bluuebunny/crossref_metadata_2025_split huggingface dataset:
Axonal neurofilaments differ in composition and morphology from those in the soma of the squid stellate ganglion
Authors: Tytell M. , Zackroff R. V. , Hill W. D. 
Published: February 2005

AbstractTriton X‐100 insoluble neurofilament (NF) fractions were obtained from two parts of the stellate ganglion and the main giant axon. These were analyzed by one‐ and two‐dimensional gradient polyacrylamide gel electrophoresis, cyclic assembly and disassembly, and electron microscopy. The NF fractions from the ganglion cell bodies (GCB) and from the part of the ganglion mainly consisting of axon initial segments (GIS) were of similar composition; neither contained detectable amounts of the 220 kda and high molecular weight ( 400 kda) NF subunits that were prominent in the axonal NF fraction. However, the GCB and GIS did contain large quantities of a set of 65 kda polypeptides that were minor constituents of the axonal NF fraction. The 65 kda‐containing NF fraction from the ganglion could be cyclically disassembled and reassembled, but only under low salt conditions, in contrast to the high salt conditions used to cycle axonal NFs. A comparison of the peptide map of the 65 kda polypeptides with that of the 60 kda axonal NF subunit showed them to be different. These biochemical differences between the ganglionic and axonal NF fractions correlated with morphologic distinctions: ganglionic NFs were relatively smooth surfaced, whereas axonal NFs had long sidearms. Such observations support the hypothesis that the NF cytoskeleton of the neuron soma is different from that of the axon. Furthermore, the change from the somal form to the axonal form of NFs appears to occur in the region where the axon initial segment increases in diameter to become the axon proper.
Microtubule‐associated organelle and vesicle transport in fibroblasts
Authors: Hayden John H. 
Published: February 2005

AbstractAllen Video‐enhanced constrast/differential interference constrast (AVEC‐DIC) microscopy was used in conjunction with video intensification immunofluorescence microscopy to demonstrate that organelles and vesicle (particles) can move in either direction along microtubular linear elements in fibroblasts [Hayden et al., 1983]. Since it is not possible to determine the number of microtubules making up a linear element with light microscopy alone, AVEC‐DIC microscopy was used in conjunction with whole‐mount electron microscopy to show bidirectional transport along a single microtubule [Hayden and Allen, 1984]. These studies demonstrate that the structural polarity of the microtubule does not determine the direction of particle motion, and since dynein is an asymetric molecule, a simple microtubule‐dynein‐particle hypothesis cannot explain bidirectional transport along a single microtubule.Very little is known about regulation of particle transport in most cell types. Human embryonic lung fibroblasts grown on glass coverslips were serum‐deprived for 24 hours and re‐fed with serumless medium; the particle translocations/5 minutes were then determined The cells were then re‐fed with either serumless medium, serum‐containing medium, or serumless medium containing some bioactive factor, and the particle translocations/5 minutes were again determined for the same cells. Medium containing 10% fetal bovine serum inhibited particle translocation by 51.8%. Of the bioactive factors tested, only vasopressin produced a significant reduction in particle translocations (38%). This suggests that protein kinase C or calcium/calmodulin kinase could be involved in regulating particle transport.
Two monoclonal antibodies to actin: One muscle selective and one generally reactive
Authors: Lessard James L. 
Published: February 2005

AbstractTwo IgG1, κ monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross‐reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino‐terminal two‐thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.
Colocalisation of acetylated microtubules, glial filaments, and mitochondria in astrocytes in vitro
Authors: Cambray‐Deakin Martin A. , Robson Susanne J. , Burgoyne Robert D. 
Published: February 2005

AbstractWe have recently shown that acetylated α‐tubulin containing microtubules (acety1‐MTs; labeled by antibody 6‐11B‐1) constitute a cold‐stable subset of the microtubule network of nonneuronal cells in rat primary forebrain cultures [Cambray‐Deakin and Burgoyne: Cell Motil. 8(3):284–291, 1987b]. In contrast, tyrosinated α‐tubulin containing MTs (tyr‐MTs; labeled by antibody YL1/2) are cold‐labile. Here we have examined the distribution of acety1‐MTs and tyr‐MTs in cultures of newborn rat forebrain astrocytes and simultaneously investigated the distribution of mitochondria and glial filaments. In double‐label immunofluorescence experiments a marked colocalisation of acetyl‐MTs and glial filament bundles was observed. Tyr‐MTs did not show a similar colocalisation with glial filament bundles. Furthermore, the distribution of mitochondria closely followed that of the acetyl‐MT and glial filament bundles. When cells were exposed to short‐term (30‐min) treatments with MT‐disrupting agents such as colchicine and nocodazole, the tyr‐MT network was removed but the distributions of acetyl‐MTs, glial filaments, and mitochondria were unchanged. Increased exposure to colchicine (9–16 hr) caused a progressive disruption of the acetyl‐MTs and the collapse of glial filaments and mitochondria to the perinuclear region. These results suggest that acetyl‐MTs and glial filaments but not tyr‐MTs may be involved in the intracellular transport of organelles and/or in the control of their cytoplasmic distribution.
Iontophoretic localization of Ca‐sensitive sites controlling activation of ciliary beating in macrocilia of Beroë: The ciliary rete
Authors: Tamm Sidney L. 
Published: February 2005

AbstractMacrocilia are thick compound ciliary organelles found on the lips of the ctenophore Beroë. Each macrocilium contains several hundred axonemes enclosed by a single common membrane around the shaft of the organelle. Macrocilia are activated to beat rapidly and continuously in the normal direction by stimulus‐triggered Ca influx through voltage‐dependent Ca channels (Tamm, 1988). Heat‐dissociated macrociliary cells are spontaneously active without depolarizing stimuli, providing Ca is present (Tamm, 1988). Here we investigate the spatial distribution of macrociliary Ca channels by iontophoretic application of extracellular Ca to different sites along quiescent, “potentially activated” macrocilia of dissociated cells in Ca‐free medium. We find that Ca sensitivity for eliciting motility is highest or resides exclusively on the basal portion of the macrociliary surface. This is the first demonstration of local differences in Ca morphologically with a reticulum of unfused ciliary membranes at the base of the macrocilium. This ciliary rete is in direct communication with the surrounding sea water. It is likely that the ciliary rete provides the necessary Ca influx to trigger beating by virtue of its greater Ca conductance (i.e., density of Ca channels) and/or greater total membrane area.
Video supplement assembly, disassembly, and movements of the microfilament‐rich ridge during the amoeboflagellate transformation in Physarum polycephalum
Authors: Pagh Kathryn I. , Adelman Mark R. 
Published: February 2005

AbstractThe amoeboflagellate transformation in Physarum polycephalum involves a series of dramatic changes in cell shape and motile behavior. This report describes the morphological and behavioral changes through which a synchronously transforming population of cells passes, stressing that, although there are a series of distinguishable stages, cells at all stages display striking plasticity. Our previous studies showed that amoeboflagellates transiently display a flattened motile extension—the ridge—that projects from a specific location on the cell surface and contains a laminar core densely packed with a series of crisscrossing arrays of actin microfilaments. Details are presented here concerning the movements of the ridge as well as the dynamics of ridge formation and disassembly in relation to other morphogenetic events of the transformation. The ridge forms at about the same time as transforming cells begin to elongate, propagates undulations parallel to the long axis of the cell as the transformation proceeds, and disassembles late in the transformation. Staining of fixed cells with the fluorescent probe rhodaminephalloidin shows that the actin of amoeboid cells is strikingly redistributed as the transformation proceeds. Amoeboflagellates contain most of the stainable actin in the ridge and in a ventral‐posterior spot that may be a site of cell‐substratum adhesion. These results provide additional insights into the possible functions of the ridge and the roles of actin during the amoeboflagellate transformation.
Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae
Authors: Bennett Holly , Condeelis John 
Published: February 2005

AbstractWe have used a polyclonal affinity‐purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod‐shaped protein 118 ± 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one‐dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.
Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization
Authors: Okuhara Koji , Murofushi Hiromu , Sakai Hikoichi 
Published: February 2005

AbstractThe localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity‐purified antibody against bovine adrenal kinesin.It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody‐stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine‐phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold‐treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37°C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.
Actomyosin organization during cytokinesis: Reversible translocation and differential redistribution in Dictyostelium
Authors: Kitanishi‐Yumura Toshiko , Fukui Yoshio 
Published: February 2005

AbstractSynchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar‐overlay technique (Yumura et al.: J. Cell Biol. 99:894–899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI.This study showed that the actin‐ and myosin‐containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring.The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.
Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts
Authors: Svitkina Tatjana M. , Surguchova Irina G. , Verkhovsky Alexander B. , Gelfand Vladimir I. , Moeremans Marc , De Mey Jan 
Published: February 2005

AbstractThe authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin‐like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin‐free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde.Electron microscopy of platinum replicas of these preparations revealed dumbbell‐shaped structures of approximately 0.28 μm in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment I and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin‐free cytoskeletons were often organized in chains and lattices formed by end‐to‐end contacts of individual filaments at their head‐containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.
Myofibril assembly is linked with vinculin, α‐actinin, and cell‐substrate contacts in embryonic cardiac myocytes in vitro
Authors: Terai Masaru , Komiyama Masatoshi , Shimada Yutaka 
Published: February 2005

AbstractThe relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double‐stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, α‐actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy.In the areas of myofibril assembly, vinculin and α‐actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell‐to‐substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross‐striated characteristics. Thus, the formation of these inferred lines has some relation to or is in fact necessary for the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins.Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.
Use of a novel Chlamydomonas mutant to demonstrate that flagellar glycoprotein movements are necessary for the expression of gliding motility
Authors: Bloodgood Robert A. , Salomonsky Nancy L. 
Published: February 2005

AbstractAs an alternative to swimming through liquid medium by the coordinated bending activity of its two flagella, Chlamydomonas can exhibit whole cell gliding motility through the interaction of its flagellar surfaces with a solid substrate. The force transduction occurring at the flagellar surface can be visualized as the saltatory movements of polystyrene microspheres. Collectively, gliding motility and polystyrene microsphere movements are referred to as flagellar surface motility. The principal concanavalin A binding, surface‐exposed glycoproteins of the Chlamydomonas reinhardtii flagellar surface are a pair of glycoproteins migrating with apparent molecular weight of 350 kDa. It has been hypothesized that these glycoproteins move within the plane of the flagellar membrane during the expression of flagellar surface motility. A novel mutant cell line of Chlamydomonas (designated L‐23) that exhibits increased binding of concanavalin A to the flagellar surface has been utilized in order to restrict the mobility of the concanavalin A‐binding flagellar glycoproteins. Under all conditions where the lateral mobility of the flagellar concanavalin A binding glycoproteins is restricted, the cells are unable to express whole cell gliding motility or polystyrene microsphere movements. Conversely, whenever cells can redistribute their concanavalin A binding glycoproteins in the plane of the flagellar membrane, they express flagellar surface motility. Since the 350 kDa glycoproteins are the major surface‐exposed flagellar proteins, it is likely that most of the signal being followed using fluorescein isothiocyanate (FITC)‐concanavalin A is attributable to these high molecular weight glycoproteins. Therefore, it is likely that the 350 kDa glycoproteins are the ones that must move laterally in the plane of the flagellar membrane in order for the cell to express whole cell gliding motility and microsphere movements along the flagellar surface. This study represents one of the first demonstrations, in any cell type, that whole cell locomotion requires glycoprotein movement within the plane of the plasma membrane.
Changes in the association of actin‐binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum
Authors: Dharmawardhane Suranganie , Warren Vivien , Hall Anne L. , Condeelis John 
Published: February 2005

AbstractTriton‐insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2′deoxy cyclic adenosine monophos‐phate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin‐binding protein (ABP‐120) in cytoskeletons following stimulation were monitored. Both actin and ABP‐120 were incorporated into the cytoskeleton at 30–40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattraciants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP‐120 and actin following the addition of 2′deoxy cAMP. The authors concluded that the association of ABP‐120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP‐120 is involved in cross‐linking newly assembled actin filaments into the cytoskeleton during chemoattractant‐stimulated pseudopod extension.
Immunocytochemical studies with antibodies to three proteins prominent in the isolated microtubule cytoskeleton of a trypanosomatid
Authors: Bramblett Gregory T. , Kambadur Ravi , Flavin Martin 
Published: February 2005

AbstractThe cytoskeleton of Crithidia fasciculata consists of a corset of paralle microtubules enclosing the cell body and closely underlying the plasma membrane. Distinct sets of crosslinks appear to connect tubules to each other and to membrane. Our objective is to determine the composition of these crosslinks and to elucidate the basis of this spectacular example of membrane‐microtubule interaction. We purified three proteins (designated COP‐33, ‐41, and ‐61 by their subunit Mr), which were consistently abundant in highly purified cytoskeletons. All three bound strongly to microtubules in vitro, and the first two induced bundles through periodic crosslinking. Polyclonal antibodies against each have been used to try to localize these proteins in thin sections of cells or whole mounts of cytoskeletons. Antibodies to COP‐41 bound sepcifically to glycosomes, organelles that encapsulate many glycolytic enzymes in these protozoa, and COP‐41 has been identified as glyceraldehyde 3‐P dehydrogenase.