ALBERT

Notizen: Although bacterial flagellar sheaths were observed over 30 years ago, they may still be characterized as structures in search of a function. In addition to true sheaths, bacterial flagella may possess other adornments that cause an increase in the organelle's cross-sectional diameter. These “complex flagella” are sharply differentiated from sheathed flagella. Immunological and chemical distinctions have been found between flagellar sheaths, flagellar cores, and LPS layers inferred to be the sheath sensu stricto. Although complex flagella may serve as specific receptors for flagellotropic phages or in allowing for more efficient swimming in viscous environments, similar functions have not yet been attributed to true sheaths. It is postulated that flagellar sheaths may allow for specific interaction between a bacterium and a surface. In addition, there is a problem as to the relationship between a rapidly rotating flagellum and the sheath.

Notizen: We have utilized a video-enhanced contrast system coupled to a DIC-equipped microscope to examine the motility of both whole coelomocytes and individual filopodia. When the cells are left in diluted coelomic fluid, they exhibit a fibroblast-like mode of translocation across the substrate. These cells extend lamellipodia at their advancing margin and develop retraction fibers at the trailing edge. Filopodia are actively extended from the lamellipodia of the advancing margin. Cells that are washed free of the coelomic fluid and placed in an isotonic buffer lose their ability to translocate. Filopodia on these stationary cells are seen to undergo a series of waving and bending motions. These motions are rapid and result in a filopodium folding back upon itself only to reextend later. Both forms of motility are discussed in light of the existing structural and biochemical knowledge of this and other cell types.

Notizen: The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero.The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.

Notizen: The flagella of Chlamydomonas reinhardtii protrude through the cell wall via short, tunnel-like openings that are lined with 11 nm × 500 nm fibers arranged in parallel array. These cylindrical collections of fibers presumably permit free movement of the flagella within the cell wall. In this report electron-microscopic evidence is presented showing that during the initial stages of the mating reaction intact collars slip off of the ends of the flagella when cell wall loss occurs. Electrophoretic analysis of isolated collars reveals one major protein and several minor species.

Notizen: The locomotion of C. elegans spermatozoa resembles, in many respects, the crawling movements of other eukaryotic cells. However, these sperm contain surprising little actin, which plays no apparent role in this cell's motility. Electron microscopy has revealed that crawling spermatozoa retain a strict morphological polarity so that the organelle-filled cell body is separated from the pseudopod by an array of cytoplasmic laminar membranes. When sperm crawl only the pseudopod contacts the substrate; the cell body is either pulled behind or carried on top of the rear portion of the pseudopod. Fingerlike projections which extend forward from the leading edge of the pseudopod initiate contact with the substrate. The underside of the pseudopod exhibits areas of close (40 nm separation) membrane-substrate association with intervening areas of wide (up to 300 nm) membrane-substrate gaps. The pseudopod cytoplasm contains 2-nm filaments but no filamentous actin has been observed. These 2-nm filaments were detected in thin sections of crawling cells and in negative-stained remnants of spermatozoa disrupted by either hypotonic buffer on Triton X-100. The filaments are found both free in the cytoplasm and closely associated with the cytoplasmic face of the plasma membrane and are usually oriented along the long axis of the cell. Neither the identity nor the function of these filaments has been established although their location and orientation suggest that they may be involved in generating propulsion.

Notizen: Changes in the state of polymerization of actin and phosphorylation of myosin have been observed in polymorphonuclear leukocytes (PMNs) soon after the addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine. At a time when the cells are observed to extend many ruffles or lamellipodia from their surface, the fraction of the cellular actin present in a monomeric form is decreased by about 25% as assayed by the ability of the G-actin to inhibit DNAase. These changes are temporally correlated with an increase in the staining by nitrobenzooxadiazole (NBD)-phallacidin, a probe that binds F-actin selectively. The NBD-phallacidin staining is observed in the surface ruffles. When the peptide concentration is decreased by addition of a tenfold excess of buffer, cells withdraw their surface ruffles and form blebs. These changes correlate with an increase in the G-actin levels detected with the DNAase inhibition assay. An increase in phosphorylation of the 20,000-dalton light chain of myosin is also observed in leukocytes stimulated by addition of chemotactic peptide. These observations of changes in cytoskeletal proteins of PMNs provide a beginning for further studies on the regulation of cell motility by chemotactic factors.

Notizen: The role of structural elements in the organization and maintenance of focal contacts was studied by microinjecting into tissue culture cells specific probes which interfere with filamentous actin or with vinculin: actin interaction. Injection of actin capping proteins from Physarum and brain resulted in breakdown of microfilament bundles starting at their distal ends and in loss of focal contacts. This process was fully reversible. Injection of a high affinity antibody against chicken gizzard vinculin led to partial breakdown of microfilament bundles concomitant with disruption of focal contacts with vinculin remaining at the plasma membrane. This process was irreversible.

Notizen: The muscle-tendon junction of murine skeletal muscles has been analyzed by a variety of extraction techniques, by myosin subfragment-1 binding experiments, and by ultrastructural immunocytochemistry. The results indicate that the muscle-tendon junction is composed of four distinct domains: an intracellular domain, the internal lamina; a domain connecting the internal lamina with the lamina densa of the external lamina, the connecting domain; the lamina densa; and a domain which attaches the lamina densa to the collagen fibers, the matrix. Each of these domains is distinct with respect to position, three-dimensional organization, and molecular composition, and is therefore considered to have a unique role in the transmission of contractile force.

Notizen: When skeletal, cardiac, and smooth muscle is exhaustively extracted with a protein-unfolding reagent such as 6 M guanidine HCl and a disulfide-reducing reagent such as 5% β-mercaptoethanol, a tissue ghost remains intact and retains the characteristic shape and dimensions of the tissue before extraction. In the case of chicken pectoral muscle, the tissue ghost contains 1% of the original muscle proteins. Guanidine HCl extraction followed by collagenase treatment of glycerol-extracted chicken pectoral muscle releases a clean preparation of elongated structures containing 0.2% of the original protein and representing the covalently cross-linked remnants of the muscle fibers. The material of these muscle fiber ghosts extends throughout the interior of the cell. Antibodies raised against the tissue ghosts of smooth muscle cross-react with glycerol extracted skeletal myofibrils, forming a banding pattern which coincides with the banding pattern observed when myofibrils are reacted with antibodies against titin. Titin, a large and soluble protein found in skeletal muscle, cross-reacts with our antigizzard antibody. However, amino acid analysis of the muscle fiber ghosts indicates that titin cannot be the only subunit of the insoluble polymer, but that one or more proteins with a very high glycine and alanine content and a very low basic and acidic amino acid content must also form part of the covalently cross-linked matrix. The possibility is presented that this matrix may be the basis of the superthin 2-3-nm filaments which have been observed in a variety of cell types.

Notizen: The sea urchin egg at fertilization is an ideal model in which to study actin-mediated surface activity. Electron microscopy of unfertilized eggs demonstrates the presence of thousands of well-arrayed short microvilli, which appear supported by cytochalasin-sensitive actin oligomers as detected with rhodamine-labeled phalloidin staining of permeabilized eggs. At insemination, the previously short microvilli elongate and cluster around the successful sperm during incorporation. Phalloidin staining demonstrates a tremendous recruitement of polymerized actin into the site of sperm incorporation, resulting in the formation of the fertilization cone. Fertilization of cytochalasin-treated eggs results in the normal activation of the metabolic and bioeletric events, but sperm incorporation does not occur since the localized actin assembly required for fertilization cone formation is precluded. After sperm incorporation, the entire fertilized surface is restructured, as a result of a massive polymerization of actin to produce a burst in microvillar elongation. Addition of cytochalasin to eggs immediately following sperm incorporation demonstrates the recruitment of actin assembly for the proper progression through the first cell cycle. During normal cell divison, the egg surface retains the long microvilli. The furrow which forms at cytokinesis does not appear as a unique new structure, but rather as a reorganization of the cortical microfilaments. Quantitative fluorescence microscopy argues against an increase in microfilaments during early cytokinesis. At the latest stages of cytokinesis, a thickening of the cortical actin is noted, which could possibly be interpreted as a contractile ring. A minor basal level of actin assembly with numerous nucleation sites in unfertilized eggs and a tremendous but localized assembly of microfilaments surrounding the sperm during incorporation, followed by a massive global microfilament assembly event to elongate the fertilized egg microvilli resulting later in the reorganization of these microfilaments to produce the forces necessary for cytokinesis, highlight the utility of the study of sea urchin eggs at fertilization for understanding actin-membrane interactions.

Notizen: In order to assess the role of cytoskeletal structure in modulating cell surface topography during cell transformation, cytoskeletal organization of 3T3 mouse cells transformed with a tsA mutant of simian virus 40 (SV40) was studied in detail by correlative light and electron microscopy. Detergent-extracted, criticalpoint dried whole cells observed in the electron microscope were seen to contain well-organized microfilament bundles (stress fibers) traversing the longitudinal axis of cells grown at the restrictive temperature (39°C). When grown at the permissive temperature (32°C), cells prepared in this manner were not observed to contain such structures. However, when semithin sections (0.5 μm) were viewed by transmission electron microscopy at 120 kV, short microfilament bundles were seen in 32°C-grown cells. There was an alteration in the morphology of these structures at sites of attachment to the substratum (focal contacts), and they were shorter in length than microfilament bundles of 39°C-grown cells. A difference was also observed between the two phenotypes in the layer of microfilaments associated with the dorsal cell surface. Since it is this layer that directly determines cell surface architecture, it is proposed that changes in microfilament bundle-generated surface tension are responsible for alterations of this layer, leading to an altered cell surface morphology. Tension may be modified by disturbances in focal contacts (or adjacent regions) or altered actin-associated protein(s).

Notizen: Measurements of the mechanical properties of the erythrocyte membrane provide a direct assessment of the proper function of its structural components. To assess the effects of alterations in molecular structure on membrane mechanical properties, measurements have been performed on cells from six individuals whose membranes contain inherited, biochemically characterized structural defects. Because the contribution of the memmbrane skeleton to the mechanical behavior of the membrane is most evident in shear deformation, mechanical experiments were performed to measure the material constants which characterize the response of the membrane to shear force resultants. The surface elastic shear modulus characterizes the elastic response of the membrane; the yield shear resultant is the maximum shear force resultant which the membrane can support elastically; and the plastic viscosity coefficient characterizes the rate of membrane deformation when the elastic limit has been exceeded.Generally, it was found that when the molecular defect is found to occur in a region of the skeleton which is stress-supporting, the maximum elastic strength of the membrane is reduced. However, the magnitude of the reduction can be quite different for membranes having similar or even identical defects. In some cases the differences can be attributed to the removal of the most fragile cells of the population by the spleen, but other results indicate that the biochemical description of the defects may be incomplete. These results emphasize the need for further refinements both in the biochemical characterization of membrane skeleton structure and in the description and measurement of membrane mechanical properties.

Notizen: Fodrin is an actin/calmodulin-binding protein with similarities to spectrin (erythrocytes) and TW 260/240 (brush border). It is concentrated beneath the plasma membranes of neurons and other cells. We have observed translocations of fodrin in both neurons and lymphocytes. Newly synthesized, radiolabeled fodrin moves down axons at a maximum velocity (about 50 mm/day) that is slower than the most rapidly axonally transported proteins (group I). A portion of fodrin appears to move more slowly at velocities (1-10 mm/day) resembling those of actin and myosin (group IV) and tubulin and neurofilament proteins (group V). In lymphocytes, when certain surface antigens are induced by cross-linking agents to migrate to one pole of the cell and form a cap, fodrin redistributes beneath the membrane and forms a subcap. The movements of fodrin in lympohocyte capping and in the axonal transport of group IV polypeptides have certain similarities. In both cases, the redistribution of fodrin is accompanied by concomitant redistributions of actin, myosin, and calmodulin, and both processes proceed at similar velocities. We consider the possibilities that these two processes are related, both being driven by a submembrane force-generating system comprising in part actin, myosin, and fodrin, and that fodrin serves to link various organelles or proteins to this system.

Notizen: To test the idea that saltatory organelle movements of nonmuscle cells might be driven by microtubule-dynein interactions, we microinjected vanadate into several different types of cultured cell. Solutions of sodium metavanadate made up in a simple buffered salt solution were pressure microinjected into fully spread cells in an open-topped culture chamber placed on the stage of an inverted microscope. The cells were observed by oil-immersion phase-contrast optics and results were recorded on movie film. Vanadate, at 10-5-10-2 M, microinjected into cultured chick embryo fibroblasts, failed to inhibit organelle movements. To test the effectiveness of vanadate's inhibitory action under living cell conditions, ciliated epithelial cells were micro-injected. In these cells even the smallest microinjection of 5 × 10-5 M vanadate caused an immediate cessation of ciliary beating. Moreover, in cells that were well spread it was found that whereas vanadate, at 5 × 10-5 × 10-3M, inhibited ciliary motion, it failed to inhibit organelle saltations in the same cell. To determine whether vanadate would inhibit a living actin-myosin system, myocardial cells were also microinjected. Following microinjection of 5 × 10-5 and 5 × 10-4M vanadate a temporary tonic contraction (which also occurred following microinjection of buffer alone) was followed by regular beating. Taken together these results demonstrate that in living cell systems microtubule-dynein interactions are as sensitive to vanadate inhibition as they are in demembranated model systems, and that a working actin-myosin system in a living muscle cell does not share this great sensitivity. In light of the pronounced differential inhibitory effects of vanadate on the movements of cilia and organelles, our results suggest that saltatory organelle movements in chick embryo fibroblasts and rabbit oviduct epithelial cells are unlikely to be brought about by microtubule-dynein interactions.

Notizen: A simple planar model of sliding can predict the amount of sliding required to form a certain degree of bend. The accuracy of this prediction relies on the assumptions that no twists occur in the axoneme and that no sliding occurs at the base. However, previous studies indicated that twists may occur.This paper explores a new method for quantitating and analyzing twists. Preliminary results using this method showed that there were twists. In order to control for possible artifacts due to fixation and other preparative procedures, the characteristic S-shaped quiescent state of Ciona spermatozoa was studied.Analyses of platinum replicas of those flagella in which this waveform is well preserved suggest that most, if not all, of the twists observed are due to the artifact of a curved shape settling onto a surface. Detailed analyses indicate that if twists do occur in quiescent sperm, they are probably less than 0.4 radian. Since axonemes are evidently easily twisted in rigor, and even after fixation, caution should be exercised in interpretation of axonemal twists.

Notizen: Association of bivalent chromosomes with the astral centers and nuclear envelope was analyzed in crane-fly spermatocytes during the final hours of diakinesis. In contrast to other systems in which movement of chromosomes during diakinesis correlates with the clustering of bivalents near the astral centers, such clustering is not prevalent in crane-fly spermatocytes. Polarization indices of bivalents calculated 5 to 10 minutes before the end of diakinesis provided evidence for polarization of only a fraction of all bivalents. Similar results were obtained in a large number of fixed cells in which asters and chromosomes were preferentially stained. Ultrastructural analysis of cells in late diakinesis revealed significant contact between bivalents and the nuclear envelope in all 46 cells that were analyzed. The extent of contact in some cells was greater than in others. Sites of contact included the telomeric ends of bivalents, and in some cases the distribution of contact sites suggested the possible involvement of centromeres in chromosome-nuclear envelope association. The results are consistent with the hypothesis that a dynamic interaction between chromosomes and nuclear envelope may exist during late prophase, when the movement of chromosomes is known to occur.

Notizen: Taxol promotes microtubule (MT) assembly in vitro and induces the reorganization of the cytoskeleton into unusual MT arrays in cultured cells. The possibility that taxol also has an indirect effect on intermediate filaments (IF) was investigated. In baby hamster kidney (BHK-21) and human skin (ENSON) fibroblasts treated with 1-10 μM taxol for 1-24 h, the drug induces changes which are similar to those produced by colchicine. These include a loss of major cellular extensions, a redistribution of organelles to a perinuclear location, and an inhibition of locomotion. Saltatory particle movements are not inhibited, however. Ruffling and filopod formation continue, indicating that cells are viable up to 24 h.Polarized light microscopy of living fibroblasts treated with taxol reveals the presence of perinuclear birefringent material which has been examined by immunofluorescence. In control cells, IF and MT radiate from a juxtanuclear region and extend to the cell periphery. In taxol-treated cells, MT and IF are excluded from cell margins, forming large central bundles.In the epithelial cell lines PtK2 and PAM, the keratin system of IF does not become redistributed; in PtK2, however, a second fibroblastlike system of IF does become redistributed to a perinuclear position during taxol treatment.Ultrastructural analyses show that taxol-treated fibroblasts contain parallel arrays of cross-bridged MT-IF as well as bundles of MT exclusive of IF. Epithelial cells contain a predominance of IF-free MT bundles which are organized into hexagonally packed arrays. In these bundles MT frequently exhibit hooks or other incomplete MT profiles and are linked by filamentous material.

Notizen: A complex of proteins with properties similar to those of erythrocyte spectrinband 4.1-actin complex has been idientified in a preparation derived from bovine brain. The complex has an apparent sedimentation coefficient of about 26S, and contains brain spectrin (also called fodrin) and actin as major components. The actin in the complex is in the oligomeric form, which nucleates assembly of actin filaments that grow from the “barbed” end. The complex cross-links actin filaments, resulting in an increase in low-shear viscosity. Whether the complex contains a protein analogous to erythrocyte band 4.1 is not known. However, it can be demonstrated that brain spectrin has the capability to interact with band 4.1 in a way which increases its ability to cross-link actin filaments.

Notizen: Talin is a recently identified cytoskeletal protein with a polypeptide molecular weight of 215,000 daltons. In cultured fibroblasts talin has been localized by immunofluorescence in adhesion plaques (focal contacts), in the ruffling membranes and leading lamellae of the cell periphery, and in fibrillar patterns that align with microfilament bundles and/or with cell surface fibronectin. These cellular locations suggest that the protein could function either in the attachment of microfilaments to the plasma membane or in the organization of microfilaments close to membrane attachment sites. Cell transformation by viruses such as Rous sarcoma virus disrupts the normal organization of talin, and in most transformed cells talin appears distributed diffusely through the cytoplasm. In a few cells talin is detected in doughnut-shaped aggregates, as a ring surrounding a central core of actin. The significance of these structures is uncertain, but in some cells the individual structures will condense to form much larger aggregates with a striking appearance when viewed by immunofluoresence microscopy.

Notizen: Further similarity between mammalian erythrocyte spectrin and pig brain spectrin has been demonstrated by (a) formation of hybrid molecules with brain α-chains and erythrocyte β-chains and by (b) identification of an ankyrin protein in brain membranes. Hybrid spectrin molecules prepared from brain α-chains and erythrocyte β-chains were visualized by low-angle rotary shadowing as double-stranded rods (dimers) 100 nM in length. 125I-labeled brain α-chain that was hybridized with erythrocyte β-subunit acquired ability to bind to ankyrin sites on erythrocyte membranes. 125I-labeled brain α-chain bound only to β-subunits of erythrocyte and brain spectrin following transfer of these polypeptides to nitrocellulose paper from sodium dodecyl sulfate (SDS) gels. Thus brain spectrin and mammalian erythrocyte spectrin have shared functional sites involved in association of their subunits. Additional evidence for similarity of brain and erythrocyte membranes is the finding of a 210,000 Mr membrane protein in brain that cross-reacts with erythrocyte ankyrin and has a water-soluble domain of 72,000 Mr that is produced by protease digestion. The 72,000 Mr domain of brain ankyrin has been isolated by affinity chromatography on erythrocyte spectrin-Sepharose, and was demonstrated to bind directly to erythrocyte and brain spectrin. The brain 72,000 Mr fragment has distinct peptide maps from the erythrocyte 72,000 Mr ankyrin fragment and thus is not a result of erythrocyte contamination.

Notizen: Recently, molecules highly related to erythrocyte spectrin have been identified in nonerythroid cells. Here we summarize our current understanding of these molecules and suggest a model for their organization. Significant differences exist between this family of proteins isolated from mammalian cells and avian cells, and this may explain the variability in antibody preparations as well as differences in peptide maps of these subunits which have been reported. We have prepared antibodies specific for the variant subunits of the spectrinlike proteins fodrin, spectrin, and TW260/240 and analyzed the distribution of these variant subunits in different chicken cell types as well as their developmental distribution in the intestine. The results suggest that fodrin is the general member of this family of proteins and can even coexist with other spectrinlike proteins in the same cells.

Notizen: Directed movement of polymorphonuclear neutrophils (PMN) requires cell polarization and the orderly making and breaking of cell-substrate contacts. We compared the movement of human PMN suspended from the underside of glass coverslips to that of PMN seen in “profile” on fibers, using brightfield, differential interference contrast and reflection interference microscopy. Images were recorded on film and videotape and analyzed in real time and time lapse. The distribution of F-actin was observed with image-enhanced fluorescence microscopy after staining with NBD-phallacidin.PMN exhibited two patterns of motility. Fifteen to twenty-five percent of cells moved in a low profile gliding pattern and exhibited cauded displacement of dorsal surface folds. Most PMN made progress by cycles of partial release of the lamellipodium from the substrate and anterior advance followed by arching or rolling and lamellipodial reassociation with the substrate. Cells stimulated with bacteria, casein, or chemotactic formyl peptide rarely spread on the coverglass but waved into the medium attached only by the uropod. Eventually, many detached completely from the substrate. Cells confined to the substrate surface with overlying agarose were able to locomote when confronted with these substances.F-actin was irregularly distributed in nonpolarized suspended cells but concentrated in the lamellipodium in polarized cells. As cells arched along a substrate, F-actin accumulated in foci corresponding to the substrate-PMN interface, particularly at the uropod and retraction fibrils. Conversely, cells that were physically restricted to movement in the plane of the substrate surface by overlying agarose exhibited diffuse F-actin along the entire cell. Suspended PMN polarized with formyl peptide and incubated with Con A accumulated F-actin at the uropod. These observations suggest that both PMN locomotion and the movement of Con A binding sites involve the caudad redistribution of F-actin.

Notizen: We have investigated the Ca2+-dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy tranfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+(∼20 μM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low con-centrations of villin (villin/actin ∼ 1:400) but is stimulated at higher concentrations (villin/actin 〉 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or upon the addition of phalloidin. The exchange of actin subunits at steady state is inhibited at low concentrations of villin (villin/actin ∼ 1:200) but is stimulated at higher concentrations (villin/actin ∼ 1:50). None of the above effects is observed at 〈 10-8 M free [Ca2+].

Notizen: Ciliary axonemes from Tetrahymena extracted by KCl to remove the dynein arms reveal an orderly array of interdoublet links connecting adjacent A-B or A-A subfibers. The links repeat every 96 nm at a stable site on the A subfiber positioned near the bases of radial spokes 2 and 3. Both links and radial spokes are in lateral register across the nine successive doublets of unbent axonemes. In contrast, bent axonemes or those reactivated by ATP to undergo partial sliding disintegration exhibit systematic displacement of the interdoublet links. The links show no evidence of having elastic or other extendable properties and, therefore, must have undergone intermittent attachment with nonstructural binding sites on the adjacent subfiber. These observations suggest a more dynamic role for the interdoublet links in ciliary motion than previously has been envisioned.

Notizen: Mouse fibroblasts chronically infected with Moloney murine leukemia virus (MuLV) were fixed using variable amounts of formaldehyde, then examined by indirect immunofluorescence light microscopy. Several antisera were employed to detect both external and internal antigens associated with the cells, eg, MuLV gp70, tubulin, vimentin, and actin. Our results indicate that the cell membranes could be partially permeabilized to IgG molecules directed against the three cytoskeletal antigens only after 3.7%, but not 1%, formaldehyde treatment. Complete permeabilization was achieved by subsequent acetone treatment of cells after 3.7% formaldehyde fixation. In such cells, normal-appearing cytoskeletal networks of microtubules and intermediate filaments were observed. Stress fibers were also seen; however, they appeared less numerous and thinner than those of uninfected mouse fibroblasts. Further, a significant amounts of F-actin fluorescence was localized in granules in the cytoplasm of infected cells. Similar observations were made using JLS-V9 mouse cells chronically infected with 334C virus, another MuLV. These results taken together suggest that subtle differences exist in the organization of actin within MuLV-infected and uninfected mouse fibroblasts.

Notizen: From 30-40 g of Hela-S3 cells grown in suspension, 0.25-0.50 mg of spectrin has been purified by conventional biochemical procedures starting from a low ionic strength extraction at alkaline pH of crude Hela membranes. Hela spectrin consists in its native form of a tetramer α2β2 of two high molecular weight polypeptides (240,000 and 230,000 daltons). Three different populations of Hela membranes depleted of both spectrin and actin have been prepared on discontinuous sucrose gradients. Surprisingly, spectrin will reassociate with only the heavier membrane fraction. This reassociation is specific for Hela spectrin, since three other purified Hela proteins as well as human erythrocyte spectrin do not reassociate under the same conditions. This binding is not due to the presence of traces of actin still present in the membrane fraction since two Hela actin-binding proteins (filamin I and II) do not show any significant binding to this fraction. The nature of the membrane-binding site for Hela spectrin is discussed.

Notizen: We have investigated the presence and localization of an α-spectrinlike protein and its potential role in the morphological transformation of sea urchin coelomocytes. In immunofluorescence images there is a diffuse fluorescence throughout the petaloid cytoplasm, indicating a random distribution of the spectrinlike protein prior to the transformation. As these cells form filopodia, there is a coincident appearance of a spectrinlike protein, as seen in fluorescent images, at the site of filopodial initiation. As the filopodia continue to form and lengthen, the spectrin localization parallels their development. There is a single polypeptide observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of whole coelomocyte lysates that cross-reacts with the anti-α-spectrin immunogen and comigrates with it at 240 kilodaltons.

Notizen: Several proteins (eg, actin, myosin, and actin-binding protein) in the Tritoninsoluble residue of thrombin-stimulated platelets are important in the formation of cytoskeletal structures. Electrophoretic analyses have shown that unidentified protein bands of 68,000, 55,000, and 48-50,000 daltons are also present in larger amounts after thrombin stimulation. Since these molecular weights correspond roughly to those of the α, β, and γ chains of fibrin, and since fibrinogen is found in platelet α-granules, these bands were compared to those obtained when purified fibrinogen was treated with thrombin, exposed to 1% Triton X-100-5 mM EGTA, and the resultant Triton-insoluble residue sedimented. Identification of the 68,000-, 55,000-, and 48--50,000-dalton bands as fibrinogen derivatives was confirmed by identifying them in comigration studies and in autoradiographs of Triton-insoluble residues of platelets that were electrophoretically transferred to nitrocellulose paper and treated with antifibrinogen antibody and 125I-protein A. Furthermore, if the platelet suspension was treated with thrombin in the presence of calcium ions, protein bands characteristic of the action of Factor XIII on fibrin were observed, active platelet Factor XIII apparently having been made available by lysis of platelets during preparation. Making use of the electrophoretic properties of tubulin recently described by Best et al [1981], comigration studies using hog brain tubulin indicated that tubulin is not present in significant amounts in the Triton-insoluble residue of platelets as previously suggested. The identification of these proteins as fibrinogen derivatives does not demonstrate a physiological interaction between fibrin and the platelet cytoskeleton, since fibrin is Tritoninsoluble and can be pelleted even in the absence of platelet cytoskeletons.

Notizen: During early anaphase PtK1 cells were briefly treated with the rapidly reversible microtubule (MT) poison nocodazole. This treatment abruptly stopped chromosome motion and effected a large decrease in spindle birefringence. On removal of the drug, chromosome to pole motion (anaphase A) returned, though at a lesser rate but not extent than untreated cells. In most cases elongation of the pole-pole distance (anaphase B) also occured, at both a rate and to an extent less than in untreated cells. During the recovery period following drug arrest spindle birefringence did not return to pretreatment levels. Electron microscopic analysis of nocodazole arrested, or arrested and released, cells revealed extensive disassembly of the nonkinetochore class of MTs (nkMTs), particularly evident in the astral region. Microtubules seen in the interzone region were largely fragments of midbody precursors. Kinetochore MTs (kMTs) appeared to be unaffected by the brief drug treatment chosen for these experiments. Analysis of MT profiles seen in transverse sections of the interzone region indicated in treated and released cells approximately 60% fewer MTs. This may suggest that chromosome motion during anaphase is not dependent on interactions between kMTs and nkMTs and separation of the spindle poles can occur in the presence of disrupted interzonal MTs.

Notizen: We report the first direct demonstration that the cytoplasmic transport of organelles and vesicles (collectively called particles) takes place along microtubules. Living keratocytes from the corneal stroma of the frog, Rana pipiens, were observed with Allen video-enhanced constrast, differential interference constrast (AVEC-DIC) microscopy [Allen et al, 1981]. In sufficiently thin regions of these cells a network of linear elements was visible. When particles were observed in motion, they always moved along these linear elements. The linear elements remained intact and in focus on the microscope when lysed in a cell lysis solution that stabilized microtubules. Preparations were then fixed in formaldehyde, washed with phosphate-buffered saline (PBS), incubated with rabbit antitubulin, washed with PBS, stained with rhodamine-conjugated goat antirabbit, and washed with PBS. The extracted cells continued to remain in place and in focus on the microscope throughout these procedures. The same cells were then observed using epifluorescence optics and a silicon-intensified target (SIT) video camera. A network of fluorescent linear elements was seen to correspond in number, form, and position to the linear elements seen in the live AVEC-DIC image. Taken together, the AVEC-DIC and fluorescence microscopy observations prove that the linear elements along which particles move are microtubules (MTLEs). The observed particle speeds, pause times, and distances moved varied widely, even for the same particle on the same microtubule. Particles were also observed to switch from one microtubule to another as they were transported. The polarity of the microtubules did not seem to affect the particle direction, since particles were observed to move in both directions on the same MTLE. When not in motion these particles behaved as if anchored to the microtubules since they showed negligible Brownian motion. Finally, it was observed that an elongate particle could move onto two intersecting linear elements such that it was deformed into an inverted “Y” shape. This indicates that there may be more than a single site of attachment between the force generator and the particle.

Notizen: Using a uniflagellate mutant of Chlamydomonas and flash photomicrography at 300 Hz, we have obtained detailed information on the forward and reverse beating modes of Chlamydomonas flagella and on the relationship between rotation of the uniflagellate cell and the bending cycle of the forward mode. Flagella ranging in length from 5 to 15.5 μm were photographed. There is a decrease in wavelength and an increase in curvature in the principal bends when the length of the flagellum is less than the normal length of 12-13 μm, but these changes are not sufficient to maintain similarity of the bending pattern. In the reverse mode, the flagellum propagates symmetrical, planar, undulatory waves with a shear amplitude which is the same as in the forward mode: there is a 19% increase in beat frequency and a similar decrease in wave length. The reorientation of the flagellar beat direction towards the axis of the cell in the reverse mode is caused both by the decrease in asymmetry of beat and by activation of sliding in the principal bends at an earlier time in the beat cycle, relative to the time of activation of sliding in reverse bends. There are additional rare modes of beating which may be related to intermediate stages in the transition between forward and reverse beating modes.

Notizen: Lateral diffusion measurements, using the photobleaching techniques, have provided unique and quantitative data on the random translational motions of proteins and lipids of membranes. Proper interpretation of this body of data can yield new insight into the structure of biomembranes. A comparative review of the lateral diffusion of membrane components in artificial lipid bilayers and of the same components in natural membranes is presented to demonstrate the effects of protein concentration and peripheral constraints on lateral mobility. Recent data on the effects of cell-substrate and cell-cell contact on lateral diffusion are reviewed. Finally, some experimental perspectives are offered in terms of emerging biophysical and biological technology.

Notizen: The dynamic state of cytoskeletal protiens actin and vinculin was studied in living cells using microinjection of fluorescently-labeled proteins combined with fluorescence photobleaching recovery (FPR). It is shown that both proteins maintain a dynamic equilibrium between their diffusible pools in the cytoplasms and their “organized” cytoskeletal fraction. These interrelationships could be simulated in model systems consisting of isolated substrate attached membranes. It was demonstrated that fluorophore bound vinculin was incorporated into the exposed focal contacts and that this binding was largely actin independent. These results are in line with the hypothesis that local contacts induce binding of vinculin to the endofacial surface of the membranes and that this region serves as a nucleation center for the assembly of actin bundles.

Notizen: Patients with Kartagener syndrome (KS) show defects in ciliary and flagellar movement that are usually associated with the partial or total absence of dynein side arms from axonemal microtubules. Dynein is essential for such movements, but its involvement in other cellular (particularly microtubule-related) processes is unknown. It has recently been reported that neutrophils from KS patients show impaired motility including responses to chemotactic stimuli, suggesting that dynein-like proteins may be generally involved in motile processes. In support of this, we have now found that spontaneous motility of cultured skin fibroblasts from KS patients is also markedly impaired. Three cell lines derived from skin explants of KS patients with deficient dynein side arms in nasal cilia and eight cell lines derived from normal volunteers were studied. Fibroblasts were seeded into dishes containing colloidal gold-coated cover glasses [Albrecht-Buehler, 1977], incubated for 24 h at 37°C, and the area of cell “phagokinetic” tracks determined.Each cell line studied in this manner reproducibly displayed an amount of spontaneous motility characteristic for that cell line. The mean track area (± SE) for all control cells studied was 14.6 ± 0.5 × 103μm2 whereas for KS fibroblasts was 8.7 ± 0.4 × 103μm2 (P 〈 0.001). Immunofluorescence microscopy using antitubulin and antihuman 210 K MAP antibodies revealed no differences in the staining patterns between control and KS fibroblasts. Pinocytic rates were identical, and the complement of tubulin and major microtubule associated proteins as seen on one-dimensional SDS polyacrylamide gel autoradio-graphs appeared similar for control and KS cells. Thus, the observed motility defect is probably not the result of alterations in the occurrence or distribution of microtubules or in the occurrence or binding of the major microtubule-associated proteins. This defect in cellular motility may be related to the absence of dynein or may reflect another independent cellular defect.

Notizen: The coordinated movement of many cells - a process called swarming - permits myxobacteria to spread rapidly over a surface. We have investigated the mechanism of swarming in Myxococcus xanthus by making time-lapse motion pictures and by measuring the dependence of cell movement and spreading rate on the concentration of cells. Motion pictures of spreading zones showed that spreading resulted from motility, not growth, and that a swarm spread outward by establishing a loose reticulum of cells, then later filling it in. The spreading rate of wildtype strains was found to be highly dependent on cell density, increasing about 8-fold as the cell density was increased from 2.5 to 200 units. Mutants swarmed if they possessed only the A-motile component (A+S-) or only the S-motile component (A-S+) of wild type (A+S+); their spreading rate increased with cell density but was always less than A+S+. Individual A+S+, A+S-, and A-S+ cells executed typical gliding movements and (when moving) progressed at approximately the same speed, as if A and S motility were different ways of engaging the same gliding machine. Photographic studies of an A-S+ strain showed that cells moved only if they were separated by less than approximately one cell length from each other. This provided further evidence that pili, which are present on A+S+ and A-S+ cells and which extend about one cell length, could be responsible for switching on movement in S-motile cells, and presumably in wild type as well.

Notizen: The outcome of contact interactions involving neurons and nonneurons varies depending on the cell types involved. When neuronal growth cones from either ciliary (motor) or dorsal root (sensory) ganglia directly contact the lamellipodium of an embryonic heart fibroblast, both neurite elongation and fibroblast locomotion are inhibited. This occurs in spite of the fact that cell-surface activity in both cells continues unabated. Such contact inhibition is not observed when homologous ganglionic nonneurons are involved in the interaction. In fact, these cells become intimately associated with growth cones and/or neuritic shafts as a result of the contact. The detailed nature of the respose to contact exhibited by nerves and nonnerves varies not only with cell type but also with the portion of the cell involved in the contact. Growth cone filopodia tend to actively palpate the fibroblast surface, whereas spread regions, termed “veils,” form areas of apposition with fibroblast lamellipodia. This latter situation resembles the “typical” contact inhibition of locomotion that occurs following embryonic heart fibroblast-fibroblast interactions. Growth cones also frequently exhibit contact guidance when interacting with nonruffling lateral surfaces of heart fibroblasts.

Notizen: Observations on the role of transformation-specific F-aggregates [Carley et al, 1981] in altering morphology, adhesion and intercellular interaction in transformed cells are reported here. The appearance and disappearance of membrane- and substrate-associated F-actin aggregates (MAG and SAG, respectively) are followed in a cell line temperature-sensitive for transformation. Since MAG structures also appear near the membrane in suspension cultures of transformed cells and in transformed cells in coculture with untransformed cells, they appear to function at cell-cell contacts. Unlike microfilament bundles in untransformed cells, MAG and SAG do not contain the F-actin regulatory protein tropomyosin. The lack of tropomyosin in these structures near the membrane is reminiscent of areas of an exceptionally active actin cytoskeleton usually associated with motile processes of the normal cell membrane. Such areas of membrane-cytoskeletal interaction may be involved in the aberrant cell-cell communication as well as the aggressive behavior often seen in transformed cells.

Notizen: Myosin-coated spheres from 0.6 to 120 μm in diameter move in vitro on a substratum of polar arrays of actin cables derived from the alga Nitella. The force for this movement is provided by skeletal muscle myosin since it is ATP-dependent, and N-ethylmaleimide (NEM) inactivation of the myosin blocks movement. These observations demonstrate that attachment of myosin in a random orientation to structures will enable those structures to move along polar arrays of actin filaments.

Notizen: Matrix vesicles, extracellular microstructures known to eb involved in endochondral calcification, are rich in alkaline phosphatase and have been shown to contain actin. The mechanism of matrix vesicle formation in chondrocytes in not well understood. Chondrocytes from the epiphyseal growth plate, when grown in primary culture, elaborate alkaline phosphatase-rich vesciles. We examined the distribution of the cytoskeletal proteins actin, myosin, tubulin, and vinculin at various time-points during culture using indirect immunofluorescent labeling. Concomitantly, the production of alkaline phosphatase-containing matrix vesicles was also followed. Cell morphology changed noticeably at two distinct stages during the 22-day culture period: Immediately after release from the growth plate the cells were founded, but after 4 days of cultre they began to spread out and acquire irregular shapes with distinct filopodia. By 13 datsm as tge cekks attaubed confluency, they reacquired a rounded, polygonal appearance. At all time-point, tubulin was seen as a dense network of microtubules radiating from the perinuclear region throughout the cytoplasm toward the cell periphery. Initially actin was seen in filamentous from, but displayed a punctate distribution focused at contact points during the cell-spreading stage of culture. After confluency, actin was concentrated at cell-cell junctions. Initially, vinculin was diffusely distributed, but became focused in multiple adhesion plaques and at the termini of filpodia during the cell-spreading stage of culture. Following confluency vinculin became concentrated at cell-cell junctions. Myosin was observed at all time-points in small, intensely localized focal points in the cytoplasmic region of the cells and was consistently absent from the nuclear and peripheral regions. The amount of myosin in the cells increased steadily with time in culture. Elaboration of alkaline phosphatase-rich vesicles, which corresponded closely with the rounded morphology of early and late stages of culture, may be correlated with contact inhibition.

Notizen: Adhesion of platelets in vitro resulted in rapid polymerization of the amorphous cytoplasmic ground substance into an organized cytoskeletal superstructure. This cytoskeleton, characterized through the use of whole-mount and stereo (3-D), high-voltage microscopy in conjunction with morphometrics and cytochemistry, comprised four major size classes of filaments organized in distinctive zones. The central matrix, or granulomere, at the center of the cell mass, was an ill-defined meshwork of 80-100-Å filaments which enshrouded granules, dense bodies, and elements of the dense tubular system as identified through peroxidase cytochemistry. Demarcasting this central matrix was a trabecular zone containing 30-50, 80-100, and 150-170 Å filaments in an open and rigid-appearing lattice. Circumscribing the trabecular zone and extending to the margins of the hyalomere was the third region, the peripheral web, in which 70-Å filaments were arranged in a tight honeycomb lattice. This organizational pattern was retained in cytoskeletons prepared by Triton x-100 extraction of the adherent cells, and was observed in basally located cells of aggregates which formed subsequent to adhesion. Our observations are consistent with biochemical studies of cytoskeletons prepared from suspended platelets and suggest a contractile protein composition for the superstructure during adhesion.

Notizen: Activation of the motile apparatus by chemokinetic factors cannot be reliably assessed in cells that are attached to a solid substratum because motility can be totally abolished by excessive adhesion. It is however, necesary to quantify the activation of the motile apparatus in order to analyze and understand chemokinetic responses.It was the purpose of the present work to establish morphological criteria that can be used to quantify motility in nonadherent (floating) neutrophils and to predict the locomotor response under conditions of limited adhesion. The proportion of neutrophils performing crawling-like movements (polarized cells) in suspension correlates very closely with stimulated locomotion at low to optimal concentration of f-Met-Leu-Phe, ie, under conditions of limited adhesion. Reduced locomotion at supraoptimal concentrations of f-Met-Leu-Phe has also morphological correlates. The major feature is the decrease in the proportion of neutrophils performing crawling-like movements and the corresponding appearance of cells that are motile but not polarized in suspension and that do not locomote on the substratum. Concentration-dependent changes in neutrophil length and in the proportion of polarized neutrophils with and without tail were also observed. The locomotor potential of neutrophils under conditions of limited contact with the substratum can be predicted on the basis of their motile behavior, in particular the proportion of cells showing crawling-like movements, in suspension. In combination with measurements of adhesion the procedure should permit a more complete analysis of the regulation of chemokinetic responses.

Notizen: On the premise that the fascia adherens of cardiac muscle cell intercalated disk membranes is a structure that is closely homologous to the focal adhesions formed by fibroblasts, a fascia adherens preparation was isolated from chicken cardiac muscle, and was analyzed for its protein composition. A prominent 200-kilodalton (kd) protein was purified from the fascia preparation and shown to be antigenically unrelated to several previously characterized cytoskeletal proteins, including cardiac myosin and vinculin. With monospecific antibodies to the 200-kd protein, an identical or closely similar intracellular protein was shown to be associated with the focal adhesion plaques of fibroblasts.

Notizen: Localization of vinculin at the sarcolemma of striated muscle fibers defines an orthogonal lattice. The costameres of the lattice are the riblike bands of vinculin that run perpendicular to the long axis of the fiber, repeat in register with I bands of the subjacent myofibrils, and seem to couple the myofibril to the sarcolemma [Pardo et al 1982, 1983a]. The colocalization studies presented in this paper show that gamma actin, spectrin, and intermediate filament antigens are additional components of this lattice of costameres. In addition, the results show that gamma actin and spectrin are also components of the internal network of collars, first visualized with antibody to desmin [Granger and Lazarides, 1978], that connects the myofibrils to each other at the level of the Z line.

Notizen: Coelomocytes from several echinoderm species undergo an actin-mediated cytoskeletal transformation once subjected to hypotonic shock. In this study, coelomocytes from the sea urchins Lytechinus variegatus and Arbacia punctulata were induced to “transform” by treatment with 〉 5 μM of the calcium ionophore A23187 in the presence of external Ca++. The dependence of ionophore transformation on external Ca++ and the lack of chlorotetracycline staining indicates that these cells rely on external Ca++ sources. NBD-phallacidin (7-Nitrobenz-2-oxa-1,3-diazole-phallacidin) staining of lysolecithin permeabilized cells and wholemount transmission electron microscopy (TEM) show that similar reorganizations of the actin cytoskeleton take place during hypotonic shock and ionophore transformation, although actin filament bundling is less apparent in A23187-treated cells. As has been shown with hypotonic shock transformation, the ionophore elicited shape change is inhibited by anticalmodulin drugs. Greater than 10 μM concentrations of W 13 inhibit filopod formation, while this drug's less active structural analogue, W 12, exhibits no effects. W 13 also appears to disrupt actin filament-membrane associations in the cells. Fluorescent localization of calmodulin using a photooxidized derivative of trifluoperazine indicates a general cytoplasmic distribution with some concentration in filopod core bundles. Coelomocyte transformation may be an example of a cellular shape change regulated by Ca++ through the action of calmodulin modulation of actin-membrane interactions.

Notizen: Cylindrical segments of extraparenchymal pulmonary artery (essentially a preparation of smooth muscle with regard to contractile capability) were isolated from adult male rats. They were mounted in an isometric muscle bath in physiological salt solution (PSS) in an environment of 95% O2/ CO2. After allowing 1 h for equilibration, the maximum force generated by the tissue in response to a depolarizing solution was determined. After relaxation, vessels were incubated for 1 h in one of several concentrations of cytochalasin D (CD) (0.01, 0.05, 0.5, 1, 10 μg/ml) and the response to stimulation retested immediately after returning to PSS, and then at 30 minute intervals up to 2 h.CD inhibited the ability of vascular smooth muscle to generate force (contract) in a concentration-dependent manner. The inhibitory effect was reversible within a short period of time. Quantitative electron microscopic examination of these vessels suggested that CD disrupts the integrity of myofilaments, especially at sites of “dense bodies.” Our results indicate that a percentage of actin in smooth muscle cells is not permanently in the filamentous “F” form, but is part of the G:F actin system of the cell, labile to polymerization:depolymerization. The ability of smooth muscle cells to generate force could depend on the proper functioning of the F:G actin “treadmill”.

Notizen: Flagellar rootlets play an important role in “primitive motile systems.” They are made of filaments able to contract by twisting and Ca+2 binding. The pusules of Dinoflagellates appear to be under the control of large bundles of 2.4 nm nonactin filaments that correspond to the striated rootlets of their two flagella.

Notizen: Taxol induces the in vitro assembly of calcium stable microtubules from flagellar tubulin solubilized from sea urchin (Strongylocentrotus purpuratus) sperm tail outer doublets by sonication. Assembly occurs in the presence or absence of exogenous GTP. The drug (10 μM) reduces the critical concentration of protein required for assembly to ≤0.04 mg/ml. 3H-Taxol binds specifically to both isolated flagellar outer doublets and to reassembled microtubules with calculated maximal binding ratios of 0.25 and 1.32 moles taxol/mole polymerized flagellar tubulin dimer, respectively. We suggest that the discrepancy in maximal binding ratios may result from the presence of an endogenous molecule(s) along the surface of outer doublet microtubules that restricts taxol binding to that structure.

Notizen: Demembranated and membrane disrupted bull sperm models exhibit an increase in motility when exposed to cAMP. Tritium-labeled cAMP was used to locate the initial site of action of cAMP in the modeled sperm preparations. cAMP did not bind selectively to the modeled cells, and the presence or absence of plasma membrane fragments on the models did not significantly alter this result. When suspension medium taken from modeled sperm preparations was subjected to gel filtration on Sephadex G25-150 columns, cAMP bound to a high molecular weight component that eluted with the void volume. The responsible binding factor is a soluble component that is released when the plasma membranes of the sperm are disrupted during the modeling procedure. To test the importance of the cAMP binding factor, modeled sperm were centrifuged, the super-natant solution was decanted, and the cells were resuspended in fresh medium. After this treat-ment the cells could be restored to motility with Mg-ATP but no longer exhibited a response to cAMP. Furthermore, addition of cAMP binding factor isolated by gel filtration partially restored the response of these sperm to cAMP. Investigation of the properties of the cAMP-binding factor have confirmed that it is specific for cAMP, with a much lower affinity for AMP and cGMP. In the pre-sence of a large excess of unlabeled cAMP the labeled complex has a half-life of approximately 1 hour. Our results indicate that the action of cAMP on the motility of modeled sperm is mediated by its attachment to a high molecular weight, soluble component of the cell cytoplasm.

Notizen: Juctions between skeletal muscle cells and tendon collagen fibers transmit forces generated by muscle cells to the skeletal system. Since force trajectories across adhesive joints partly determine the stresses at the joint (eg, shear or tensile), the geometry of actin filament-membrane-collagen fiber associations has been modeled based on ultrastructural data, and force trajectories at the junction have thereby been established. Measurements show that in healthy twitch cells, actin filaments lie at a mean angle of 4.3° (standard deviation = 0.95°; 15 cells analyzed) to the plasma membrane. Calculations indicate that maximum isometric loading is seen by the junctional membrane almost entirely as a shear stress. In disuse-atrophied muscle cells, the mean angle between actin filaments and the membrane is 9.1° (standard deviation = 3.3°; 11 cells analyzed). The shear component of loading for the junctions of atrophied cells is only 1% less than that in healthy cells. The tensile component of the stress at atrophied junctions is more than doubled, however. These data are used to interpret patterns of myotendinous junction mechanical failure in terms of adhesive joint mechanics. An increased occurrence of failure of the atrophied junction is observed at physiological loads and can be attributed to a reduction of adhesive strength under increased tensile load component.

Notizen: The organization of microvillus actin and its associated proteins have been investigated in sublines of mammary ascites tumors (MAT) with mobile (MAT-B1) and immobile (MAT-C1) cell surface receptors. Microvilli isolated from these sublines differ in morphology (branched for MAT-C1 versus unbranched for MAT-B1) and the presence of a 58,000-dalton polypeptide (58K). 58K is found associated with MAT-C1 microvilli, microvillar cytoskeletons obtained by nonionic detergent extractions, and microvillar membranes prepared under conditions which depolymerize actin microfilaments. By extraction with actin-stabilizing buffers (isotonic Triton-Mg-ATP) microvillar actin can be fractionated into four forms. About 40% of the actin is sedimented at low speed (7,500g, 15 min). The pellets contain microfilaments; actin and α-actinin are the predominant proteins. High-speed pellets from these low-speed supernates contain about 10% of the actin as a transmembrane complex with a cell surface glycoprotein (cytoskeleton-associated glycoprotein, [CAG] 75-80,000 daltons) in MAT-B1 cells or with CAG and 58K in MAT-C1 cells. Transmembrane complexes can be purified from MAT-B1 and MAT-C1 microvillar membranes in Triton-containing buffer by gel filtration or sucrose density gradient centrifugation. The presence of only CAG and actin in the MAT-B1 transmembrane complex strongly suggests the direct interaction of actin and a cell surface component. The high-speed supernates contain soluble actin. By gel filtration or rate-zonal sucrose density gradient centrifugation about 30% of the microvillar actin is found as small oligomers and about 10% as G-actin in this extraction buffer. We suggest that the actin-containing transmembrane complexes may serve as membrane-association sites for oligomeric actin segments and microfilaments and as initiation sites for actin polymerization.

Notizen: HMWP (high molecular weight protein), a high molecular weight actin binding protein, was previously isolated from HeLa cells; its physical properties, amino acid composition, and intracellular localization indicated its homology with actinbinding protein and filamin [Weihing, 1982, 1983]. We now report the identification of HMWP in striated paracrystals. Purified HMWP is incubated at 25° C and subjected to negative staining with uranyl acetate. Examination by electron microscopy reveals long, striated paracrystals formed from filaments a few nanometers in diameter that lie parallel to the long axis of the paracrystal. At intervals of about 200 nm, the filaments are crossed by granular aggregates, accounting for the striated appearance. Treatment of the paracrystals with an affinity-purified antibody to HMWP decorates the filaments; such decorations are not observed if nonimmune goat IgG or phosphate-buffered saline are substituted for the antibody. Electron microscopic and electrophoretic analysis of paracrystals sedimented onto grids by centrifugation at 864 g reveals that the grids are covered with paracrystals and the major polypeptide present on grids centrifuged in parallel is HMWP. Taken together, these data indicate that the filaments of the paracrystals contain elongated molecules of HMWP. Additional experiments are needed to decide if the paracrystals from by self-association between HMWP molecules or by association with one or more of the minor polypeptides that remain in the purified HMWP.

Notizen: Membrane-associated mouse brain spectrin is a 972,000 Mr, 10.5S, (αβ)2 tetramer containing two ∼ 240,000 Mr subunits and two ∼ 235,000 Mr subunits. Two-dimensional [125I]tryptic peptide mapping indicates that these subunits share only limited and equivalent overlap with the α- and β-subunits of red blood cell (RBC) spectrin. Both the 220,000 Mr β-subunit of RBC spectrin and the 235,000 Mr β-subunit of brain spectrin are phosphorylated in the intact mouse. In vitro analysis suggests that both are phosphorylated by a cAMP-independent protein kinase. Antibodies against pure native mouse red blood cell spectrin cross-react with brain spectrin, and antibodies against pure brain spectrin cross-react with both the α-and β-subunits of mouse RBC spectrin. Both antibodies have been utilized to localize brain spectrin within distinct cellular entities of the mouse cerebellum. Granule cell neurons of the internal granule layer and Purkinje cell neurons demonstrated intense fluorscence of the cortical cytoplasm immediately adjacent to the plasma membrane and unstained nuclei, when either RBC or brain spectrin antibodies were utilized for staining. The molecular layer of the cerebellum stained only lightly, and oligodendrocytes and astrocytes appeared to have little fluorescence. Therefore, while brain is a tissue rich in nonerythroid spectrin, the concentration of these immunoreactive analogues is quite variable within distinct cellular entities of the cerebellum.

Notizen: The sperm of the shiner surfperch are packaged into high density aggregations which are introduced into the female genital tract at insemination. Germ cell differentiation occurs within cysts formed by nongerminal Sertoli cells. In late spermiogenesis, spermatozoa within the cysts come to lie parallel to each other and become more densely packed. These sperm packets (spermatophores), containing approximately 600 spermatozoa, then are released into the efferent sperm ducts.The exact nature of the spermatophore binding material is not known, but a major component is proteinaceous and is synthesized in the rough endoplasmic reticulum of the efferent sperm duct epithelial cells. The mechanism by which the spermatophores pass from cysts into ducts is not clear. It appears that whereas many Sertoli cells degenerate causing the cyst wall to break down, many Sertoli cells do not degenerate, but rather assume the configuration of columnar duct cells. The spermatophores remain intact within the testicular ducts, but rapidly dissolve within the female ducts in response to increased pH.

Notizen: The telemetered electromyographic activity (EMG) of select hindlimb muscles of unrestrained cats during standing, walking, trotting, and galloping have been recorded. Simultaneous cinematographic records permitted close correlation of muscle activity and locomotor behavior. In general, the pattern of extensor activity of the ankle, knee, and hip during locomotion is fairly consistent, while that of the flexors is more variable.Changes in basic EMG patterns from walk, to trot, to gallop are most evident in the two-jointed muscles associated with the knee and hip. Progressively greater variation of activity onset and cessation can be seen among extensor muscle groups from the walk, to trot, to gallop. Co-activation of the joint extensors and flexors, especially of the hip, at the end of the stance phase (E3) is slight in the walk, moderate in the trot, and considerable in the gallop. These EMG changes are necessary to meet the demands imposed upon the musculature at the faster gaits, particularly galloping, which include limb rigidity as related to loading, momentum as related to the limb's directional change from the stance phase to the swing phase, and lower spinal movements.The peroneal muscles of the ankle and the gluteal muscles of the hip show extensor activity and act as joint stabilizers during locomotion. Both biceps femoris anterior muscle and biceps femoris posterior muscle show consistent hip extensor patterns at all gaits. During quiet standing, extensor activity about the knee, ankle, and metatarsophalangeal joints is evident; but the hip extensor and flexor musculature is remarkably silent.EMG data for unrestrained cats are compared to those of dogs on a treadmill (Tokuriki, '73a,b, '74; Wentink, '76) and those recorded from decerebrate cats (mesencephalic preparation) during controlled locomotion (Gambaryan et al., '71). The EMG patterns from decerebrate cats are more consistent at the walk and gallop within functional groups of muscles at the ankle, knee, and hip than the EMG patterns observed in unrestrained cats or animals moving on a treadmill.

Notizen: End-plate distributions have been determined for three frog muscles of different morphology in order to relate end-plate topography to spatial muscle structure and nerve branching. Koelle's cholinesterase technique was applied, both on whole muscles and frozen sections. The end-plates of the short parallel-fibered cutaneus pectoris muscle appeared to be located in short bands along the nerve branches. The nerve tree is restricted to a zonal area across the middle part of the muscle. Depending on the way the nerve branches, the end-plate bands form innervation patterns, varying from one single continuous band to multiple distributed bands. In the latter case one frequently observes that different end-plate bands do not run across the same longitudinal muscle fiber area, although the respective nerve branches run parallel across this area. The long parallel-fibered sartorius muscle has a wider nerve tree and exhibits the same phenomenon for close parallel nerve branches, but end-plate bands along parallel nerve branches far apart cover the same muscle fiber area. The end-plate distribution in the bipennate, short-fibered gastrocnemius is zonal throughout the muscle except in certain compartments containing tonic fibers. The end-plate zone centers around the inner aponeurosis about half-way between the muscle tendon junctions of the fibers and is visible only at the muscle surface where muscle fibers run over their entire length at that surface. The results are of general use in the electrophysiology of neuromuscular transmission because they illustrate how in certain twitch muscles neuromuscular morphology may help to localize end-plates.

Notizen: When a larva of Haplothrips verbasci is ready to feed, it grasps the surface of the leaf with its pretarsi, sinks down between its front legs, lifts its head, and places the tip of its mouthcone against the surface. It then shortens its mouthcone and punches a hole in the epidermis by rapidly and repeatedly protracting and retracting its left mandibular stylet. The thrips then inserts its two maxillary stylets as a unit into the wound with a series of rapid thrusts and withdrawals, salivating continuously while doing so. When a food source in the epidermis or mesophyll is found, probing and salivation stop and cibarial pumping begins. Cytoplasm is sucked into the opening at the tip of the protracted stylets, up the food canal between them and into the cibarium.Probing and feeding can occur without mandibular intervention but uptake of liquid seems to require use of the mutually coadapted maxillary stylets, even when these are fully retracted.Prior to molting, the larva protracts its maxillary stylets maximally and, in the pharate state, seems incapable of feeding or drinking.Structures used in feeding are fully described and are shown to resemble those of Hemiptera except for the presence of maxillary and labial palpi and the absence of the loral lobes, right mandible and of a salivary canal between the protracted maxillary stylets. Seven single and 18 paired muscles function in the feeding act, nine less than in adults of the same species.Differences in the feeding mechanism of terebrantian and tubuliferous thrips are discussed and evidence is presented to suggest that the simplified and more highly specialized mouthparts of the latter insects are adaptations for feeding in confining spaces.

Notizen: In contemporary entomology the morphological characters of insects are not always treated according to their phylogenetic rank. Fossil evidence often gives clues for different interpretations. All primitive Paleozoic pterygote nymphs are now known to have had articulated, freely movable wings reinforced by tubular veins. This suggests that the wings of early Pterygota were engaged in flapping movements, that the immobilized, fixed, veinless wing pads of Recent nymphs have resulted from a later adaptation affecting only juveniles, and that the paranotal theory of wing origin is not valid. The wings of Paleozoic nymphs were curved backwards in Paleoptera and were flexed backwards at will in Neoptera, in both to reduce resistance during forward movement. Therefore, the fixed oblique-backwards position of wing pads in all modern nymphs is secondary and is not homologous in Paleoptera and Neoptera. Primitive Paleozoic nymphs had articulated and movable prothoracic wings which became in some modern insects transformed into prothoracic lobes and shields. The nine pairs of abdominal gillplates of Paleozoic mayfly nymphs have a venation pattern, position, and development comparable to that in thoracic wings, to which they are serially homologous. Vestigial equivalents of wings and legs were present in the abdomen of all primitive Paleoptera and primitive Neoptera. The ontogenetic development of Paleozoic nymphs was confluent, with many nymphal and subimaginal instars, and the metamorphic instar was missing. The metamorphic instar originated by the merging together of several instars of old nymphs; it occurred in most orders only after the Paleozoic, separately and in parallel in all modern major lineages (at least twice in Paleoptera, in Ephemeroptera and Odonata; separately in hemipteroid, blattoid, orthopteroid, and plecopteroid lineages of exopterygote Neoptera; and once only in Endopterygota). Endopterygota evolved from ametabolous, not from hemimetabolous, exopterygote Neoptera.The full primitive wing venation consists of six symmetrical pairs of veins; in each pair, the first branch is always convex and the second always concave; therefore costa, subcosta, radius, media, cubitus, and anal are all primitively composed of two separate branches. Each pair arises from a single veinal base formed from a sclerotized blood sinus. In the most primitive wings the circulatory system was as follows: the costa did not encircle the wing, the axillary cord was missing, and the blood pulsed in and out of each of the six primary, convex-concave vein pair systems through the six basal blood sinuses. This type of circulation is found as an archaic feature in modern mayflies. Wing corrugation first appeared in preflight wings, and hence is considered primitive for early (paleopterous) Pterygota. Somewhat leveled corrugation of the central wing veins is primitive for Neoptera. Leveled corrugation in some modern Ephemeroptera, as well as accentuated corrugation in higher Neoptera, are both derived characters. The wing tracheation of Recent Ephemeroptera is not fully homologous to that of other insects and represents a more primitive, segmental stage of tracheal system.Morphology of an ancient articular region in Palaeodictyoptera shows that the primitive pterygote wing hinge in its simplest form was straight and composed of two separate but adjoining morphological units: the tergal, formed by the tegula and axillaries; and the alar, formed by six sclerotized blood sinuses, the basivenales. The tergal sclerites were derived from the tergum as follows: the lateral part of the tergum became incised into five lobes; the prealare, suralare, median lobe, postmedian lobe and posterior notal wing process. From the tips of these lobes, five slanted tergal sclerites separated along the deep paranotal sulcus: the tegula, first axillary, second axillary, median sclerite, and third axillary. Primitively, all pteralia were arranged in two parallel series on both sides of the hinge. In Paleoptera, the series stayed more or less straight; in Neoptera, the series became V-shaped. Pteralia in Paleoptera and Neoptera have been homologized on the basis of the fossil record.A differential diagnosis between Paleoptera and Neoptera is given. Fossil evidence indicates that the major steps in evolution, which led to the origin first of Pterygota, then of Neoptera and Endopterygota, were triggered by the origin and the diversification of flight apparatus. It is believed here that all above mentioned major events in pterygote evolution occurred first in the immature stages.

Notizen: Oocytes and nurse tissue of Bruchidius differentiate from germ cells during the extended period of pupal development (7.2 ± 0.6 days). A system of 15 pupal stages correlates ovarian development with changes in pigmentation of the eyes, maxillae, alae and tarsalia. The ovarioles grow in length at a constant rate, though their width does not change.A differentiating zone, consisting of germ cells and the basal layer of interstitial cells, arises at the base of the tropharium and separates presumptive oocytes and nurse cells. Early in pupal development the germ cells are arranged in primary syncytia with the cells connected by persisting intercellular bridges filled with fusomal material, never with larger particles, such as mitochondria. At later stages membrane disintegration changes the primary syncytium into a secondary one including all nurse cell nuclei.Nutritive cords are first noticeable when differentiation of oocytes and nurse cells starts. The cords seem to be of primary origin, i.e., they are connections between sister cells which become elongated as these cells are separated during growth. This is indicated by the persistence of intercellular bridges which are sometimes found as part of the membrane of growing nutritive cords connecting young oocytes with the nurse cell syncytium.

Notizen: The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.

Notizen: Testis structure in four species of goodeid teleosts is described. Testicular tubules terminate blindly at the testis periphery where spermatogonia are located. In goodeid teleosts, development of sperm takes place synchronously within cysts whose periphery is made up of a single layer of Sertoli cells. Upon completion of spermiogenesis, spermiation ensues wherein sperm are shed, as spermatozeugmata, into the testis efferent duct system. Subsequently, Sertoli cells, which comprised the cyst periphery, transform into efferent duct cells.Sertoli cells phagocytize residual bodies and are involved in the formation of spermatozeugmata. The structure of the goodeid spermatozeugmatum is quite different from that observed in the related poeciliids. It is concluded, in view of this and other considerations, that the goodeids and poeciliids have independently evolved solutions to the problems of internal fertilization and gestation.

Notizen: The papillae basilares of 12 species of lizards from seven different families were studied by SEM. The iguanids, Sceloporus magister and S. occidentalis, have typical “iguanid type” papillae with central short-ciliated unidirectional hair cell segments and apical and basal long-ciliated bidirectional hair cell segments. These species of Sceloporus are unique among iguanids in that the bidirectional segments consist of but two rows of hair cells. The agamids, Agama agama and Calotes nigrolabius, have an “agamid-anguid type” papilla consisting of an apical short-ciliated unidirectional hair cell segment and a longer basal bidirectional segment. Agama agama is unusual in having a few long-ciliated hair cells at the apical end of the apical short-ciliated segment. The agamid, Uromastix sp., has an “iguanid type” papilla with a central short-ciliated unidirectional segment and apical and basal bidirectional segments. The anguid, Ophisaurus ventralis, has an “iguanid” papillar pattern with the short-ciliated segment centrally located. All the short-ciliated hair cells of the above species are covered by a limbus-attached tectorial network or cap and the long-ciliated hair cells, only by loose tectorial strands.The lacertids, Lacerta viridis and L. galloti, have papillae divided into two separate segments. The shorter apical segment consists of opposingly oriented, widely separated short-ciliated cells covered by a heavy tectorial membrane. The apical portion of the longer basal segment consists of unidirectionally oriented hair cells, while the greater part of the segment has opposingly oriented hair cells.The xantusiids, Xantusia vigilis and X. henshawi, have papillae made up of separate small apical segments and elongated basal segments. The apical hair cells are largely, but not exclusively, unidirectional and are covered by a heavy tectorial cap. The basal strip is bidirectional and the hair cells are covered by sallets. The kinocilial heads are arrowhead-shaped.The papilla of the cordylid, Cordylus jonesii, is very similar to that of Xantusia except that the apical segment is not completely separated from the basal strip.The papilla of the Varanus bengalensis is divided into a shorter apical and a longer basal segment. The hair cells of the entire apical and the basal three quarters of the basal segment are opposingly oriented, not with reference to the midpapillary axis but randomly to either the neural or abneural direction. The apical quarter of the basal segment contains unidirectional, abneurally oriented hair cells. The entire papilla is covered by a dense tectorial membrane.The functional correlations of the above structural variables are discussed.

Notizen: The circulatory systems of four polystyelids, Botryllus schlosseri, B. primigenus, Botrylloides violaceus and Symplegma reptans, were compared. The palleal buds are connected to the parent zooid by a peduncle and to the colonial vascular system by connecting vessels. The peduncle of S. reptans disappears at an earlier stage of bud development than in B. primigenus; it survives the dissolution of the parent zooid in B. schlosseri and B. violaceus. The connecting vessel is formed by anastomosis between an epidermal outgrowth from the bud and a neighboring colonial vessel, and is characterized by the presence of a sphincter. The number of connecting vessels formed in a palleal bud is three in S. reptans, two in B. primigenus and one each in B. schlosseri and B. violaceus. In each species, the larva has eight rudiments of ampullae. In B. primigenus, the original ampullae degenerate soon after metamorphosis and new ampullae extend from the ventral epidermis of the oozooid. In the other species, the colonial vascular system is derived from the original ampullae.The whole colonial vascular system contracts and expands periodically, with regionally different phases. During each expansion cycle, the sphincter contracts once in B. primigenus and twice in S. reptans. The correlation may be due to blood pressure and the propagation of excitation through the colonial vascular system.

Notizen: The mammalian ovary has been studied by optical microscopy and by scanning and transmission electron microscopy with the purpose of presenting an integrated view of the differentiating mammalian follicle. During follicular development, changes in the granulosa cells are particularly noteworthy and include dramatic modifications in cell shape coincident with antrum formation. The cytoplasmic processes of those granulosa cells immediately surrounding the oocyte, as well as the more peripheral granulosa cells comprising a second and third layer, traverse the zona pellucida, infrequently interdigitate with the microvilli of the egg, and make both desmosomal and gap junction contacts with the oocyte. The zona pellucida is thus distinguished by numerous fenestrations of varying diameters. The membrana limitans (basal lamina) is a bipartite structure composed of (a) a homogeneous stratum upon which the peripheral layer of granulosa cells rests, and (b) an outer region of collagen-like fibers. The specific advantages and limitations of the different methodologies utilized to study folliculo-genesis are discussed.

Notizen: Papillary projections along the anterolateral margin of the tongue were observed in fetal and young stages of the striped dolphin, Stenella coeruleoalba. Papillary projections appear in the prenatal period and attain maximum development in the early postnatal period. They almost disappear by weaning. Vestigial eminences remain at the corresponding region, but they completely disappear in the adult. The papillary projections observed differ markedly from the lingual papillae of the general mammalian tongue as they are temporary, localized at the anterolateral margin and large in size. The projections were also present in young individuals of some other dolphins. No taste buds could be seen on the projections in any of the stages of all specimens observed. Such projections may have important mechanical functions during suckling in these mammals.

Notizen: The fiber composition of the distal accessory flexor muscle (DAFM) and the branching pattern of its excitor axon were compared in several species of crabs, in the lobster and the crayfish. The muscle is composed exclusively of long sarcomere (〉 6 μm) fibers and therefore of the slow type. In all the crab species, except one, there is a distal to proximal gradient of fibers with increasing sarcomere lengths; this gradient is reverse in lobsters and crayfish. A proximal to distal gradient of increasing fiber diameters occurs in the DAFM of all crab species but not in the lobster and crayfish, in which all the fibers are approximately equal in diameter. The single excitatory axon traverses the width of the DAFM and gives off primary branches on either side in the lobster and crayfish but on only one side in crabs. The hypothesis that the axonal branching pattern may govern the regional distribution of fibers with differing sarcomere lengths in proposed.

Notizen: The ultrastructure of the pinealocyte in the woodchuck, Marmota monax, was studied during the four seasons of the year. Fall cells have a fairly uniform cytoplasmic density, organelles consistent with synthetic and/or secretory activity and rather extensive pericapillary and intercellular spaces. Many winter pinealocytes are nearly devoid of ribosomes and granular endoplasmic reticulum but contain lipid droplets associated with mitochondria. Pericapillary and intercellular spaces are minimal. Spring glands have the greatest variation in cytoplasmic density with intercellular and pericapillary spaces similar to that seen in fall glands. Cells containing electron dense cytoplasm have Golgi zone associated, secretory granules, free ribosomes, short sections of granular endoplasmic reticulum and dense bodies. Cells with a more electron lucent cytoplasm are similar to the most frequently observed summer pinealocytes which have numerous Golgi zones but few associated secretory granules. Microtubules are prominent in the cytoplasm of these cells, the plasma membranes are smooth and intercellular and pericapillary spaces are minimal. A yearly rhythm or cyclic activity of the pinealocyte is suggested.

Notizen: The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations.It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.

Notizen: The developmental morphology of regenerating male breast feathers of the jungle fowl was studied at the ultrastructural level. The process of keratinization was observed in the three types of cells which form feather barbs: barbule cells, cortical cells, and medulla cells. Keratinization first became evident in the barbule cells and resembled the process of keratinization as observed in hair cortical cells and embryonic down feathers. Eventually the whole cytoplasmic area of the barbule cell was occupied by keratin.The barb cortex cells became keratinized in a similar fashion as the barbule cells but not until they were developmentally twice as old as the barbule cells. When keratinization was complete in these cells, the keratin was in the form of large agglomerates scattered in the cytoplasm.The barb medulla cells showed no obvious signs of keratinization until they were developmentally three times as old as the barbule cells. Keratin filament bundles were first seen near the plasma membranes of the medulla cells. Large empty vacuoles appeared in the cytoplasm which also contained moderate amounts of glycogen.

Notizen: The ultrastructure of neuromuscular junctions in the twitch fibers of the stapedius muscle of Gallus gallus (domesticus) was investigated as part of a series of neurophysiological studies. Among the morphological features observed were elongated end-plates with numerous large and clear synaptic vesicles mixed with larger dense core vesicles and irregular or aperiodic “active sites” in the presynaptic membrane where synaptic vesicles were focused. The most remarkable features of these junctions were large synaptic clefts (50-80 nm) and the absence of junctional folds in the sarcolemmal surface. Unlike the large periodic junctional folds seen in the neuromuscular junctions of frogs and in the fast twitch fibers of the mammalian stapedius, the preparations studied only show small aperiodic invaginations (primitive folds) in the postsynaptic membranes. This morphological feature remains essentially constant from newly hatched to adult chickens. While these smooth junctions are consistent with earlier findings of inconspicuous junctional folds in the twitch fibers of the chicken posterior latissimus dorsi they are unlike those seen in the fast twitch fibers of the mammalian stapedius muscle, or other twitch fibers in general. The morphological findings of the present study may also suggest that the simple, unmodified neuromuscular junctions in the stapedius of Gallus may be a useful preparation for studies of synaptic membrane structures that employ the freeze-fracture technique.

Notizen: A skeletal neomorph - the preglossale - is described from the tip of the tongue in Passer. This medial unpaired skeletal element is a dorsally open trough articulating with the anterior tips of the paraglossalia and supporting the heavy epidermal pad of the seed-cup. The large paired Mm. hypoglossus anterior originate from the posterior half of the preglossale and insert onto the anterior bodies of the paired paraglossalia; they serve to depress the anterior portion of the preglossale. A regular pattern of dermal papillae is present in the seed-cup; these are arranged in about 20 rows of six to 8 papillae per row. Each papilla contains a series of Merkel cells and associated nerve endings (touch receptors). The seed-cup serves to orient and hold the seed in place while it is being husked; the battery of tactile receptors provides information on the position of the seed on the tongue. The preglossale serves to support the seed-cup and to change its shape - the curvature of the dorsal surface - as it is depressed relative to the paraglossalia. The paraglossale and associated features of the seed-cup in Passer would provide a valuable preparation to study a diversity of problems such as developmental interactions between endomesodermal and ecto-mesenchymal skeletal features, the ontogenetical development of Merkel cells, and the sensory physiology of Merkel cells and their associated nerve endings as tactile corpuscles.

Notizen: The structure of the longitudinal zebra stripes on the thorax of adult Zaprionus vittiger has been investigated by light-, polarization-, transmission electron-, and scanning electron microscopy. Each stripe consists of a central white stripe of about 50 μm width and two lateral dark brown stripes about 30 μm wide. Three different types of trichomes occur: Very long bent trichomes of the grooved-type, long bent trichomes of the crested-type, and short straight trichomes. The central white stripe contains neither bristle organs nor short straight trichomes but carries many long bent trichomes most of which are of the grooved type, contain two cavities and polarize the light in the polarization microscope. The dark brown stripes carry bristle organs and many trichomes of the short and straight-type. Bent trichomes of the crested-type are found on the whole zebra stripe at about equal frequencies; they contain no cavities and do not polarize the light. The cuticle of the dark stripes is underlain by pigment cells. It is suggested that the pigment granules in the epidermal cells cause the dark color of the dark brown stripes, whereas the form and structure of the bent grooved type trichomes cause the white color of the central stripe.

Notizen: New data on the brain of Latimeria indicate that previous estimates of the brain weight were too high by a factor of two. Our data suggest a brain weight of 1.1-1.5 grams for a specimen with a body weight of 30 kilograms. Quantitative data on major divisions of the brain are presented for the first time, and the relative size of the major brain divisions is similar to that of sturgeons and generalized sharks (such as hexanchids and squalids). Examination of brain component weight (s): body weight plots in a sample of non-teleost actinopterygian fishes indicates that all major divisions of the brain, except the telencephalon, are larger than in Latimeria. Brain component sizes in Latimeria are more similar to those extrapolated for amphibian brains than to those for actinopterygians. However, the cerebellum of Latimeria is considerably larger than that of amphibians.

Notizen: The Champy-Maillet osmium tetroxide-zinc iodide technique and a new method using azur B-sodium thioglycolate were used to study the general nervous tissue structure in planarians. A subepidermal and a submuscular nerve plexus, partially reported by earlier authors, are described, and a gastrodermal plexus is reported for the first time in triclads. The possible functions for each one of these plexuses are discussed. By the Champy-Maillet method, the innervation within the parenchyma appears as an array of numerous single nerve fibers that course between the parenchyma cells making apparent synaptic contacts. The pharynx has outer and inner nerve nets similar in structure to the submuscular nerve plexus. Both nerve nets are connected to each other by radial nerves.The central nervous system has a sponge-like structure with many lacunae filled with cell bodies, dorso-ventral muscle fibers, parenchymal cell processes and excretory ducts. The existence of this sponge-like nervous tissue structure is discussed in relation to the still incomplete centralization of the nervous tissue in these organisms, to the lack of a true vascular system and to the acoelomate level of organization. A comparison with the nervous tissue structure of more advanced groups like polyclads and nemertines is suggested.

Notizen: The ultrastructure of the adenohypophysis (AH) in the larval anadromous sea lamprey, Petromyzon marinus L., was examined. The AH is subdivided into three regions, the pro-, meso-, and meta-AH. Cells of the nasopharyngeal stalk extend directly beneath the pro- and meso-AH to form the ventral surface of the gland. Some cells in the pro- and meso-AH are arranged into small follicles. Each region of the AH is characterized by a single granulated (secretory) cell type. Granulated cells constitute 80-90% of the pro-AH and contain secretory granules that range from 800 to 2400 Å in diameter. Only 10-20% of the cells in the meso-AH are granulated and they contain much smaller secretory granules (400 to 1250 Å diameter) than those in the pro-AH. Granulated cells constitute 80-90% of the meta-AH and contain only a few secretory granules, ranging from 1000 to 2500 Å in diameter, and many vesicles containing either a loose flocculent or dense granular material. Nongranulated (stellate) cells are found in all regions. They are characterized by their long cell processes, abundant cytoplasmic filaments, and variable electron density. The appearance of organelles in these cells suggests they are nonsecretory. They may play a role in maintaining the structural integrity of the gland and the regulation of granule release in the pro-AH. Two types of nongranulated cells make up 80-90% of the meso-AH. Type I are stellate cells, type II may be undifferentiated cells. The functional significance of the secretory cells in the larval AH is discussed.

Notizen: Measurements have been made of those changes which lead to increases in the surface area of the intestine during the metamorphosis of three species of lampreys. Although the intestine of the Southern Hemisphere lamprey, Geotria australis, increases in length by 1.13 times and in diameter by 1.12 times, the main factor influencing the 5.71 times increase in surface area is the development of longitudinal folds. The contribution of the typhlosole to the internal perimeter of the intestine is less in most life cycle stages of G. australis than in Lampetra spp. The changes in the various intestinal measurements of the nonparasitic species L. planeri parallel those of the presumed ancestral parasitic species, L. fluviatilis, during the first six stages of metamorphosis. However, the longitudinal folds, but not the typhlosole, subsequently start regressing in L. planeri just after the time when the rate of gonadal development increases markedly. An account is also given of the pattern of fold formation and the development of the typhlosolar vein in G. australis.

Notizen: No Abstracts.

Notizen: The formation of the alimentary canal, nervous system, and of other ectodermal derivatives in the embryo of the primitive moth, Neomicropteryx nipponensis Issiki, is described. The stomodaeum is formed from an invagination in the medioposterior portion of the protocephalon. The proctodaeum arises as an extension of the amnioproctodaeal cavity. The midgut epithelium orginates from anterior and posterior rudiments in blind ends of the stomodaeum and proctodaeum. The decondary dorsal organ is formed in developing midgut. The development of the brain is typical of insects. The ventral nerve cord originates in large part from neuroblasts arising in 3 gnathal, 3 thoracic, and 11 abdominal segments. Intrasegmental median cord cells probably differentiate into both ganglion cells and glial elements of the ventral nerve cord; intersegmental cells appear not to participate in the formation of the nervous system. The stomatogastric nervous system develops from three evaginations in the dorsal wall of the stomodaeum, and consists of the frontal, hypocerebral, and ventricular ganglia, the recurrent nerve, and corpora cardiaca. Five stemmata arise from the epidermis on each side of the head. Five pairs of ectodermal invaginations are formed in the cephalognathal region to produce the tentorium, mandibular apodemes, corpora allata, and silk glands. Prothoracic glands orginate in the prothorax. Mesothoracic spiracles shift anteriorly to the prothorax during development. Oenocytes arise in the first seven abdominal segments. Invaginated pleuropodia are formed in the first abdominal segment.

Notizen: The arrangement and external morphology of the rodlike setae and associated structures located on the dactylopodites of the walking legs of six species of decapod crustaceans are compared. The dactyls of littoral species, represented by the rock crab, Cancer antennarius, and the spiny lobster, Panulirus interruptus, have dense tufts and bands of rodlike setae, as is typical of many decapods, and additionally only a few small plumed setae. The arrangement of setae on the dactyls of the recently discovered Galapagos vent crab, Bythograea thermydron, closely resembles that of C. antennarius. Rodlike and long plumed setae occur in about equal numbers on the dactyls of the pelagic anomuran, Pleuroncodes planipes. The dactyls having the fewest rodlike setae are those of the terrestrial hermit crab, Coenobita perlatus, and those of the kelp crab, Pugettia producta, where flat setae typical of Majidae have replaced most rodlike setae. The presence and structures of the terminal pores in rodlike setae vary intra- and interspecifically, possibly as a function of molt stage. Variations in some features of rodlike setae, such as tip acuity and presence of microsetae and surface sculpting, appear to be related to development. Serrated setae occur on the dactyls of megalopal P. producta but not in later stages. The topography and typology of setae located on the ambulatory dactyls of decapod crustaceans are considered in light of recent interest in using setal characteristics to determine the sensory functions of sensilla and to clarify the phylogeny of arthropod groups.

Notizen: In Pieris rapae the external structure of meso- and metathoraces includes intersegmental folds as well as 4 transverse shallow grooves on the dorsal side and 2 on the ventral side in addition to several leg segments. The musculature of both segments is very similar, but has some segment-specificity. Sixty-seven muscle are common to both hemi-mesothorax and hemimetathorax. Four are specific for the mesothorax and 3 for the metathorax. Moreover, thickness and number of subdivisions of some common muscles are specific for one segment. Attachments areas of all muscles are clearly indicated on the pattern of cuticular grooves. They have a tendency to pile up or line up to form various sizes of united attachment sites, most of which are located on or near the cuticular groove. On the other hand all grooves have some muscle attachment sites. Thus, attachments of larval muscles may relate to formation of the grooves. Comparison of the musculature with that previously reported for some lepidopteran larvae shows a major common basic plan and minor interspecific variation. Its attachment sites allow the role of each muscle to be inferred for body contraction, bending, and twisting, and for leg direction and flexion.