bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoskeleton of the mouse egg and embryo: Reorganization of planar elements
Authors: Ian Gallicano G. , McGaughey Robert W. , Capco David G.
Published: February 2005

Examination of detergent‐extracted mouse eggs and embryos reveals the existence of two cytoskeletal networks. One network is the typical thin filament network observed in somatic cells while the other is composed of large planar elements. These latter cytoskeletal structures, with individual widths of 60.0±6.8 nm, alter their spatial organization in a developmental stage‐specific manner. The planar elements are composed of filaments with a diameter of 10 nm aligned side‐by‐side with these filaments exhibiting a linear periodicity of 20.0±1.6 nm. A biochemical fraction containing components of the planar elements has been prepared from different stages of development and disappearance of prominent polypep‐tides from this fraction correlates with the altered spatial organization of the planar elements. Ultrastructure and biochemistry of cytoskeletal planar elements in eggs and embryos of the mouse are comparable with cytoskeletal sheets of Syrian hamster eggs and embryos, suggesting these cytoskeletal components may have a functional role in mammalian embryogenesis. Because such structures have not been identified in eggs or embryos of species other than mammals, their function may be unique to mammalian embryogenesis.
Direct binding of F‐actin to ponticulin, an Integral plasma membrane glycoprotein
Authors: Chia Catherine P. , Hitt Anne L. , Luna Elizabeth J.
Published: February 2005

We have developed an 125I‐labeled F‐actin blot overlay assay for the identification of F‐actin‐binding proteins after transfer to nitrocellulose from SDS‐polyacryl‐amide gels. Two major F‐actin‐binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F‐actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin‐binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J. [1987]: J. Cell Biol. 105:1741–1751). The binding of F‐actin to ponticulin on blots is specific because unlabeled F‐actin competes with 125I‐labeled F‐actin and because G‐actin does not bind. Nitrocellulose‐bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F‐actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125I‐labeled F‐actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2‐D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F‐actin in proportion to their abundance. Thus the actin‐binding activities of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin‐binding activity of nitrocellulose‐bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I‐labeled F‐actin blot overlay assay also may enable us to identify F‐actin‐binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.
Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia
Authors: Macrae Thomas H. , Langdon Carrie M. , Freeman John A.
Published: February 2005

In many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.
Cell locomotion: New research tests old ldeas on membrane and cytoskeletal flow
Authors: Heath Julian P. , Holifield Bruce F.
Published: February 2005

Recent studies on the mobility of membrane markers on crawling cells indicate that there is no long‐range centripetal flow of membrane proteins or lipids during cell locomotion. In this article we reflect on the history of ideas about membrane flow in cells, and we discuss how these new findings will shift the focus of research in cell locomotion away from the cell surface to the molecular interactions and dynamics of the actin cytoskeleton.
Microtubule binding and translocation by inner dynein arm subtype i1
Authors: Smith Elizabeth F. , Sale Winfield S.
Published: February 2005

Structural, biochemical, and genetic evidence has demonstrated there are three inner dynein arm subforms, I1, I2, and I3, which differ in organization and composition (see Piperno et al.: J. Cell Biol. 110:379–389, 1990). Using dynein extracted from Chlamydomonas outer dynein armless mutant pf28, we have begun to define the structural and functional properties of isolated inner arm subforms. Inner dynein arm I1 was purified either by sucrose density gradient centrifugation or microtubule binding affinity. I1, composed of heavy chains 1α and 1β, sedimented at 21S and selectively bound to and cross‐linked purified microtubules in and ATP‐sensitive manner. Deep etch electron microscopy revealed that the 21S sedimenting fraction contained two‐headed structures in which large globular heads are connected by long, flexible‐stem domains. In contrast, components derived from I2 and I3 sedimented as a mixture of 11S particles with single globular heads which did not bind to purified microtubules. Both the 21S and 11S sedimenting fractions supported microtubule translocation in in vitro motility assays. In 1 mM MgATP the I1‐containing fraction produced very slow microtubulegliding velocities (0.76 μm/sec) compared to the I2, I3‐containing fraction (4.1 μm/sec).
Flagellar photoresponses of Chlamydomonas cells held on micropipettes: II. Change in flagellar beat pattern
Authors: Rüffer Ursula , Nultsch Wilhelm
Published: February 2005

In response to step‐up as well as step‐down blue or white light stimuli, changes of beat pattern were observed in the two flagella of Chlamydomonas. The front amplitude was either increased or decreased, always in reverse in the two flagella. Again, two opposite combinations of step‐up and step‐down responses were found roughly in parallel to the two types of beat frequency changes. It is shown that positive phototaxis is probably achieved by the first type [called type (+)] and negative phototaxis by the second one [called type (−)]. Comparative measurements have revealed that frequency is not only related to the rate of flagellar movement, but also to the beat pattern. The rate of movement may change in different ways in the recovery and in the effective stroke. Though beat frequency and pattern changes are opposite in the two types, the rates of movement of the two flagella during the effective stroke are not always. In type (−) divergent changes were found in the rates of effective stroke movement, perhaps indicating the involvement of an additional mechanism.
Post‐mitotic cellular reorganisation in the diatom Cymatopleura solea: The role of microtubules and the microtubule centre
Authors: Pickett‐Heaps Jeremy D.
Published: February 2005

Cell division in Cymatopleura requires precise and major relocations of the nucleus and chioroplast which have been followed by time‐lapse cinematography and with the electron microscope. These movements are (1) the premitotic nucleus migrating to one end of the cell; (2) after cytokinesis, the daughter nuclei moving back to the cell centre, often oscillating several times while establishing their final location; (3) the single chloroplast folding over and sandwiching the central nucleus; and (4) the folded end of the chloroplast stretching back to fill the empty half of the cell. In all cases, straight, actively moving, transient strands of cytoplasm are associated with the movement of the nucleus and chloroplast, and these often appear to be pulling on the surface or the fold of the chloroplast which undergoes transient distortion.

These movements are rapid and colchicine‐sensitive. Ultrastructurally, they appear to be mediated by the prominent microtubule centre (MC) and its associated cytoskeleton of microtubules (MTs) although MTs do not attach directly to either nucleus or chloroplast. The MC is located close to the moving nucleus. Later, it moves ahead of the moving chloroplast and its MTs ensheath the tip. Later still, it is seen embedded in the fold of the chloroplast. In all three situations, MTs from it are seen in the strands of cytoplasm radiating from this area across the vacuole. After these events, the MC resumes its usual interphase situation on the nuclear surface.
Actin in the developing stomatal complex of winter rye: A comparison of actin antibodies and Rh‐phalloidin labeling of control and CB‐treated tissues
Authors: Cho Soon‐Ok , Wick Susan M.
Published: February 2005

Actin localization during stomatal complex formation in rye leaf epidermis was compared by three different labeling procedures. When leaf segments are fixed with formaldehyde prior to staining microfilament (MF) patterns visualized with actin antibodies and those with rhodamine‐phalloidin (Rh‐ph) are basically identical in controls. Likewise, on tissues treated with cytochalasin B (CB), actin antibodies and Rh‐ph produce very similar labeling patterns. Compared to MF alignments in fixed samples, additional sets of MFs are observed at the very cortical regions of epidermal cells that are stained with Rh‐ph without aldehyde fixation. Cortical MFs are also present in a variety of mitotic cells; MFs of meristematic cells and guard mother cells are more concentrated near the walls facing spindle poles, whereas a fine meshwork of MFs is observed along the entire periclinal surface of subsidiary mother cells. Although exactly how MFs are involved in control of the division site in higher plant cells is still to be determined, the presence of MFs during mitosis and the abnormal division observed in some stomatal cells after treatment with CB suggest that MFs are necessary for normal orientation of division in these cells, and thus normal morphogenesis.
Characterization of a nucleotide‐sensitive high molecular weight microtubule‐associated protein in the ovary of a hemipteran insect
Authors: Anastas Angela , Hunt Cherryl , Stebbings Howard
Published: February 2005

We have identified a 260 kD polypeptide as being the major microtubule‐associated protein (MAP) in the ovaries of the hemipteran insect, Oncopeltus fasciatus. The 260 kD insect ovarian MAP resembles certain mammalian brain MAPs by remaining soluble after boiling and promoting the assembly of tubulin into microtubules. It differs from most MAPs by exhibiting nucleotide‐sensitivity, being removed from microtubules by both ATP and GTP. Antibodies specific for the 260 kD MAP allowed its immmunofluorescent localization to the massive micro‐tubule aggregates forming the translocation systems which in hemipterans link the developing oocytes with anteriorly positioned nutritive cells. Such antibodies, in conjunction with electrophoretic methods, also demonstrated the 260 kD MAP to be species‐ and, to an extent at least, tissue‐specific. The Oncopeltus 260 kD MAP was not present in the ovaries of either Notonecta or Corixa, hemipterans which have similar microtubule systems to Oncopeltus but MAPs of slightly different molecular weight. The 260 kD MAP from the ovaries of Oncopeltus was not present in neuronal ganglia of the same species. The significance of the species‐ and tissue‐specificity of the 260 kD MAP, as well as its nucleotide‐sensitivity, are speculated upon and discussed.
Site‐specific antibodies block kinase a phosphorylation of desmin in vitro and inhibit incorporation of myoblasts into myotubes
Authors: Tao Jing‐Xiang , Ip Wallace
Published: February 2005

Desmin and vimentin are two type III intermediate filament (IF) proteins, which can be phosphorylated in vitro by cAMP‐dependent kinase (kinase A) and protein kinase C, and the in vitro phosphorylation of these proteins appears to favor the disassembled state. The sites of phosphorylation for desmin and vimentin have been mapped to their amino‐terminal headpiece domains; in chicken smooth muscle desmin the most kinase A‐reactive residues are ser‐29 and ser‐35. In this study we have examined the phosphorylation of desmin by the catalytic subunit of kinase A by using anti‐peptide antibodies directed against residues 26–36. The antibodies, which we call anti‐D26, recognize both native and denatured desmin and can discriminate between intact desmin and those derivatives that do not possess residues 26–36. Pre‐incubation of desmin with affinity purified anti‐D26 blocks total kinase A catalyzed incorporation of 32P into desmin by 75–80%. When antibody‐treated IFs are subjected to phosphorylation, no filament breakdown is observed after 3 hours. Thus anti‐D26 antibodies block phosphorylation of IF in vitro. We have also explored the role of desmin phosphorylation in skeletal muscle cell differentiation using these antibodies. Quail embryo cells, induced to differentiate along the myogenic pathway by infection with avian SKV retroviruses expressing the ski oncogene, were microinjected with affinity purified anti‐D26 at the mononucleated, myoblast stage. By 24 h post‐injection, the vast majority of uninjected cells had fused into multinucleated myotubes, but all microinjected cells were arrested in the process of incorporating into myotubes and remained mononucleated. This observation suggests that kinase A phosphorylation‐induced dynamic behavior of the desmin/vimentin IF cytoskeleton may be one of the many cytoskeletal restructuring events that must take place during myoblast fusion.
Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis
Authors: Conrad Abigail H. , Clark William A. , Conrad Gary W.
Published: February 2005

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1‐day posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross‐reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross‐reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin.

Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to form the contractile ring for cytokinesis.
Comparative kinetics of short and long sperm in sperm dimorphic Drosophila species
Authors: Bressac C. , Joly D. , Devaux J. , Serres C. , Feneux D. , Lachaise D.
Published: February 2005

All species of the Drosophila obscura group exhibit within‐ejaculate sperm length dimorphism. The present work is a contribution to the understanding of sperm competition through a comparative study of sperm kinetic parameters in four of these species. Videomicrographic observations at 200 frames per second of sperm from males and females, out of the storage organ, prior or after storage were made. Drosophila sperm display both major and minor waves. The former is analysed by measuring coiling diameter (μm) and the latter by recording both beat frequency (s−1) and wave propagation velocity (μm·s−1). Results show that the ‘behaviour’ of short and long spermatozoa noticeably differ: short sperm kinetics remains unaltered after storage while both major and minor waves of long spermatozoa are markedly modified. Thus, evidence is provided here of a sort of “differential activation” which is assumed to result in different survival abilities of short and long sperm within the storage organ of females.
Diversity of microtubular doublets in insect sperm tails: A computer‐aided image analysis
Authors: Afzelius B. A. , Bellon P. L. , Dallai R. , Lanzavecchia S.
Published: February 2005

Axonemal doublets of some insect spermatozoa were fixed in a mixture of glutaraldehyde and tannic acid, post‐fixed in uranyl acetate, and examined by electron microscopy, in order better to characterize the protofilament pattern. Most species had outer and inner dynein arms; others had only the inner one or none. Electron micrographs show the individual protofilaments to be well resolved and to be separated by an electron dense material. A certain “noise” inherent in the electron‐microscopical technique was found and is believed to be due to irregularities in fixation, embedding, and section staining, and to beam damage. The noise level was reduced by using a computer program in which similar picture elements are averaged. The resulting averaged images of the axonemal doublets show a few widened “gaps” in the wall of protofilaments. These widened gaps coincide with the location of dynein arms, spokes, or intertubular material. There were, on the other hand, no widened gaps at the level of attachement of the accessory tubules. We tentatively conclude that at least some of the proteins that associate with microtubules are inserted deep inside the microtubular wall rather than having a superficial attachement. The internal structure of the A‐subtubule is rather constant in species where both sets of dynein arms are present, whereas that of the B‐tubule is more variable.
Control of ciliary orientation in ciliated sheets from Paramecium–differential distribution of sensitivity to cyclic nucleotides
Authors: Noguchi Munenori , Nakamura Yoichi , Okamoto Ken‐Ichi
Published: February 2005

Ciliated sheets of cell cortex were prepared from Triton‐glycerol‐extracted Paramecium to observe directly the change of ciliary orientation. The observation of the ciliary responses revealed the modes of ciliary control by Ca2+ and cyclic nucleotides. The cilia changed their pointing direction clockwise from 11–12 to 5 o'clock (with the anterior of the cell defined as 12 o'clock) in the horizontal plane of cell surface when Ca2+ concentration was decreased from 10−6 M to 10−7 M. Cyclic AMP competed with Ca2+ ion in determining the orientation of the cilia. On the other hand, cGMP tended to change the ciliary orientation toward 3 o'clock. Ciliary sensitivity to cyclic nucleotides depended on their location on the cell surface. The cilia on the left‐hand field of the cell were more sensitive to cyclic nucleotide than those on the right‐hand field. The differential distribution of ciliary sensitivity within a single cell seems to be functional in the sophisticated turning mechanism in the behavioral response of Paramecium.
Cloning, sequencing, and mapping of an α‐actinin gene from the nematode Caenorhabditis elegans
Authors: Barstead Robert J. , Kleiman Lawrence , Waterston Robert H.
Published: February 2005

The dense‐bodies in the body wall muscle of the nematode Caenorhabditis elegans function to anchor the actin thin filaments to the adjacent sarcolemma. One of the major components of the dense‐bodies is the actin‐binding protein α‐actinin. To facilitate a genetic analysis of α‐actinin, we have cloned a cDNA encoding the nematode protein, identified its position on the nematode physical map, and developed a unique PCR based approach to test the position of the cloned gene relative to known genetic deletions. The peptide sequence deduced from the cDNA shows that, apart from a few exceptional regions, the nematode protein shows strong similarity to other known α‐actinins. Its position on the genetic map shows that none of the known muscle affecting mutations identified in C. elegans are in this α‐actinin gene. This gene has been given the name atn‐1 (α‐actinin‐1).
Dictyostelium discoideum essential myosin light chain: Gene structure and characterization
Authors: Pollenz Richard S. , Chisholm Rex L.
Published: February 2005

We have used a Dictyostelium essential myosin light chain (EMLC) cDNA clone to isolate additional cDNA clones which supply a different 3′ sequence from that previously described. The revised cDNA sequence encodes a polypeptide of 150 amino acids. Amino acid residues 147–167 of the previously reported sequence are replaced by new residues 147 to 150. The new cDNA encodes a polypeptide with 66% amino acid sequence identity with the Physarum polycephalum EMLC, and approximately 30% identity with mammalian EMLC sequences. These new cDNA clones were used to isolate two genomic DNA fragments which contain the entire EMLC gene. The Dictyostelium EMLC gene contains a single intron located immediately 3′ of the translation initiation codon and encodes a product most similar to MLC3 isoform of vertebrates. Primer extension analysis places the transcription initiation site approximately 90 nucleotides upstream of the translation initiation site. A DNA fragment containing 350 bases of sequence upstream of the putative transcription initiation site is sufficient to drive expression of a reporter gene upon reintroduction into growing Dictyostelium cells. In addition, the CAT reporter mRNA produced by this construct showed a pattern of developmental regulation similar to that previously reported for the endogenous EMLC mRNA. Based on comparison with published EMLC sequences from a variety of sources, the Dictyostelium EMLC shows slightly higher similarity to vertebrate EMLCs from striated musele sources than nonmuscle sources. While Dictyostelium and human nonmuscle sequences display only 28% identity over their entire sequence, the region from residue 88 to 108 shows much higher identity (67%). The high evolutionary conservation of this region of the EMLC suggests it may play an important role in EMLC function, and as such, represents a good target for future mutagenesis studies.
The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera
Authors: Balczon Ron , West Kelly
Published: February 2005

Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.
Analysis of inducible contractile rings suggests a role for protein kinase C in embryonic cytokinesis and wound healing
Authors: Bement William M. , Capco David G.
Published: February 2005

A semi‐in vitro system derived from Xenopus oocytes which allows induction of contractile ring (CR) formation and closure is described and exploited to elucidate regulatory and structural features of cytokinesis. The inducible CRs (ICRs) are composed of actin filaments and closure is actin filament‐dependent as is cytokinesis in vivo. ICR closure in this system is calcium‐dependent and pH‐sensitive, as is cytokinesis in permeabilized cells (Cande: Journal of Cell Biology 87:326, 1980). Closure of ICRs proceeds at a rate and with a kinetic pattern similar to embryonic cytokinesis. Collectively, these data demonstrate that this system is a faithful mimic of cytokinesis in vivo. ICR formation and closure is protein kinase C (PKC)‐dependent and neomycin‐sensitive, indicating that the PKC branch of the polyphosphoinositide pathway regulates formation of the actomyosin ring which is the effector of cytokinesis. Kinetic measurements show that the rate of ICR closure reaches a peak of 4–8 μm/sec. Since the maximum measured velocity of actin filament translocation by vertebrate, non‐muscle myosins is 0.04 μm/sec, the later observations support a model in which the CR is segmented, containing multiple sites where filaments overlap in a “sliding filament” fashion. Because the rate decreases after reaching a peak, the results also suggest that the number of overlap sites decrease with time.
α‐, β‐, and γ‐tubulins: Sequence comparisons and structural constraints
Authors: Burns Roy G.
Published: February 2005

Comparison of ≏ 160 α‐, β‐, and γ‐tubulins, and excluding the highly divergent C‐terminal peptide, indicates that the three subclasses have similar tertiary structures. Conserved sequences within or between the subclasses have been identified, together with the locations of known epitopes, chemical modifications, and mutations. Evidence is also reviewed concerning the identity of the GTP‐binding sites, about which residues are exposed in the assembled microtubule and at subunit:subunit interfaces. These characteristics constrain the possible tertiary structure of the tubulin subunit.
Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum
Authors: Roberts Thomas M , King Karen L.
Published: February 2005

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55–66, (1989)). Examination by video‐enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled reaward at the same rate (10–50 μm/ min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as, weak acids and protonophores, caused the fiber complexes to disassemble completely in 4–5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30–60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3−. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.
Properties of the spectrin‐like structural element of smooth‐muscle α‐actinin
Authors: Kahana Edith , Gratzer W. B.
Published: February 2005

The fragment of smooth muscle α‐actinin, comprising the four spectrin‐like structural repeating units, has a high α‐helix content, similar to that of spectrin, and a hydrodynamic frictional coefficient, indicative of an elongated, probably bent or kinked rod‐like structure, as found for spectrin dimer and tetramer. The fragment exists in solution as an extremely stable dimer, which is dissociated only under denaturing conditions and is much more resistant to dissociation by urea than is the spectrin heterodimer. High‐resolution proton magnetic resonance spectra reveal that a part of the polypeptide chain gives rise to sharp resonances; this is also true of spectrin and it implies that the individual structural repeating units contain segmentally mobile elements, which may be required to generate the elastic properties of the spectrin family of proteins. Again like spectrin, the α‐actinin fragment contains multiple binding sites for long‐chain fatty acids, as revealed by quenching of tryptophan fluorescence by 2‐bromostearate (though not by 9(10)‐bromostearate). The results point to extensive structural and functional similarities between the repeating units of all the proteins of the spectrin family.
Conserved β‐tubulin binding domain for the microtubule‐associated motors underlying sperm motility and fast axonal transport
Authors: Goldsmith Matthew , Connolly Joe A. , Kumar Norm , Wu Jie , Yarbrough Lynwood R. , van der Kooy Derek
Published: February 2005

An antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma β‐ (but not α‐) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine β‐tubulin C terminus. Furthermore, addition of this β‐tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule‐based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the β‐tubulin peptide and we suggest a distinct but similarly located α‐tubulin domain) mediating the attachment of tubulin‐associated motors.
Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility
Authors: Wessels Deborah , Murray John , Jung Goeh , Hammer John A. , Soll David R.
Published: February 2005

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB−). DMIB− cells generate elongated cell shapes, form particulate‐free pseudopodia filled with F‐actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB− cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F‐actin in pseudopodia, and a concomitant increase in cortical F‐actin. DMIB− cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB− cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB− cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB− cells.
Modulation of growth cone morphology by substrate‐bound adhesion molecules
Authors: Payne H. R. , Burden S. M. , Lemmon Vance
Published: February 2005

The growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position‐specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N‐cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N‐cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N‐cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate composition.
The role of β1 integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro
Authors: Hilenski Lula L. , Xuehui Ma , Vinson Nancy , Terracio Louis , Borg Thomas K.
Published: February 2005

The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I + III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN > LN > C I+III > plastic > BSA. Addition of anti‐β1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration.

Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of α‐actinin aggregates into Z bands. At later times, vinculin at 8 hours and β integrin at 22 hours became co‐localized with α‐actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM‐integrin‐cytoskeleton associations.
End‐stabilized microtubules observed in vitro: Stability, subunit interchange, and breakage
Authors: Dye Rick B. , Flicker Paula F. , Lien David Y. , Williams Robley C.
Published: February 2005

We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such “end‐stabilized” microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When sub‐units are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End‐stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.
Fertilization alters the orientation of pigment granule saltations in Arbacia eggs
Authors: Allen P. G. , Baltz J. M. , Begg D. A.
Published: February 2005

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time‐lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or pathlength of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non‐radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.
Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells
Authors: Gillard Baiba Kurins , Thurmon Lisa T. , Marcus Donald M.
Published: February 2005

We reported recently that two glycosphingolipids (GSLs), globoside (Gb4)and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Doublelabel immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, keratin and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.
Microtubule assembly protects the region 28–38 of the β‐ tubulin subunit
Authors: Chène Patrick , Mazarguiland Honoré , Wright Michel
Published: February 2005

Polyclonal antibodies have been raised against the peptide 28–38 of the β‐subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all β'‐tubulin subunits, except for β‐tubulin isotypes, and did not recognize the α‐tubulin subunit. The 28–38 region does not play a role in the interaction between the α‐and β‐subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubule associated proteins, the region 28–38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28–38 region of β‐tubulin is also protected in cellular structures. These observations suggest that the 28–38 region of the β‐tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly. © 1992 Wiley‐Liss, Inc.
Dynamics of polar filament discharge and sporoplasm expulsion by microsporidian spores
Authors: Frixione Eugenio , Ruiz Lourdes , Santillán Moisés , de Vargas Lourdes V. , Tejero José M. , Undeen Albert H.
Published: February 2005

Spores of the microsporidium Nosema algerae were stimulated to germinate in vitro while observed with video‐enhanced contrast microscopy. Field‐by‐field playback of tape‐recorded sequences yielded the first serial illustrations and kinematic analysis of the explosive discharge of the polar filament and the sporoplasm. The filament emerges from the anterior pole of the spore in a regularly pitched helicoidal course along a nearly straight axis, with a mean maximum instant velocity of 105 μm/s. Just before elongation is completed the filament tip follows a tortuous path that often results in a curved or spiralling terminal configuration. Then elongation stops and, after a lag that may vary from less than 15 to over 500 ms, the sporoplasm pours out at the filament tip forming a globule that quickly grows up to a size larger than its original volume within the spore. Concomitantly, the helical filament becomes straightened and frequently the spore body is pulled forward. Thereafter a relaxed filament, usually 5–10% shorter than when maximally extended, remains connecting the empty spore case and the sporoplasmic droplet. Experiments with hyperosmolar media produced a considerable slowdown of filament extrusion and often precluded sporoplasm discharge. The present results are fully consistent with the hypothesis of a hydrostatic pressure‐triggered mechanism of spore germination, and revealed that the process is composed of two discrete phases separated by a variable lag: (1) complete eversion of the polar filament, and (2) passage of the main sporoplasm mass along the tube. The data provide a preliminary basis toward the conception of a quantitative physical model of microsporidian spore germination. © 1992 Wiley‐Liss, Inc.
Distribution of profilin in fibroblasts correlates with the presence of highly dynamic actin filaments
Authors: Buß Folma , Temm‐Grove Constance , Henning Sabine , Jockusch Brigitte M.
Published: February 2005

We have used polyclonal and monoclonal antibodies raised against calf thymus profilin to localize the corresponding protein in translocating, spreading, and stationary rat fibroblasts. Immunofluorescence of whole cells and immunogold labeling on ventral membranes of lysis‐squirted cells showed that profilin was markedly enriched in the highly dynamic lamellipodia or pseudopodial lobes. Within these regions, a significant fraction was colocalized with dynamic actin filaments organized in actin ribs, cortical filaments, or stress fiber‐like bundles, and little profilin was found in membrane areas appearing free of actin. In contrast, stress fibers of stationary cells as well as actin arcs and ring‐like bundles of spreading and migrating cells showed very little label. These results are discussed in context with the proposed role of profilin in regional membrane dynamics typical for fibroblasts and are compared to previous data (Hartwig et al.: J. Cell Biol. 109:1571–1579, 1989) on profilin distribution in platelets and granulocytes. © 1992 Wiley‐Liss, Inc.