumption that each type of axonemal dynein may have a specific function. At least three distinct functions can now be identified: bend initiation, maintenance of the angle of propagating bends, and generation of power to overcome viscous resistances. Only the last of these three functions is an outer arm dynein function. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 195-198 
    ISSN: 0886-1544
    Keywords: baculovirus ; tau ; MAP2 ; neuronal cytoskeleton ; growth cones ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The phenotypes induced by the expression of neuronal microtubule-associated proteins (MAPs) in Sf9 cells have provided data on the in situ function of these proteins. Both MAP2 and tau can induce long processes in Sf9 cells, and the processes contain bundles of microtubules. In both cases the microtubules are aligned with their plus ends distal. Tau expression usually induces a single process that is unbranched and of uniform caliber. Processes can form even when the cells are grown in suspension. Microtubules do not extend all the way to the tip; instead the terminal region contains an actin-rich meshwork. Taxol treatment of Sf9 cells also induces the assembly of microtubules into bundles but does not induce process formation in Sf9 cells. Therefore the in vitro properties of tau as a molecule capable of assembling, stabilizing, and bundling microtubules do not fully account for the in vivo ability of tau alone to transduce microtubule assembly into a change in cell shape. The morphological features of the processes induced by MAP2 differ in highly informative ways. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 205-212 
    ISSN: 0886-1544
    Keywords: microtubule motor ; organelle transport ; vesicle transport ; liposomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytoplasmic dynein is the putative motor protein for retrograde organelle transport along microtubules in cells and, thus, must be capable of binding to organelle membranes. Such an attachment may occur via receptor proteins or through a direct interaction of dynein with the membrane phospholipids. We show here that cytoplasmic dynein-synaptic membrane binding does not require a receptor protein and that this binding is mediated by an electrostatic interaction with acidic phospholipids. The properties of cytoplasmic dynein binding to NaOH-extracted synaptic membranes are not significantly affected when those membranes are treated with trypsin to digest endogenous integral membrane proteins. Moreover, purified cytoplasmic dynein is capable of binding to liposomes composed of pure phospholipids. Dynein binds to liposomes with a profile remarkably similar to that of dynein binding to native membranes. Dynein-liposome binding is dependent upon the presence of acidic phospholipids and is disrupted by NaCl. Thus, these studies suggest that electrostatic interactions can effect dynein-membrane binding. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 213-230 
    ISSN: 0886-1544
    Keywords: axoneme-plasmalemma cross-linkers ; cytoskeleton-linked glycoconjugates ; cytoskeleton-membrane interactions ; hydrophobic interactions ; lectin cytochemistry ; SDS-resistance ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-membrane cross-linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross-linkers co-fractionate and maintain their in situ location after Triton X-100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross-linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23°, 60°, 95°C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross-linker glycoconjugates are complex, SDS-resistant multi-molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole-mounted axonemes, in conditions aimed to selectively solubilize the cross-linkers. The cross-linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4 or NaI. The results indicate that the connecting cilium-specific cross-linker complexes are bound via high-affinity interactions to both axoneme and overlying plasma membrane. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 231-242 
    ISSN: 0886-1544
    Keywords: squid axoplasm ; organelle movement ; calmodulin ; actin filaments ; axonal transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley-Liss, Inc.
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  • 52
    ISSN: 0886-1544
    Keywords: cytoskeleton ; actin binding ; transgelin sequence ; gelation ; gene family ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065-1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT-PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987: J. Biol. Chem. 262:5985-5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518-530], and human WS3-10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1-7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal-Ginard, 1991: J. Biol. Chem. 266:13284-13288], and Drosophila mp20 [Ayme-Southgate et al., 1989: J. Cell Biol. 108:521-531] suggest that all of these proteins may be classified as members of a new transgelin multigene family. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 256-264 
    ISSN: 0886-1544
    Keywords: taxol ; cytochalasin ; polarity ; microtubule-associated proteins ; lammelopodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Insect Sf9 cells usually elaborate a highly characteristic single process when infected with a baculovirus encoding recombinant human tau. The processes are unbranched, of uniform caliber, and contain bundles of microtubules. Because taxol treatment alone does not induce process outgrowth in these cells, it is believed that tau confers properties on microtubules that permits the conversion of microtubule assembly into the formation of processes. Here we have analyzed the reorganization of both actin filaments and microtubules during process initiation. A zone of organelle exclusion representing the focal reorganization of actin at one pole of the cell anticipated process emergence. A relationship between actin organization and process emergence was also suggested by a shift from single to multiple process formation after treatment with cytochalasin D. The rate of process elongation doubled after cytochalasin treatment of tau-expressing cells. The increase in rate was due to the inhibition of the growth arrest phases which occur in the absence of cytochalasin. In contrast, Sf9 cells treated with cytochalasin after more than 20 h of tau expression were relatively resistant to the drug's effects. We conclude that actin and microtubules are specifically reorganized during tau-induced process outgrowth and that a dynamic relationship between actin filaments and microtubules effects process formation. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 265-277 
    ISSN: 0886-1544
    Keywords: intermediate filament proteins ; vimentin ; domain function ; filament assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 279-284 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 285-302 
    ISSN: 0886-1544
    Keywords: PMN ; 3-D video-microscopy ; quasielastic laser light scattering ; chemotaxis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three-dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three-dimensional cell edges outlined in the optical sections were analyzed qualitatively in time-lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 μm/min with a period of 50-55 seconds. Within the periodic bursts of speed, a fibroblast-like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2-3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two- to 2.5-fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward.Three-dimensional movies of PMN cells are included in the video supplement. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 327-332 
    ISSN: 0886-1544
    Keywords: tubulin ; isoforms ; Atlantic cod ; Antarctic fish ; evolutionary aspects ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynamic instability of microtubules free of microtubule-associated proteins from two genera of cold-living fishes was measured, by means of video-enhanced differential interference-contrast microscopy, at temperatures near those of their habitats. Brain microtubules were isolated from the boreal Atlantic cod (Gadus morhua; habitat temperature ∽ 2-15°C) and from two austral Antarctic rockcods (Notothenia gibberifrons and N. coriiceps neglecta; habitat temperature ∽ -1.8 to + 2°C). Critical concentrations for polymerization of the fish tubulins were in the neighborhood of 1 mg/ml, consistent with high interdimer affinities. Rates of elongation and frequencies of growth-to-shortening transitions (“catastrophes”) for fish microtubules were significantly smaller than those for mammalian microtubules. Slow dynamics is therefore an intrinsic property of these fish tubulins, presumably reflecting their adaptation to low temperatures. Two-dimensional electrophoresis showed striking differences between the isoform compositions of the cod and the rockcod tubulins, which suggests that the cold-adapted microtubule phenotypes of northern and southern fishes may have arisen independently. © 1994 Wiley-Liss, Inc.
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  • 59
    ISSN: 0886-1544
    Keywords: maytansine ; vinblastine ; diphenylpyridazone ; colchicine ; taxol ; tubulin ; microtubule ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0°C and at 37°C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 μM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 26-40 
    ISSN: 0886-1544
    Keywords: cleavage furrows ; cytokinesis ; actin ; phalloidin ; myosin ; filamin ; talin ; attachment plaques ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 59-68 
    ISSN: 0886-1544
    Keywords: fertilization ; nucleus ; chromosomes ; cytoskeleton ; mitosis ; sulfhydryl groups ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule-based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT-treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg-derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 69-78 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microtubule-associated protein (MAP) ; marine egg extracts ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Alkaline pH favors the assembly of microtubules (MTs) in marine egg extracts [Suprenant and Marsh, 1987: J. Cell Sci. 184:167-180; Suprenant, 1989: Exp. Cell Res. 184:167-180; 1991: Cell Motil. Cytoskeleton 19:207-220] and mammalian brain extracts [Tiwari and Suprenant, 1993: Anal. Biochem. 215:96-103], even though the assembly of purified microtubule protein (MTP) from both of these sources is favored at slightly acidic pH. The present investigation examines whether alkaline pH has a direct or indirect effect on MT nucleation and growth in soluble brain extracts. Cell-free extracts were prepared from bovine cerebral cortex, and a nucleated assembly assay was used to demonstrate that MT assembly in brain extracts is favored at slightly acidic pH. The increase in MT mass found at alkaline pH is due to an increase in the solubility of tubulin not an increase in the extent of assembly On average, 47.7 ± 11.3% of the total tubulin is soluble at pH 7.2, while only 30.9 ± 8.9% of the tubulin is soluble at pH 6.8. A model is proposed that indicates how microtubule proteins from both mammalian brain and marine eggs may be associated with pH-dependent factors. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 59-68 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; paracrystal ; coiled ribbons ; microtubule-associated proteins ; assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Isolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18°C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37°C. However, at 30°C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule-associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium-binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (〉4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP-dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins. © 1994 Wiley-Liss, Inc.
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  • 65
    ISSN: 0886-1544
    Keywords: kinesin ; brain mitochondria ; motility ; membrane-associated ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 108-118 
    ISSN: 0886-1544
    Keywords: Xenopus MAPs ; microtubule cycling ; 230 kDa heat-stable MAP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe the purification of microtubule proteins from Xenopus egg extracts by temperature-dependent assembly and disassembly in the presence of dimethyl sulfoxide and identify a number of presumptive microtubule-associated proteins (MAPs). One of these proteins has a molecular weight of 230 kDa and is immunologically related to HeLa MAP4. We show that this MAP is heat stable and phosphorylated, and that it promotes elongation of microtubules from axonemes. © 1994 Wiley-Liss, Inc.
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  • 67
    ISSN: 0886-1544
    Keywords: Listeria ; actin ; alpha-actinin ; vinculin ; talin ; filopodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: After the infectious bacterium, Listeria monocytogenes, is phagocytosed by a host cell, it leaves the lysosome and recruits the host cell's cytoskeletal proteins to assemble a stationary tail composed primarily of actin filaments cross-linked with alpha-actinin. The continual recruitment of contractile proteins to the interface between the bacterium and the tail accompanies the propulsion of the bacterium ahead of the elongating tail. When a bacterium contacts the host cell membrane, it pushes out the membrane into an undulating tubular structure or filopodium that envelops the bacterium at the tip with the tail of cytoskeletal proteins behind it. Previous work has demonstrated that alpha-actinin can be cleaved into two proteolytic fragments whose microinjection into cells interferes with stress fiber integrity. Microinjection of the 53 kD alpha-actinin fragment into cells infected with Listeria monocytogenes, induces the loss of tails from bacteria and causes the bacteria to become stationary. Infected cells that possess filopodia when injected with the 53 kD fragment lose their filopodia. These results indicate that intact alpha-actinin molecules play an important role in the intracellular motility of Listeria, presumably by stabilizing the actin fibers in the stationary tails that are required for the bacteria to move forward. Fluorescently labeled vinculin associated with the tails when it was injected into infected cells. Talin antibody staining indicated that this protein, also, is present in the tails. These observations suggest that the tails share properties of attachment plaques normally present in the host cells. This model would explain the ability of the bacterium (1) to move within the cytoplasm and (2) to push out the surface of the cell to form a filopodium. The attachment plaque proteins, alpha-actinin, talin, and vinculin, may bind and stabilize the actin filaments as they polymerize behind the bacteria and additionally could also enable the tails to bind to the cell membrane in the filopodia. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 119-134 
    ISSN: 0886-1544
    Keywords: microtubules ; vinculin ; desmin ; sarcolemmal damage ; free radicals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Damage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4-hydroxynonenal (4-HNE), a free radical--induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4-HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin-containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4-HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4-HNE, does not involve Ca2+ -dependent proteases. However, 4-HNE-induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes.Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 169-179 
    ISSN: 0886-1544
    Keywords: calcium-binding proteins ; myonemes ; centrin ; contractility ; ciliates ; protozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 359-359 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 333-345 
    ISSN: 0886-1544
    Keywords: ciliary beat frequency ; metachronal wave ; ciliary coupling ; extracellular ATP ; acetylcholine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated.On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5-9 μm, and the direction of the wave propagation was in the range of 90°-125° clockwise to the ESD.When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10°-30°, and the metachronal wavelength remained unaltered. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 155-164 
    ISSN: 0886-1544
    Keywords: microfilamentous cytoskeleton ; actin binding proteins ; formyl peptides ; ionic extraction ; immunoblots ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: F-actin is a major component of the neutrophil (PMN) cytoskeleton. In basal PMNs, F-actin exists in two structurally and functionally distinct pools: Triton insoluble F-actin (TIF)-cold insensitive, not depolymerizable by dilution, and distributed in pseudopods and submembranous locations; and Triton soluble F-actin (TSF)-unstable in cold, diffusely distributed, and gelsolin enriched. The element(s) conferring these unique properties to the Triton insoluble F-actin pool are unknown, but logically include distinct actin regulatory proteins. To study the morphologic and functional determinants of the Triton insoluble F-actin pool, the distribution and quantity of three candidate regulatory proteins, α-actinin, tropomyosin (TM), and actin binding protein (ABP-280), were compared in F-actin (Triton insoluble and Triton soluble) and G-actin pools isolated from basal and chemotactic factor activated human PMNs in suspension, using immunoblots and ionic extraction. F-actin content was measured by NBDphallacidin binding and gel scans. The results show that: (1) α-actinin, actin binding protein 280, and tropomyosin are localized to TIF and excluded from TSF; (2) TM, α-actinin, and ABP 280 are required to stabilize fractions of Triton insoluble F-actin in PMNs; and (3) chemotactic factor activation results in release of a fraction of TM from the Triton insoluble F-actin pool in temporal association with F-actin polymerization in the Triton insoluble F-actin pool. Shifts in ABP 280 or α-actinin do not occur. The results suggest that TM, α-actinin, and ABP 280 provide structure to TIF and that TM release from TIF is involved in chemotactic factor induced actin polymerization in PMNs. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 165-178 
    ISSN: 0886-1544
    Keywords: WISH ; Keratin ; 3-D reconstruction ; mitosis ; intermediate filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Three dimensional (3-D) reconstruction of four mitotic WISH cells from ultrathin sections gave an informative representation of the spatial distribution of keratin densities in these cells. The correspondence between the densities as studied by transmission electron microscopy (TEM) and the Keratin bodies initially revealed by immunoflourescent colabeling of cultures, was confirmed by immunoelectron-microscopy. The smaller, and sometimes more elongated densities, were relatively abundant just beneath the subplasmalemmal microfilament band; and at certain levels of the mitotic cell they were observed to be connected to neighboring densities by intact intermediate filaments (IFs). The larger and more spherical densities appeared to be somewhat more discrete and randomly distributed. Other observed associations of the keratin densities included the telophase contractile ring of microfilaments, chromosomes, the reformed telophase nucleus, and desmosomal junctions with neighboring interphase cells. Cytochalasin D (CD) treatment of cells displaced the peripheral keratin densities toward the cell membrane. The density volume constituted 0.52% to 1.57% of the total cell volume, and the proportional density size was decreased in the cells that had progressed into anaphase and telophase. The observed formation and subsequent dissolution of keratin densities during mitosis may represent a dynamic mechanism of restructuring the keratin cytoskeleton in an unpolymerized form in order to allow for rapid reformation of interphase cell junctions. The physical associations observed between intact IFs and the keratin densities may provide support at certain depths of the mitotic cell, and the juxtaposition of densities with nuclear components suggests a possible source of and role for keratin IFs during nuclear events. © 1994 Wiley-Liss, Inc.
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  • 74
    ISSN: 0886-1544
    Keywords: myofibril assembly ; myoblasts ; muscle specific proteins ; skeletal muscle ; desmin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic “birthdate.” All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a α-actin, troponin-I, α-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were α-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but α-actinin- or α-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of ∼ 2 μ long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of rnyoblasts stained positively for desmin and rnyofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in rnyoblasts of the myotome and limb bud in vivo, as well as in nonrnuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized rnyogenic cell lines. © 1994 Wiley-Liss, Inc.
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  • 75
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    Keywords: unconventional myosins ; tropomyosin isoform diversity ; myosin regulation ; in vitro motility ; MgATPase ; actin binding ; villin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In this report, we have compared the physical properties and actin-binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-II and brush border (BB) myosin-I. All of the bacterially produced nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced α-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity of F-actin than a tissue-purified striated muscle α tropomyosin. The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle α-tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activity of muscle myosin S1 by ∼ 70-85%, comparable to the inhibition seen with tissue-purified striated muscle α tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase activity of muscle myosin S1 approximately two- to threefold. The in vitro sliding of actin filaments translocated by muscle myosin-II (2.4 μm/sec at 19°C, 5.0 μm/s at 24°C) increased 25-65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility. The actin-activated MgATPase activity of BB myosin-I was inhibited 75-90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin-I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 82-93 
    ISSN: 0886-1544
    Keywords: trichomonads ; cytoskeleton ; antibodies ; electrophoresis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The production of monoclonal antibodies and the use of biochemical techniques revealed that B-type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A-type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein of Trichomonas vaginalis also recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A- or B-type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root-forming proteins in addition to centrin, giardin, and assemblin. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 25-33 
    ISSN: 0886-1544
    Keywords: actin structures ; vinculin ; focal contacts ; spontaneous metastasis ; extracellular pH ; cell motility ; malignancy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three-dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three-dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 μm2 to 0.4 μm2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time-lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three-dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 1-24 
    ISSN: 0886-1544
    Keywords: myofibrillogenesis ; directionality ; non-muscle myosin II ; myosin ; α-actinin ; Z-bodies ; zeugmatin ; titin ; C-protein ; premyofibril ; nascent myofibril ; mature myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When cardiac muscle cells are isolated from embryonic chicks and grow in culture they attach to the substrate as spherical cells with disrupted myofibrils, and over several days in culture, they spread and extend lamellae. Based on antibody localizations of various cytoskeletal proteins within the spreading cardiomyocyte, three types of myofibrils have been identified: 1) fully formed mature myofibrils that are centrally positioned in the cell, 2) premyofibrils that are closest to the cell periphery, and 3) nascent myofibrils located between the premyofibrils and the mature myofibrils. Muscle-specific myosin is localized in the A-bands in the mature, contractile myofibrils, and along the nascent myofibrils in a continuous pattern, but it is absent from the premyofibrils. Antibodies to non-muscle isoforms of myosin IIB react with the premyofibrils at the cell periphery and with the nascent myofibrils, revealing short bands of myosin between closely spaced bands of α-actinin. In the areas where the nascent myofibrils border on the mature myofibrils, the bands of non-muscle myosin II reach lengths matching the lengths of the mature A-bands. With the exception of a small transition zone consisting of one myofibril, or sometimes several sarcomeres, bordering the nascent myofibrils, there is no reaction of these non-muscle myosin IIB antibodies with the mature myofibrils in spreading myocytes. C-protein is found only in the mature myofibrils, and its presence there may prevent co-polymerization of non-muscle and muscle myosins. Antibodies directed against the non-muscle myosin isoforms, IIA, do not stain the cardiomyocytes. In contrast to the cardiomyocytes, the fibroblasts in these cultures stain with antibodies to both non-muscle myosin IIA and IIB. The premyofibrils near the leading edge of the lamellae show no reaction with antibodies to either titin or zeugmatin, whereas the nascent myofibrils and mature myofibrils do. The spacings of the banded α-actinin staining range from 0.3 to 1.4 μm in the pre- and nascent myofibrils and reach full spacings (1.8-2.5 μm) in the mature myofibrils. Based on these observations, we propose a premyofibril model in which non-muscle myosin IIB, titin, and zeugmatin play key roles in myofibrillogenesis. This model proposes that pre- and nascent myofibrils are composed of minisarcomeres that increase in length, presumably by the concurrent elongation of actin filaments, the loss of the non-muscle myosin II filaments, the fusion of dense bodies or Z-bodies to form wide Z-bands, and the capture and alignment of muscle myosin II filaments to form the full spacings of mature myofibrils. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 225-230 
    ISSN: 0886-1544
    Keywords: Weber's Law ; retinal ; retinal analogs ; photoreception ; alga ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The unicellular green alga Chlamydomonas reinhardtii maintains sensitivity of its phototaxis response (alignment of swimming direction along the axis of a light beam) over several orders of magnitude of light intensities. It is widely accepted that the rotation of the swimming cell provides temporal comparisons of light intensities via periodic contrast generated by its asymmetrically positioned refractile eyespot organelle. The cells also exhibit a second behavioral response to light called the photophobic (or stop) response, which is a brief cessation of swimming caused by a temporal change in light intensity. The cells are desensitized to photophobic stimuli by light exposure. Through comparative measurements of both responses, we explain the behavioral basis of the large dynamic range of phototaxis in terms of precise desensitization of the photophobic response. The basis of the explanation is that the flagellar beat changes which cause phototactic orientation are the residual of the photophobic response after desensitization (i.e., “mini-photophobic” reactions which cause brief reorienting motions without a full stop). This interpretation predicts quantitatively the dependence of the extent of desensitization on light intensity and the dependence of onset and maintenance of phototaxis on extent of desensitization. These predictions are tested and confirmed in this report. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 241-249 
    ISSN: 0886-1544
    Keywords: immunofluorescence ; microinjection ; mitotic apparatus ; monoclonal antibodies ; sand dollar egg ; tubulin isotypes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect on fixation on the reactivities of mitotic microtubules with monoclonal anti-tubulin antibodies was investigated by the indirect immunofluorescence procedure. All of the seven antibodies used intensely stained mitotic microtubules in sea urchin eggs lysed and fixed with methanol at -20°C, whereas only two of them stained the stabilized microtubules in the lysed eggs before the fixation. The other five did not stain the mitotic microtubules even after microtubule components other than tubulin were removed by treating the lysed eggs with 0.4 M KCl solution containing taxol. These results exclude the possibility that the fixation affects proteins, which interact with microtubules including microtubule-associated proteins (MAPs) and interfere with the binding of monoclonal antibodies with tubulin, and strongly suggest that the fixation directly affects the three-dimensional conformation of tubulin Furthermore, microinjection of these antibodies indicated the results as follows [combining the results reported previously; Oka et al., 1990: Cell Struct. Funct. 15: 373-378]: The antibodies which stained mitotic microtubules stabilized in the lysed eggs induced disassembly of native mitotic microtubules in the living eggs, but those which did not stain the stabilized microtubules did not disassemble the native microtubules. From these results, it is suggested that the monoclonal antibodies which stain microtubules in the eggs lysed but not fixed are useful for microinjection experiments. © 1994 Wiley-Liss, Inc.
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  • 81
    ISSN: 0886-1544
    Keywords: binding of caldesmon to myosin ; actin-activated ATPase activity of myosin ; actin-myosin interaction with in vitro motility assay ; myosin-binding domain of caldesmon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We reported previously that smooth muscle caldesmon stimulates the ATP-de-pendent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurment and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 259-270 
    ISSN: 0886-1544
    Keywords: calsequestrin ; calreticulin ; sarcoplasmic reticulum ; skeletal muscle ; myofibril ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A major Ca2+-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca2+-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, antidesmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 280-290 
    ISSN: 0886-1544
    Keywords: mitosis ; phosphorylation ; protein phosphatase ; okadaic acid ; mitotic apparatus ; sea urchin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein component of isolated mitotic apparatus having a relative molecular mass of 62,000 (p62) is a substrate of a calcium/calmodulin dependent protein kinase, and the phosphorylation of p62 in vitro correlates directly with microtubule disassembly. In vivo experiments have determined the phosphorylation of p62 increases after fertilization; maximum incorporation of phosphate occurs during late metaphase/early anaphase and decreases thereafter. Because the level of p62 is constant throughout the cell cycle [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-54] the decrease in phosphorylation of p62 observed after anaphase onset is most likely due to the action of a phosphatase. By examination of the relative amount of phosphorylated p62 which remained radiolabeled as a function of time using a standard in vitro phosphorylation assay, the activity of a phosphoprotein phosphatase capable of dephosphorylating p62 in the isolated mitotic apparatus was observed. To characterize the p62 phosphatase, okadaic acid and calyculin A were used to inhibit the dephosphorylation of p62 in vitro. It was found that specific concentrations of okadaic acid (50-500 nM) and of calyculin A (10-100 nM) were effective at inhibiting the dephosphorylation of p62 in vitro. Lower concentrations of either inhibitor had a negligible effect on dephosphorylation of p62. These data indicate the presence of phosphoprotein phosphatase type 1 activity associated with mitotic apparatus isolated from sea urchin embryos using the procedures described here. The implications of these findings relative to our understanding of the regulation of mitosis and cytokinesis are discussed. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 321-338 
    ISSN: 0886-1544
    Keywords: protrusive activity ; adherens junctions ; stress fibers ; permeabilized cell models ; myosin light chain kinase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Addition of protein kinase inhibitor H-7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169-178] it was demonstrated that intercellular junctions in H-7-treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H-7-treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact-attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N-cadherin associated with cell-cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H-7 suppresses cellular contractility. Thus, short pretreatment with H-7 leads to strong inhibition of the ATP-induced contraction of saponin permeabilized cells. Comparison of H-7 effects with those of other kinase inhibitors revealed that H-7-induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31-8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H-7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell-cell junctions in low-calcium medium. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 339-344 
    ISSN: 0886-1544
    Keywords: microfilament ; phalloidin ; immunoblotting ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Anti-actin monoclonal antibodies were prepared using phalloidin-stabilized actin that was purified from pea roots by DNase I affinity chromatography. One monoclonal antibody, designated mAb3H11, bound plant actin in preliminary screenings and was further analyzed. Immunoblot analysis showed that this antibody had a high affinity for plant actin in crude and purified preparations but a low affinity for rabbit muscle actin. In immunoblots of plant extracts separated on two-dimensional gels it appeared to bind all actin isoforms recognized by the JLA20 anti-chicken actin antibody. Using immunofluorescent cytochemistry, the antibody was used to observe actin filaments in aldehyde-fixed and methanol-treated tobacco protoplasts. These results indicate that mAb3H11 should be a useful reagent for the study of plant actins. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994), S. 345-353 
    ISSN: 0886-1544
    Keywords: IgE receptors ; receptor activation ; myosin II phosphorylation ; PKC ; RBL 2H3 cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat basophilic leukemia cells secrete histamine and serotonin in response to cross-linking of the IgE receptor by multivalent antigen [Metzger et al., 1986: Ann. Rev. Immunol. 4:419-470]. Receptor crosslinking also induces phosphorylation of the light and heavy chains of myosin II with kinetics similar to that of secretion [Ludowyke et al., 1989: J. Biol. Chem. 264:12492-12501]. Here we show that myosin II localization changes after activation with similar kinetics. Furthermore, these changes are coincident with changes in cell shape and increase in motile activity induced by activation. Within 2 min, activated cells begin to flatten, spread on their substratum, and extend lamellipodia which show active ruffling. Quantitation of the extent of cell spreading from video micrographs shows that 48% of the cells increase significantly in surface area by 5 min and 71% by 15 min. Myosin II is uniformly distributed in unactivated cells but is deficient in newly formed lamellipodia that start to appear at 2 min after activation. In contrast these lamellipodia show strong staining for actin. Further changes in myosin organization are detected by 15 min after activation when myosin reappears in the cell periphery, is concentrated in the perinuclear area, and is also organized in punctate linear arrays that extend from the nucleus to the cell periphery. The kinetics of the early cell shape changes and formation of the myosin-deficient lamellipodia correlate well with, and may relate to, the increase in the level of myosin II phosphorylation reported by Ludowyke et al. [1989: J. Biol. Chem. 264:12492-12501]. Changes in the distribution of cell surface-bound IgE also occur upon antigen activation, and they correlate with the myosin distribution in a manner that suggests that they may be driven by myosin II. © 1994 Wiley-Liss, Inc.
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  • 87
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    Keywords: spermatozoa ; centriole ; axoneme ; immunogold ; acetylated tubulin ; tubulin heterogeneity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti-acetylated tubulin antibody (6-11B-1), confirming previous results of Hermo et al. [Anat. Rec. 229:31-50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2-D PAGE of mouse and hamster sperm extracts showed a high level of α and β-tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous α as well as β-tubulin isoforms. This suggests that the major testis-specific tubulin isotypes (mα3/7 and mβ3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 69-78 
    ISSN: 0886-1544
    Keywords: kinesin ; dynein ; MAP-motor interactions ; microtubule arrays ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Bundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark-field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent-extracted and reactivated. We have studied the nucleotide-specificity and the drug-sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 88-96 
    ISSN: 0886-1544
    Keywords: cell movement ; speed ; persistence time ; colcemid ; alveolar macrophage ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of microtubules in random cell migration was investigated using time-lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long-term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley-Liss, Inc.
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  • 90
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    Keywords: cleavage furrow ; cytokinesis ; intercellular bridge ; polar lobe constriction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag+-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag+-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag+-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 161-168 
    ISSN: 0886-1544
    Keywords: fluorescent nucleotide analogs ; methylanthraniloyl ATP ; anthraniloyl ATP ; Chlamydomonas ; axonemal mutants ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Substrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose-modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivate wild-type Chalmydomonas axonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset of Chlamydomonas outer arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 248-261 
    ISSN: 0886-1544
    Keywords: myogenesis ; protein isoforms ; muscle types ; Z-disc ; phosphorylation ; chicken muscle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin-binding protein (ABP) filamin is associated with stress fiber-like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z-disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z-disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two-dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non-striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14-somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 27 (1994), S. 286-286 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 27 (1994), S. 150-160 
    ISSN: 0886-1544
    Keywords: axoneme ; bend propagation ; computer simulation ; flagella ; microtubule sliding ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate, Ciona, are reactivated in the presence of 2 mM Li+ has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occuring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid-region of the flagellum.Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 135-142 
    ISSN: 0886-1544
    Keywords: bidirectional swimming ; flagellar movement ; helical bends ; 9+0 axoneme ; planar bends ; viscosity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spermatozoa of the small myzostomid worm Myzostomum cirriferum usually swim with the flagellum foremost but occasionally stop and then swim with the head foremost. The spermatozoa have axoneme of the 9+0 type; thus each lacks the central pair microtubules. The flagellum emerges in the anterior end of the cell body and attaches to it with junctions. To understand the mechanism regulating the swimming direction of the spermatozoa, we recorded the sperm and their flagellar movements using a video camera with a high-speed shutter. The effects of calcium and viscosity on these movements were also examined.The cell body with the flagellum attached to it formed a curved plate during beating, while the free portion of the flagellum beats with small helical bends. Motive force to propel a spermatozoon was mainly due to the bends in the cell body. The spermatozoa reversed the direction of their swimming as a result of a change in the direction of bend propagation. The direction of bend propagation was regulated by calcium; the bends in the cell body propagated from the end of the head toward the free portion of the flagellum at low concentrations of Ca2+, whereas the direction of bend propagation was reversed at high concentrations of this ion. High viscosity of the medium stimulated a change in the direction of bend propagation. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 28 (1994), S. 143-154 
    ISSN: 0886-1544
    Keywords: microtubule-associated proteins ; microtubule nucleation ; tubulin ; cytoskeleton ; axon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The neuronal microtubule-associated protein tau has been implicated in the development of axonal morphology including the organization of microtubules into a uniformly oriented array of microtubules commonly referred to as “bundle.” Determination of the functional organization of tau has revealed that regions of tau protein which flank the microtubule-binding domain affect the bundling of microtubules in vitro with a microtubule-binding fragment of tau being most effective [Brandt and Lee, 1993: J. Biol. Chem. 268:3414-3419]. In order to study the relation of microtubule bundles that form in vitro to those observed in the axon, we determined the orientation of individual microtubules in bundles and the effects of bundling on microtubule assembly and stability in cell-free assembly reactions. Here we report that bundles induced by a microtubule-binding fragment of tau contain randomly oriented microtubules as determined by using the difference in growth rates at microtubule plus and minus ends. We demonstrate that in vitro bundling increases microtubule growth (about 30%), stabilizes microtubules against dilution- and cold-induced disassembly, and allows microtubule nucleation despite the absence of a tau region which has previously been shown to be required for tau-dependent microtubule nucleation. We conclude that conditions that stabilize microtubules can lead to bundle formation and allow microtubule assembly by a mechanism different from that employed by microtubule-associated proteins. The data also support the view that additional mechanisms besides the action of tau and tubulin exist in order to organize microtubules in the axon. © 1994 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 29 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 28 (1994), S. 346-358 
    ISSN: 0886-1544
    Keywords: Listeria monocytogenes ; actin ; alpha-actinin ; actin polymerization ; assembly ; disassembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Listeria monocytogenes can penetrate and multiply within a variety of cell types, including the PtK2 kidney epithelial line. Once released within the cytoplasm, L. monocytogenes acquires the capacity for rapid movement through the host cell [Dabiri et al., 1990: Proc. Natl. Acad. Sci. 87:6068-6072]. In the process, actin monomers are inserted in proximity to one end of the bacterium, forming a column or tail of actin filaments [Sanger et al., 1992: Infect. Immun. 60:3609-3619]. The rate of new actin filament growth correlates closely with the speed of bacterial migration. In this study we have used fluorescently labeled actin and alpha-actinin to monitor the movement and turnover rate of actin and alpha-actinin molecules in the tails. The half-lives of the actin and alpha-actinin present in the tails are approximately the same: actin, 58.7 sec; alpha-actinin, 55.3 sec. The half-life of alpha-actinin surrounding a dividing bacterium was 30 sec, whereas its half-life in the tails that formed behind the two daughter cells was about 20-30% longer. We discovered that the speeds of the bacteria are not constant, but show aperiodic episodes of decreased and increased speeds. There is a fluctuation also in the intensities of the fluorescent probes at the bacterium/tail interface, implying that there is a fluctuation in the number of actin filaments forming there. There was no strong correlation, however, between these fluctuating intensities and changes in speed of the bacteria. These measurements suggest that while actin polymerization at the bacterial surface is coupled to the movement of the bacterium, the periodic changes in intracellular motility are not a simple function of the number of actin filaments nucleating at the bacterial surfaces. © 1994 Wiley-Liss, Inc.
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  • 99
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 20-28 
    ISSN: 0886-1544
    Keywords: proliferation ; large T antigen ; peripheral nervous system ; cytoskeleton ; microtubules ; myelination ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Schwann cells (SC), the myelinating cells of the peripheral nervous system, show a remarkable capacity to switch from a differentiated state to a proliferative state both during development and peripheral nerve regeneration. In order to better understand the regulatory mechanisms involved with this change we are studying a Schwann cell line transfected with the SV-40 large T gene (TSC). Serum-free medium combined with elevating intra-cellular cAMP levels produced a slower proliferating TSC whose morphology changed from pleiomorphic to process bearing, reminiscent of primary SC in culture. This change was abrogated by colcemid but was unaltered by cytochalasin D, indicating a major role for microtubules. Ultrastructural studies demonstrated numerous microtubules in the cellular extensions which correlated with strong immunocytochemical staining for tubulin in the processes. Analysis of cytoskeletal fractions from the treated cells revealed a greater proportion of tubulin in the polymerized state compared with untreated cells which closely resembled the distribution in primary SC. The cytoskeletal changes observed in the TSC as a result of elevating the intra-cellular cAMP levels may reflect the earliest cellular changes in the induction of myelination. © 1994 Wiley-Liss, Inc.
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  • 100
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 110-116 
    ISSN: 0886-1544
    Keywords: high-molecular weight MAPs ; microfilaments ; microtubules ; low-shear viscometry ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: High molecular weight microtubule-associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G- and F-actin. Using a solid-phase immunoassay we show that MAP1A binds in a dose-dependent manner to both G-actin and F-actin. Addition of MAP1A to F-actin causes gelation of F-actin and SDS-PAGE analysis shows that MAP1A co-sediments with the gelled network, under conditions where F-actin alone does not pellet. The low apparent viscosity of F-actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F-actin. Co-incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non-neuronal cellular morphology. © 1994 Wiley-Liss, Inc.
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