bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH
Authors: King Karen L. , Essig Juliane , Roberts Thomas M. , Moerland Timothy S.
Published: February 2005

The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly‐disassembly‐reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re‐establishment of the pH gradient, and re‐initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH‐sensitive MSP‐membrane interaction. © 1994 Wiley‐Liss, Inc.
Functional analysis of a cardiac myosin rod in Dictyostelium discoideum
Authors: LeBlanc‐Straceski Janine M. , Fukui Yoshio , Sohn Regina L. , Spudich James A. , Leinwand Leslie A.
Published: February 2005

Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament‐forming, or light meromyosin (LMM) domain of the myosin molecule in cyto‐kinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1–11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677–688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat β cardiac LMM was transfected into both wild‐type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild‐type and Dd mhc null cells. Although null cells expressing chimeric mhc at ∼10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility. © 1994 Wiley‐Liss, Inc.
Two‐step mechanism for actin polymerization in human erythroleukemia cells induced by phorbol ester
Authors: Niu M. Y. , Nachmias V. T.
Published: February 2005

Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin [Jarvinen et al., 1987: Eur. J. Cell Biol. 44:238–246]. We observed an early (20–30 min) stage of spreading in which F‐actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs. By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize. The cells also spread on RGD polymer. DiC8 (1,2‐dioctanoyl‐sn‐glycerol, C8:0, Sigma Chemical Co., St. Louis, MO) induced spreading but only if DAG kinase inhibitor and A‐23187 were also present; in their absence cells adhered but did not spread. Spreading was ∼85% inhibited by 100 nM staurosporine. PKC‐β was shown to be present in the cells by immunoblotting. In cells spread for 1 h with PMA, F‐actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G‐actin‐thymosin β4 decreased significantly.

To determine whether the F‐actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment. Under these conditions PMA‐treated suspended cells still increased their F‐actin to 126–137% of controls, a significant increase over control levels. Staurosporine inhibited F‐actin increases under all the conditions studied.

Permeabilized cell suspensions, incubated with rhodamine labelled G‐actin, incorporated the labelled actin along cell membranes at a low level. A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation. This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 μM).

We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC‐like molecule. In suspended cells, the polymerization occurs along the membrane. When cells spread, F‐actin increased to a significantly greater extent. This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms. © 1994 Wiley‐Liss, Inc.
Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335
Authors: Bré Marie Hélène , de Néchaud Béatrice , Wolff Annie , Fleury Anne
Published: February 2005

Microtubular networks are extensively developped in many ciliate species. In several of them, we investigate the occurrence of the post‐translational glutamylation of tubulin [Eddé et al., 1990: Science 247:82–85; Eddé et al., 1991: J. Cell. Biochem. 46:134–142] using as a probe for such modified tubulin, the monoclonal antibody GT335 [Wolff et al., 1992: Eur. J. Cell Biol. 59:425–432]. Results obtained in Paramecium strongly suggest that both axonemal and cytoplasmic tubulin are glutamylated. As in the vertebrate brain tubulin so far tested, the GT335 epitope is located at the carboxy‐terminal fragment of cytoplasmic tubulin removed by subtilisin treatment. Immunoblotting and immunofluorescence experiments reveal that, unlike tubulin acetylation, glutamylation is not restricted to cold‐resistant microtubules. In addition, immunofluorescence studies performed on dividing cells show that glutamylation takes place soon after the polymerization of microtubules.

Finally, glutamylated tubulin is also detected in the ciliate species Euplotes, Tetrahymena, and Paraurostyla. Together with results obtained on flagellate species, this suggests that tubulin glutamylation came out early in the course of eukaryotic evolution and has been widely exploited in various cellular strategies. © 1994 Wiley‐Liss, Inc.
Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria
Authors: Jellali Abdeljelil , Metz‐Boutigue Marie‐Hélène , Surgucheva Irina , Jancsik Veronika , Schwartz Christian , Filliol Dominique , Gelfand Vladimir I. , Rendon Alvaro
Published: February 2005

Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria‐associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane‐associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol‐free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria‐associated kinesin is discussed. © 1994 Wiley‐Liss, Inc.
Purification of microtubule proteins from Xenopus egg extracts: Identification of a 230K MAP4‐like protein
Authors: Faruki Shamsa , Karsenti Eric
Published: February 2005

We describe the purification of microtubule proteins from Xenopus egg extracts by temperature‐dependent assembly and disassembly in the presence of dimethyl sulfoxide and identify a number of presumptive microtubule‐associated proteins (MAPs). One of these proteins has a molecular weight of 230 kDa and is immunologically related to HeLa MAP4. We show that this MAP is heat stable and phosphorylated, and that it promotes elongation of microtubules from axonemes. © 1994 Wiley‐Liss, Inc.
Cytoskeletal alterations in cultured cardiomyocytes following exposure to the lipid peroxidation product, 4‐hydroxynonenal
Authors: VanWinkle W. Barry , Snuggs Mark , Miller Joseph C. , Buja L. Maximilian
Published: February 2005

Damage to the cardiac myocyte sarcolemma following any of several pathological insults such as ischemia (anoxia) alone or followed by reperfusion (reoxygenation), is most apparent as progressive sarcolemmal blebbing, an event attributed by many investigators to a disruption in the underlying cytoskeletal scaffolding. Scanning electron microscopic observation of tissue cultured rat neonatal cardiomyocytes indicates that exposure of these cells to the toxic aldehyde 4‐hydroxynonenal (4‐HNE), a free radical‐‐induced, lipid peroxidation product, results in the appearance of sarcolemmal blebs, whose ultimate rupture leads to cell death. Indirect immunofluorescent localization of a number of cytoskeletal components following exposure to 4‐HNE reveals damage to several, but not all, key cytoskeletal elements, most notably microtubules, vinculin‐containing costameres, and intermediate filaments. The exact mechanism underlying the selective disruption of these proteins cannot be ascertained at this time. Colocalization of actin indicated that whereas elements of the cytoskeleton were disrupted by increasing length of exposure to 4‐HNE, neither the striated appearance of the myofibrils nor the lateral register of neighboring myofibrils was altered. Monitoring systolic and diastolic levels of intracellular calcium ([Ca2+]i) indicated that increases in [Ca2+]i occurred after considerable cytoskeletal changes had already taken place, suggesting that damage to the cytoskeleton, at least in early phases of exposure to 4‐HNE, does not involve Ca2+ ‐dependent proteases. However, 4‐HNE–induced cytoskeletal alterations coincide with the appearance of, and therefore suggest linkage to, sarcolemmal blebs in cardiac myocytes.

Although free radicals produced by reperfusion or reoxygenation of ischemic tissue have been implicated in cellular damage, these studies represent the first evidence linking cardiomyocyte sarcolemmal damage to cytoskeletal disruption produced by a free radical product. © 1994 Wiley‐Liss, Inc.
Characterization of keratin densities in mitotic wish cells
Authors: Meek William D. , Henderson David A.
Published: February 2005

Three dimensional (3‐D) reconstruction of four mitotic WISH cells from ultrathin sections gave an informative representation of the spatial distribution of keratin densities in these cells. The correspondence between the densities as studied by transmission electron microscopy (TEM) and the Keratin bodies initially revealed by immunoflourescent colabeling of cultures, was confirmed by immunoelectron‐microscopy. The smaller, and sometimes more elongated densities, were relatively abundant just beneath the subplasmalemmal microfilament band; and at certain levels of the mitotic cell they were observed to be connected to neighboring densities by intact intermediate filaments (IFs). The larger and more spherical densities appeared to be somewhat more discrete and randomly distributed. Other observed associations of the keratin densities included the telophase contractile ring of microfilaments, chromosomes, the reformed telophase nucleus, and desmosomal junctions with neighboring interphase cells. Cytochalasin D (CD) treatment of cells displaced the peripheral keratin densities toward the cell membrane. The density volume constituted 0.52% to 1.57% of the total cell volume, and the proportional density size was decreased in the cells that had progressed into anaphase and telophase. The observed formation and subsequent dissolution of keratin densities during mitosis may represent a dynamic mechanism of restructuring the keratin cytoskeleton in an unpolymerized form in order to allow for rapid reformation of interphase cell junctions. The physical associations observed between intact IFs and the keratin densities may provide support at certain depths of the mitotic cell, and the juxtaposition of densities with nuclear components suggests a possible source of and role for keratin IFs during nuclear events. © 1994 Wiley‐Liss, Inc.
Complex intermolecular interactions maintain a stable linkage between the photoreceptor connecting cilium axoneme and plasma membrane
Authors: Muresan Virgil , Besharse Joseph C.
Published: February 2005

Microtubule‐membrane cross‐linkers in motile and nonmotile cilia are supramolecular structures, held together by strong interactions between the constituent molecules. We have characterized these interactions in the photoreceptor connecting cilium, where cross‐linkers co‐fractionate and maintain their in situ location after Triton X‐100 extraction of axonemes. In bovine photoreceptor cells, the transmembrane assemblage that is cross‐linked to the connecting cilium axoneme contains three high molecular mass glycoconjugates of 425, 600, and 700 kDa (Horst et al., 1987). The relative amounts of the three glycoconjugates, as judged from band intensity in electrophoretograms, depend strongly on sample treatment prior to electrophoresis. The electrophoretic pattern was reproducible after several weeks of storage of the axoneme fraction in extraction buffer containing 50% sucrose. Removal of sucrose from the buffer by dialysis eliminated the 600 kDa and 700 kDa, and decreased the detected amount of the 425 kDa glycoconjugate. When samples were incubated in Laemmli sample buffer at increasing temperatures (23°, 60°, 95°C), a gradual reduction in the intensity of the three bands was observed. The quantitative reduction of high molecular mass glycoconjugates was accompanied by the appearance of novel protein species of lower molecular mass, as detected by lectin and antibody overlays of axonemal transblots. These results suggest that the previously characterized cross‐linker glycoconjugates are complex, SDS‐resistant multi‐molecular conglomerates. We have further used fluorescent lectins to monitor the presence of glycoconjugates on whole‐mounted axonemes, in conditions aimed to selectively solubilize the cross‐linkers. The cross‐linker complexes could not be dissociated from the axoneme by incubation with buffers containing 1 M of either Na2SO4 or NaI. The results indicate that the connecting cilium‐specific cross‐linker complexes are bound via high‐affinity interactions to both axoneme and overlying plasma membrane. © 1994 Wiley‐Liss, Inc.
Movement of axoplasmic organelles on actin filaments from skeletal muscle
Authors: Kuznetsov Sergei A. , Rivera Domingo T. , Severin Fedor F. , Weiss Dieter G. , Langford George M.
Published: February 2005

It was recently shown that, in addition to the well‐established microtubule‐dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722‐725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video‐enhanced contrast differential interference contrast (AVEC‐DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine‐phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC‐DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley‐Liss, Inc.
Cloning and sequencing of cDNAs encoding the actin cross‐liking protein transgelin defines a new family of actin‐associated proteins
Authors: Prinjha R. K. , Shapland C. E. , Hsuan J. J. , Totty N. F. , Mason I. J. , Lawson D.
Published: February 2005

We have used degenerate oligonucleotides, derived from the amino acid sequence of transgelin peptides [Shapland et al., 1993: J. Cell Biol. 121:1065‐1073], to isolate and sequence overlapping cDNA clones encoding this actin gelling protein. Primers with 5′ restriction enzyme sites directed against the N and C terminal amino acids present in these clones were then used to amplify and clone the entire transgelin coding region from reverse transcribed rat small intestine cDNA (RT‐PCR). These studies have shown that transgelin is the product of a single gene which is conserved between yeast, Drosophila, molluscs, and humans. Transgelin is expressed as a single message that is regulated at the level of transcription in SV40 transformed 3T3 cells. Our data have shown that transgelin and several other proteins of unknown function, SM22α [Pearlstone et al., 1987: J. Biol. Chem. 262:5985‐5991], mouse p27 [Almendral et al., 1989: Exp. Cell Res. 181:518‐530], and human WS3‐10 [Thweatt et al., 1992: Biochem. Biophys. Res. Commun. 187:1‐7], share extensive homology. More limited regions of homology shared between transgelin and other proteins such as rat NP25 (unpublished), chicken calponins α and β [Takahashi and Nadal‐Ginard, 1991: J. Biol. Chem. 266:13284‐13288], and Drosophila mp20 [Ayme‐Southgate et al., 1989: J. Cell Biol. 108:521‐531] suggest that all of these proteins may be classified as members of a new transgelin multigene family. © 1994 Wiley‐Liss, Inc.
Human neutrophil motility: Time‐dependent three‐dimensional shape and granule diffusion
Authors: Felder Stephen , Kam Zvi
Published: February 2005

The locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three‐dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three‐dimensional cell edges outlined in the optical sections were analyzed qualitatively in time‐lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 μm/min with a period of 50–55 seconds. Within the periodic bursts of speed, a fibroblast‐like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2‐3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two‐ to 2.5‐fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward.

Three‐dimensional movies of PMN cells are included in the video supplement. © 1994 Wiley‐Liss, Inc.
Differential regulation of skeletal muscle myosin‐II and brush border myosin‐I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms
Authors: Fanning A. S. , Wolenski J. S. , Mooseker M. S. , Izant J. G.
Published: February 2005

In this report, we have compared the physical properties and actin‐binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin‐II and brush border (BB) myosin‐I. All of the bacterially produced nonmuscle tropomyosins bind to F‐actin with the expected stoichiometry and with affinities comparable to that of a tissue produced α‐tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity of F‐actin than a tissue‐purified striated muscle α tropomyosin. The bacterially produced isoforms also protect F‐actin from severing by villin as effectively as tissue‐purified striated muscle α‐tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin‐activated MgATPase activity of muscle myosin S1 by ∼ 70–85%, comparable to the inhibition seen with tissue‐purified striated muscle α tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin‐activated MgATPase activity of muscle myosin S1 approximately two‐ to threefold. The in vitro sliding of actin filaments translocated by muscle myosin‐II (2.4 μm/sec at 19°C, 5.0 μm/s at 24°C) increased 25–65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin‐II motility. The actin‐activated MgATPase activity of BB myosin‐I was inhibited 75–90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin‐I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo. © 1994 Wiley‐Liss, Inc.
Differential localization of α‐actinin and the 30 kD actin‐bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum
Authors: Furukawa Ruth , Fechheimer Marcus
Published: February 2005

Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, α‐actinin and the 30 kD actin‐bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for α‐actinin is not apparent in this region. By contrast, α‐actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of α‐actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and α‐actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18–27]. α‐actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that α‐actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium. © 1994 Wiley‐Liss, Inc.
Fluorescence studies of spectrin and its subunits
Authors: Subbarao Nanda K. , MacDonald Robert C.
Published: February 2005

To better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2‐p‐toluidinyl napthalene‐6‐sulfonic acid (TNS). It therefore appears that low ionic strength, α‐β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994 Wiley‐Liss, Inc.
Novel touch‐induced, Ca2+‐dependent phobic response in a flagellate green alga
Authors: Kreimer Georg , Witman George B.
Published: February 2005

The biflagellate green algaSpermatozopsis similisexhibits a remarkable avoidance reaction in addition to the photophobic or stop response characteristic of such algae.S. similisnormally swims forward with its anteriorly attached flagella directed posteriorly and propagating sine‐like waves from base to tip. Upon contact with surfaces or other cells,S. similisresponds with rapid backward swimming, covering distances of up to 50 μm in 140 to 220 msec. This reaction, which we term the mechanoshock response, also can be triggered by vigorous mechanical stimulation, but not by physiological light intensities. It consists of 3 phases: (1) a rapid acceleration phase with average duration of 31 msec; (2) a phase of about 66 msec with constant high speed (maximal velocities of > 600 μm·sec−1) or slow deceleration; and (3) a deceleration phase of ∼ 83 msec, followed by a stop or short period of circling. The cells then resume forward swimming in a random direction. Prior to the mechanoshock response the flagella rapidly are brought together into a close parallel configuration extending anteriorly of the cell body. They then appear to propel the cell by undulatory beating, while the cell describes a pronounced helical path. Small decreases in the extracellular Ca2+concentration, as well as low concentrations of Ba2+, strongly suppress the probability of this phobic reaction. We conclude that this mechanoshock response involves large Ca2+influxes, probably mediated by mechanosensitive and/or stretch‐activated ion‐channel(s). © 1994 Wiley‐Liss, Inc.
Microtubule associated protein MAP1A is an actin‐binding and crosslinking protein
Authors: Pedrotti Barbara , Colombo Roberto , Islam Khalid
Published: February 2005

High molecular weight microtubule‐associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G‐ and F‐actin. Using a solid‐phase immunoassay we show that MAP1A binds in a dose‐dependent manner to both G‐actin and F‐actin. Addition of MAP1A to F‐actin causes gelation of F‐actin and SDS‐PAGE analysis shows that MAP1A co‐sediments with the gelled network, under conditions where F‐actin alone does not pellet. The low apparent viscosity of F‐actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F‐actin. Co‐incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non‐neuronal cellular morphology. © 1994 Wiley‐Liss, Inc.
22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes
Authors: Fok Agnes K. , Wang Hali , Katayama Akiko , Aihara Marilynn S. , Allen Richard D.
Published: February 2005

In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three‐headed while the 12S is a one‐headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three‐headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia. © 1994 Wiley‐Liss, Inc.
Effect of protein kinase inhibitor H‐7 on the contractility, integrity, and membrane anchorage of the microfilament system
Authors: Volberg Tova , Geiger Benjamin , Citi Sandra , Bershadsky Alexander D.
Published: February 2005

Addition of protein kinase inhibitor H‐7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992: J. Cell Biol. 117:169–178] it was demonstrated that intercellular junctions in H‐7‐treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H‐7‐treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact‐attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N‐cadherin associated with cell‐cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H‐7 suppresses cellular contractility. Thus, short pretreatment with H‐7 leads to strong inhibition of the ATP‐induced contraction of saponin permeabilized cells. Comparison of H‐7 effects with those of other kinase inhibitors revealed that H‐7‐induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31‐8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H‐7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell‐cell junctions in low‐calcium medium. © 1994 Wiley‐Liss, Inc.
Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon
Authors: Schwuchow Jochen , Sack Fred D.
Published: February 2005

Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos‐methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30–60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load‐bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. © 1994 Wiley‐Liss, Inc.
NI2+ inhibition induces asymmetry in axonemal functioning and bend initiation of bull sperm
Authors: Lindemann Charles B. , Walker Jay M. , Kanous Kathleen S.
Published: February 2005

Bull sperm extracted with 0.1% Triton X‐100 can be reactivated to full motility with 0.33 mM Mg‐ATP (sperm models). When motile sperm models are treated with 0.66 mM NiSO4, spontaneous motility is lost. During the transition to motility arrest, the beat becomes progressively more asymmetric, finally arresting at one extreme of the beat cycle. After spontaneous motility has been lost, the flagellum retains the ability to respond to mechanical stimulation. If a microprobe is used to bend the flagellum in the direction opposite to its own prevailing curvature and released, the recoil is rapid and overshoots the equilibrium position. When the same flagellum is manipulated in the opposite direction (into a tighter bend of the existing curve), the recoil is slower and does not exceed the initial bend. If a microprobe is used to carefully bend the whole flagellum into a curve, the flagellum will resume continuous beating, but only if the imposed bend is in the direction opposite the natural curvature. The reinstated beating activity (mechanical reactivation) is sustained as long as the flagellum is held by the microprobe. The rate of change of the shear angle in these mechanically reactivated, Ni2+ ‐inhibited sperm suggests an impaired rate of sliding on one side of the axoneme compared to similarly restrained control sperm. It appears that Ni2+ has a selective inhibitory effect on the dynein arms that bend the flagellum in one direction. Furthermore, the remaining functional arms activate only when the flagellum is bent in the direction opposing their own action. © 1995 Wiley‐Liss, Inc.
Phospholipid membrane‐associated brush border myosin‐I activity
Authors: Zot Henry G.
Published: February 2005

Brush border myosin‐I (BBMI) is associated with the membrane of intestinal epithelial cells where it probably plays a structural role. BBMI also has been identified on Golgi‐derived vesicles in intestinal epithelial cells where it may translocate vesicles into the brush border. However, the mechanochemical activity of BBMI bound to a phospholipid membrane has not been described. This study reports that phospholipid membrane‐associated BBMI displays ATPase activity when bound to phospholipids, but does not move actin filaments when associated with a phospholipid bilayer. BBMI does not bind significantly to brush border membrane lipids, which contain about 16% phosphatidylserine (PS), in either a pelleting or planar membrane assay. Similarly, planar membranes containing 20% PS do not bind a significant amount of BBMI. Increasing the concentration of PS to 40% does result in the binding of BBMI to both vesicles and planar membranes. This binding is enhanced with increased Ca2+ concentrations. BBMI retains its ATPase activity when bound to phospholipid vesicles containing 40% PS. However, BBMI attached to a phospholipid bilayer surface does not move actin filaments, even though the amount of BBMI bound to the lipid surface, as reflected by the number of actin filaments associated with bilayer‐bound BBMI, is sufficient to observe motility in control experiments. When membrane fluidity is reduced by adding cholesterol to the membrane lipids containing 40% PS, BBMI still binds to the membrane, but again no actin filament motility is observed. The lack of binding by BBMI to brush border membrane lipids and the absence of membrane‐associated BBMI mechanical activity suggest that factors in addition to membrane lipids are necessary for membrane‐associated myosin‐I motility. © 1995 Wiley‐Liss, Inc.
Actin cytoskeleton of fibroblasts organizes surface proteoglycans that bind basic fibroblast growth factor and lipoprotein lipase
Authors: Fernáandez‐Borja Mar , Bellido David , Makiya Ricardo , David Guido , Olivecrona Gunilla , Reina Manuel , Vilaró Senén
Published: February 2005

Cell surface proteoglycans participate in molecular events that regulate cell adhesion, migration, and proliferation. To investigate the organization of these molecules at the cell surface, the distribution of two well‐known proteoglycan ligands has been studied. These ligands, lipoprotein lipase and basic fibroblast growth factor, showed a characteristic binding pattern consisting of highly organized parallel arrays that crossed the upper surface of human skin fibroblasts. The proteoglycan nature of the binding sites was evident from their susceptibility to heparinases, and from ligand displacement by heparin. Parallel localization of the ligands and actin, and treatment of the cells with cytochalasin, showed that the binding proteoglycans are organized by the actin cytoskeleton. The ligands induced a different behaviour of the binding sites on incubation of the cells at 37°C. Lipoprotein lipase produced a movement of the binding proteoglycans along the actin filaments towards the cell center. In contrast, after binding of basic fibroblast growth factor the binding proteoglycans remained spread over the cell surface and actin depolymerization was induced. Since an increasing number of ligands appear to depend on proteoglycans for their interactions with their high affinity receptors, distribution and movement of proteoglycans at the cell surface that is organized by the actin cytoskeleton could direct and enhance the encounters between the ligands and their specific receptors. © 1995 Wiley‐Liss, Inc.
Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells
Authors: Pasdar Manijeh , Li Zhi , Chan Honey
Published: February 2005

Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin‐Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylared on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell‐cell contact, in the presence of protein kinase inhibitor, H‐7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H‐7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid‐treated cultures. These results suggest that via reversible protein phosphorylation involving both protein kinase and protein phosphatases. © 1995 Wiley‐Liss, Inc.
Quantitation of cap Z in conventional actin preparations and methods for further purification of actin
Authors: Casella James F. , Barron‐Casella Emily A. , Torres Michelle A.
Published: February 2005

Gel‐filtration is commonly used to remove contaminants from conventional actin prepared by the method of Spudich and Watt. It has been shown that this procedure removes the majority of a factor that reduces the low‐shear viscosity of actin. We have previously reported that this factor is Cap Z, a barbed end capping protein. We now establish that, even after gel‐filtration, enough Cap Z can be present in conventionally prepared actin to affect events occurring at the barbed ends of actin filaments. We also demonstrate that the concentration of Cap Z can be reduced to more than a log below the KD for binding of Cap Z to actin by either (1) immunoabsorbtion of conventionally prepared actin with anti‐Cap Z antibodies, or (2) an additional cycle of polymerization/depolymerization followed by repeat gel‐filtration. © 1995 Wiley‐Liss, Inc.
Purification of microtubule associated protein MAP1B from bovine brain: MAP1B binds to microtubules but not to microfilaments
Authors: Pedrotti Barbara , Islam Khalid
Published: February 2005

A simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion‐exchange chromatographic steps and results in >95% pure MAP1B with a typical recovery of about 25–30 mg/kg of brain tissue. SDS‐PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti‐MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co‐sediments with taxol‐stabilised microtubules. Co‐incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G‐actin nor F‐actin nor co‐sediments with F‐actin, suggesting that it is not an actin‐binding protein.
Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells
Authors: King‐Smith Christina , Bost‐Usinger Laurie , Burnside Beth
Published: February 2005

To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well‐characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule‐dependent processes in RPE.