bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractCellular 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

AbstractThe 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

AbstractThe 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

AbstractWe 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

AbstractUnfertilized 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

AbstractWe 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

AbstractPolyclonal 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

AbstractSpores 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

AbstractWe 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.
Actin‐binding proteins regulate the work performed by myosin II motors on single actin filaments
Authors: Janson Lee W. , Sellers James R. , Taylor D. Lansing 
Published: February 2005

AbstractRegulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299–313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, “The Enzymes,” Vol. 18, Orlando, FL: Academic Press, 1987, pp. 381–418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin‐binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353–402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987–1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57–143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272–6276, 1986] by including filamin or α‐actinin on the substrate with myosin II to examine how actin‐crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin‐crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation‐contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185–197, 1982] which proposes that increased crosslinking of actin inhibits myosin‐based contraction. This study also illustrates the potentially varied roles of different actin‐crosslinking proteins and offers a novel method to examine actin‐binding protein activity and their regulation of motility at the single molecule level. © 1992 Wiley‐Liss, Inc.
Association of kinesin with characterized membrane‐bounded organelles
Authors: Leopold Philip L. , McDowall Alasdair W. , Pfister K. Kevin , Bloom George S. , Brady Scott T. 
Published: February 2005

AbstractThe family of molecular motors known as kinesin has been implicated in the translocation of membrane‐bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane‐bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane‐bounded organelles in the cell. In addition, immuno‐electron microscopy of mitochondria revealed a patch‐like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility. © 1992 Wiley‐Liss, Inc.
Establishment of division plane and mitosis in monoplastidic guard mother cells of Selaginella
Authors: Cleary Ann L. , Brown Roy C. , Lemmon Betty E. 
Published: February 2005

AbstractStomatogenesis, the determinate developmental pathway leading to formation of a pair of specialized guard cells, was studied in monoplastidic cells ofSelaginella. Observations of living cells followed by immunofluorescence microscopy of the same cells made it possible to correlate changes in cytoskeletal organization with developmental events. The guard mother cell divides in a plane perpendicular to previous divisions and this shift in polarity is marked by morphogenetic plastid migration, as well as by extensive reorganization of cytoskeletal arrays. The single plastid divides and daughter plastids move to a position opposite each other (incipient spindle poles). The axis defined by the opposing plastids rotates in the cell before becoming fixed in position with polar plastids adjacent to the lateral anticlinal walls. Plastid polarity predicts spindle orientation and the plane of division. Once division polarity is defined by plastid position, which will remain unchanged throughout mitosis and cytokinesis, cortical microtubules become reorganized from radial to longitudinal (relative to the long axis of the leaf). The initially random cortical F‐actin also becomes aligned longitudinally. A wide preprophase band of microtubules and F‐actin is formed at right angles to the spindle axis. Plastid‐based microtubules establish the preprophase spindle and also connect to the preprophase band. The mitotic spindle remains anchored at the polar plastids. After mitosis, a phragmoplast that forms among microtubules emanating from plastids and nuclei develops in the plane marked previously by the preprophase band. Mitosis is completed in 1 h 15 min ± 3 min (mean ± S.E.). © 1992 Wiley‐Liss, Inc.
Antikinetochore antibodies interfere with prometaphase but not anaphase chromosome movement in living PtK2 cells
Authors: Wise Dwayne A. , Bhattacharjee Lakshmisri 
Published: February 2005

AbstractInjection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate‐buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP‐B, ‐C, and ‐D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement. © 1992 Wiley‐Liss, Inc.
LC20 and kinetics of gizzard myosin subfragment‐1: Digestion with papain vs. S. aureus protease
Authors: Drew Jean S. , White Marianne P. , Moos Carl , Stein Leonard A. 
Published: February 2005

AbstractPrevious reports have shown that papain‐digested gizzard subfragment‐1 (PAP‐S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) Greene et al., Biochemistry 22:530–535, 1983; Sellers et al., J. Biol. Chem. 257:13880‐13883, 1982; Umemoto et al., [J. Biol. Chem. 264:1431–1436, 1989], while S. aureus protease‐digested S‐1 (SAP‐S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment‐ 1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP‐S1 and SAP‐S1. SDS‐PAGE showed that SAP‐S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP‐S1 (15 minutes digestion at 20°C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22–24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP‐S1 and SAP‐S1 had Vmax similar to phosphorylated HMM (1.1–1.5 s−1). SAP‐S1 had the same KATPase as phosphorylated HMM (38 μM actin). but KATPase for PAP‐S1 was 3‐fold stronger (11 μM actin). Subsequent digestion of SAP‐S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3‐ to 5‐fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding. © 1992 Wiley‐Liss, Inc.