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  • 1
    Electronic Resource
    Electronic Resource
    A calcium- and pH-regulated actin binding protein from D. discoideum (1982)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 2 (1982), S. 287-308 
    Details
    ISSN: 0886-1544
    Keywords: actin-binding protein ; Dictyostelium ; cytoskeleton ; amoeboid movement ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein from Dictyostelium discoideum with an apparent subunit molecular weight of 95,000 daltons (95K protein) was previously identified as an actin-binding protein ‘Hellewell and Taylor, 1979’. In this paper, we present a method for purifying the protein, and characterize some important aspects of its structure and function. Purification of the 95K protein is achieved by fractionation with ammonium sulfate followed by chromatography on DEAE-cellulose, gel filtration on 6% agarose, and final purification on hydroxyapatite. The 95K protein is a dimer, composed of apparently identical subunits. It is a rod-shaped molecule, 38 nm in length, with a Stokes radius of 74 Å. In these structural properties, the 95K protein is similar to muscle and nonmuscle α-actinins. The 95K protein and filamin are equally competent, when compared on a weight basis, to enhance the apparent viscosity of actin as determined by falling ball viscometry. The apparent viscosity of mixtures of the 95K protein and actin is dramatically reduced at pH greater than 7.0 or free ‘Ca2+’ greater than 10-7 M. We also examine the mechanism by which calcium regulates the interaction of the 95K protein and actin. A change in free ‘Ca2+’ induces no detectable change in the quaternary structure of the 95K protein. Our experiments indicate that the 95K protein does not dramatically alter the length distribution of actin filaments in the presence of micromolar free ‘Ca2+’. A large fraction of the 95K protein cosediments with actin in the presence of low free ‘Ca2+’ (ca. 3 × 10-8M), but not in the presence of high free ‘Ca2+’ (ca. 4 × 10-6M). We conclude that increased free ‘Ca2+’ inhibits gelation of actin by the 95K protein by reducing the affinity of the 95K protein for actin. We propose that 95K protein is an important component of the cytoskeletal/contractile system in D. discoideum amoebae.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970020308
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  • 2
    Electronic Resource
    Electronic Resource
    Induction of shape transformation in sea urchin coelomocytes by the calcium ionophore A23187 (1984)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 57-71 
    Details
    ISSN: 0886-1544
    Keywords: actin ; calcium ; coelomocytes ; ionophore ; pH ; shape transformation ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the ability of the Ca+ + ionophore A23187 to induce the transformation of petaloid sea urchin coleomocytes to the filopodial form. The response of individual cells to different media was observed with time-lapse phasecontrast video microscopy. In the presence of 1 mM CaCl2, isotonic medium containing 1-5 μM A23187 produces a similar shape transformation to that caused by hypotonic shock. Higher concentrations of ionophore (10-20 μM) induce the formation of filopodia that are thinner and less rigid than those generated by hypotonic shock or low doses of ionophore. A23187 also induces shape transformation in highly flattened cells that do not respond fully to hypotonic shock. The induction of cytoplasmic alkalinization by NH4Cl, methylamine-HCl, or the Na+ ionophore monensin does not induce shape transformation, suggesting that increased intracellular pH is not the stimulus for this process. Ultrastructural changes in cytoskeletal organization were examined in negatively stained detergent-extracted cells. Low doses of ionophore produce filopodia that are indistin-guishable from those of hypotonically shocked cells, with actin filament bundles that are straight and cohesive along their entire length. High concentrations of ionophore produce filopodia with filament bundles that branch repeatedly and splay apart near their tips, forming loops and irregular curves. These results suggest that an increase in intracellular free Ca+ + concentration acts as the trigger that stimulates coelomocyte shape transformation, but that abnormally high concentrations of intracellular Ca+ +, produced by high doses of ionophore, interfere with actin filament bundling.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970040107
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  • 3
    Electronic Resource
    Electronic Resource
    Bending patterns of Chlamydomonas flagella: II. Calcium effects on reactivated Chlamydomonas flagella (1985)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 53-60 
    Details
    ISSN: 0886-1544
    Keywords: calcium ; Chlamydomonas ; flagella ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ca2+ has profound effects on the movement of cilia and eukaryotic flagella, including those of Chlamydomonas. Two clear changes seen in Chlamydomonas flagella with changes in Ca2+ are beat frequency and symmetry. Photographic and computer assisted analysis of flagellar bending patterns on a uniflagellate mutant of Chlamydomonas have been used to examine details of the effects of Ca2+ on the movement of ATP-reactivated, demembranated flagella. In addition to the forward mode bending pattern seen at low Ca2+ concentrations (10-9 M), which has a frequency of about 50 Hz and the reverse mode bending pattern seen at high Ca2+ concentrations (10-4 M) with a frequency around 70 Hz, we carefully examined bending patterns in the intermediate Ca2+ concentration range of 1-6.5 × 10-6 M. In this intermediate range, the bending patterns have significantly reduced asymmetry and slightly increased frequency, compared to the motility observed at low Ca2+ concentrations. These observations indicate that changes in these two parameters of motion do not occur in parallel and suggest that the effects of Ca2+ may be a multicomponent process. Physiologically, these changes in the beat pattern at intermediate Ca2+ may signal either (1) the beginning stages of transition to the symmetrical, high-frequency beating seen at high Ca2+, or (2) a more normal forward mode motility for the trans flagellum as suggested by Kamiya and Witman [1984]. No large amplitude bending patterns associated with transitions between forward and reverse mode beating in intact cells were seen at the intermediate Ca2+ concentrations.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970050105
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  • 4
    Electronic Resource
    Electronic Resource
    cAMP-mediated inhibitory effect of calmodulin antagonists on ciliary reversal of ParameciumA part of this work was presented as a poster session at the 3rd International Congress on Cell Biology, Tokyo, 1984. (1987)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 154-159 
    Details
    ISSN: 0886-1544
    Keywords: calcium ; protein phosphorylation ; TFP ; Triton-extracted model ; ciliary orientation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To explore possible roles of calmodulin in Ca2+-induced ciliary reversal, we tested the effects of calmodulin antagonists on Triton-extracted models of Paramecium. In the extracted models prepared by the method of Naitoh and Kaneko [Science 176:523-524, 1972], the Ca2+ -induced ciliary reversal was not inhibited by calmodulin antagonists, trifluoperazine (TFP), or 5-chloro-l-naphthalenesulphone amide (W-7). However, in the presence of adenosine 3′,5′-cyclic mono-phosphate (cAMP), whose concentration is below the one that alters the ciliary direction, TFP inhibited ciliary reversal and the models swam forward at 10-5 M Ca2+. When the washing medium in the preparation of the extracted models was replaced with one containing MgCl2, the extracted model showed sensitivity to calmodulin antagonists without addition of cAMP; at 10-5 M Ca2+, 40 μM TFP or 100 μM W-7 inhibited the ciliary reversal and the models swam forward. Such effect of antagonists was abolished by an inhibitor of cAMP-dependent protein kinase. On the other hand, addition of cAMP enhanced the inhibitory effect of antagonists. These results suggest that calmodulin antagonists act to increase the extent of cAMP-dependent protein phosphorylation that inhibits the Ca2+ -induced ciliary reversal.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970070207
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  • 5
    Electronic Resource
    Electronic Resource
    Trifluoperazine-induced changes in swimming behavior of paramecium: Evidence for two sites of drug action (1984)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 4 (1984), S. 249-267 
    Details
    ISSN: 0886-1544
    Keywords: Paramecium ; trifluoperazine ; cilia ; calmodulin ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted “models” of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 μM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the “barium dance”), but cells preincubated in TFP did not “dance.” Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 ≥ pCa ≥ 6) were not affected by addition of up to 40 μM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa 〈 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970040404
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  • 6
    Electronic Resource
    Electronic Resource
    Regulation of activation state and flagellar wave form in epididymal rat sperm: Evidence for the involvement of both CA2+ and cAMP (1987)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 8 (1987), S. 324-332 
    Details
    ISSN: 0886-1544
    Keywords: sperm motility ; procaine ; calcium ; cAMP ; flagellum ; epididymis ; TMB-8 ; hyperactivation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine-HCI or 0.1 mM 8-(N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X-100-extracted sperm models will exhibit the same pattern of motility and curvature as procaine- or TMB-8-activated cells, but only when calcium is removed by a strong chelating agent, and in the pesence of cAMP (3 μM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine-treated intact cells was not accompained by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompained by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970080405
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  • 7
    Electronic Resource
    Electronic Resource
    Intracellular particle motions (cytoplasmic streaming) in staminal hairs of Setcreasea purpurea: Effect of azide and low temperature (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 305-313 
    Details
    ISSN: 0886-1544
    Keywords: cytoplasmic streaming ; Setcreasea purpurea ; intracellular particle movements ; intercellular transport ; azide ; low temperature ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytoplasmic streaming and its response to azide and low temperature were examined by using high-resolution video-enhanced light microscopy in Setcreasea purpurea staminal hair cells of immature flowers. Particles and organelles examined moved along well-defined pathways, in repeated and unequal saltatory steps, at different rates and sometimes against the main direction of flow (bidirectionally) in both transvacuolar strand and peripheral cytoplasm. Particle movements were reversibly inhibited with azide. Low temperatures caused transvacuolar strands to shift or break. This cytoplasm accumulated in areas outside of the vacuole where spherosomes continued to saltate, but not along well-defined pathways. In the peripheral cytoplasm, however, the spherosomes continued to move normally, amyloplasts became swollen, and they plus the other organelles (except spherosomes) were stationary. Normal particle movements were obtained when chilled cells were rewarmed to 27°C for ca 15 min.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060307
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  • 8
    Electronic Resource
    Electronic Resource
    Ultrastructure and motion analysis of permeabilized paramecium capable of motility and regulation of motility (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 73-84 
    Details
    ISSN: 0886-1544
    Keywords: cilia ; metachronal waves ; electron microscopy ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 ± 19 μm/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick-fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X-100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 ± 20 μm/second. When these cells are quick-fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 ± 7 μm/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090108
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  • 9
    Electronic Resource
    Electronic Resource
    Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 184-189 
    Details
    ISSN: 0886-1544
    Keywords: cell locomotion ; cell motility ; calcium ; polymorphonuclear leukocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N-formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time-lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 μM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 μM) plus external calcium inhibited locomotion.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090210
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  • 10
    Electronic Resource
    Electronic Resource
    Roles of actin filaments and three second-messenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth (1991)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 20 (1991), S. 7-29 
    Details
    ISSN: 0886-1544
    Keywords: calcium ; cAMP ; protein kinase C ; growth cone ; filopodia ; lamellipodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In a previous study (J. Cell Biol. 109:1229-1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin-stabilizing drug phalloidin could block or reverse calcium-ionophore-induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium-induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second-messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium-permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2-AM-measured calcium levels indicated that the effects of cAMPmanipulations could be partially explained by a cAMP-induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent mechanism.
    Additional Material: 16 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970200103
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