bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stress fiber reformation after ATP depletion
Authors: Glascott Peter A. , McSorley Karen M. , Mittal Balraj , Sanger Jean M. , Sanger Joseph W. 
Published: February 2005

AbstractFlurescently labeled heavy mermoyosin, alpha‐actinin, and vinculin were used to localize actin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP‐depleting medium (20 mM sodium azide and 10 mM 2‐deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha‐actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha‐actinin, and vinculin in the cell became localized in one perinuclear aggregate, with a diameter of approximaterly 15–25 μm. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 μm/minute and grew to as large as 70 μm in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diamater of te rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytoplasmic motility.
Invited review: Fine (2–5‐nm) filaments: New types of cytoskeletal structures
Authors: Roberts Thomas M. 
Published: February 2005

AbstractOver the past 30 years filaments 2–5 nm in diameter have been foun in a number of different types of eukaryotic cells. As a group, these fine filaments lack the similarity of composition and function that characterize the three major classes of cytoskeleta elements—microfilaments, microtubules, and intermediate filaments. Six different proteins that form fine filaments have been identified; proposed functions for these fibers range from cell motility to cytoarchitecture. Recent studies, however, have revealed filaments with similar compositions and/or functions in otherwise different cells, sugesting that the fine filaments may eventually fit into a limited number of subgroups.
Cell type‐specific association between two types of spectrin and two types of intermediate filaments
Authors: Langley Robert C. , Cohen Carl M. 
Published: February 2005

AbstractWe have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside‐out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such association may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.
“Pull” and “push” in neurite elongation: Observations on the effects of different concentrations of cytochalasin B and taxol
Authors: Letourneau Paul C. , Shattuck Terri A. , Ressler Alice H. 
Published: February 2005

AbstractNeurite elongation involves two distinct cytoskeletal functions the “push” of anterograde transport of the cytoskeleton and associated organelles to the neurite tip, and the “pull” exerted by protrusion and generation of tensions in the growth cone. We investigated the roles of these two activities in neurite elongation via the drugs taxol and cytochalasin B (CB), which act on the key cytoskeletal components, microtubules and actin filaments, respectively. When neurons are treated with concentrations of CB below 0.2 μg/ml, neurite elongation, growth cone protrusion, and neurite tension are all inhibited in a similar concentration dependent manner. Protrusive activity and tensions are absent at CB concentrations above 0.3 μg/ml, yet neurite elongation continues at a plateau level. Thus, “pull” does modulate, but it is not required for neurite elongation. Surprisingly, the inhibitory effects of taxol on neurite elongation are removed by the addition of CB at levels that substantially disrupt the actin filaments of neurites. The neurites extended by taxol‐CB neurons are unbranched and curiously unattached to the substratum. When CB is added to taxol‐treated neurons, neurite extension begins rapidly, even if protein synthesis is severely reduced. We propose that taxol inhibits microtubule transport in neurites, and this inhibition of “push” is reversed by the disruptive effects of CB on the cytoplasmic matrix, allowing taxol‐induced microtubule bundles to be transported distally.
Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes
Authors: Paulin‐Levasseur Micheline , Brown David L. 
Published: February 2005

AbstractWe have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through in the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearragements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.
Analysis of the flagellar bending waves of ejaculated ram sperm
Authors: Chevrier C. , Dacheux J. L. 
Published: February 2005

AbstractThe variability of flagellar movement, illustrated by the highly heterogenous nature of the ejaculated sperm population of the ram, was analyzed by the use of a stroboscopic technique and an adapted microphotographic 24 × 36 camera system. The multiple‐moving‐exposures (MME) records give very distinct successive sequences of the flagellar beats and are particularly suitable for the analysis of bend development and propagation along the tail. With this technique, the parameters of the flagellar bending waves of ejaculated ram sperm have been determined. Most of the sperm have planar flagellar beatings; few are rolling under the conditions of observation. The trajectories of the gametes are mostly linear; nevertheless, some have circular paths. The analysis of bending has been focused on two examples for which the difference in the progressiveness ratio was maximum. The circular pathways for ram spermatozoa are linked to an asymmetry between principal and reverse bend probably induced by differences in wave propagation evidenced along the flagellum. A typical sperm flagellar movement may be related either to the conditions of the observations or to some differences in the maturation process of the sperm.
Acetylated and detyrosinated α‐tubulins are co‐localized in stable microtubules in rat meningeal fibroblasts
Authors: Cambray‐Deakin Martin A. , Burgoyne Robert D. 
Published: February 2005

AbstractWe have examined the distribution of acetylated α‐tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meaninges. Meningeal fibroblasts showed heterogenous staining patterns with a monoclonal antibody against acetylated α‐tubulin ranging from staining of primary cilia or microtubule‐organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti‐α‐tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double‐labeling experiments using an antibody against acetylated α‐tubulin (6–11B‐1) and antibodies against either tyrosinated or detyrosinated α‐tubulin, it was found that acetylated α‐tubulin and tyrosinated α‐tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated α‐tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated α‐tubulin and was cold stable, and the other contained tyrosinated α‐tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of α‐tubulin are involved in the specification of stable microtubules.
Flagellar movement of intact and demembranated, reactivated ram spermatozoa
Authors: Ishijima Sumio , Witman George B. 
Published: February 2005

AbstractThe flagellar movement of intact ejaculated ram sperm, and of demembranated models reactivated with ATP, has been studied using high‐speed, high‐resolution video microscopy.Intact sperm attached to the coverslip by their heads had an average beat frequency of 20.9 Hz and an average wave amplitude of 20.2 μm. There was little difference in the beat frequency or waveform of these sperm and sperm swimming freely near the coverslip or captured by their heads with a micropipette and held far from the coverslip, inducationg that the flagellar waveform of ram sperm is relatively resistant to distorition as a result of immobilization of the head or proximity to a surface. The beat envelope was nearly planar as determined by observations of free‐swimming sperm and sperm captured by their head and oriented so they were beating either parallel or perpendicular to the plane of focus.The effect of various conditions for demembranation and reactivation of the sperm were examined. Treatment of sperm with 0.2 % Triton X‐100 removed most of their plasma membrane. Under optimal conditions, nearly 100 % of the demembranted sperm reactivated at MgATP2− concentrations ranging from ∼4 μM to ∼20 mM. From ∼ 1 mM to ∼ 10 mM MgATP2−, their beat pattern closely resembled that of intact sperm; beat frequency depended on MgATP2− concentration. Percent motility was maximal between pH 7.5 and 8.0 and decreased sharply below pH 7.0 and avove pH 8.5. The addition of 50 μM cAMP to the reactivation medium had no effect on percent motility or the beat pattern and did not accelerate the initiation of movement.
Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states
Authors: Spudich Annamma , Wrenn Joan T. , Wessells Norman K. 
Published: February 2005

AbstractChanges in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold‐labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1‐μm‐thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin‐filament phalloidin‐binding sites by some cellular actin‐binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.
Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels
Authors: Zigmond Sally H. , Slonczewski Joan L. , Wilde Mary W. , Carson Monica 
Published: February 2005

AbstractChemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N‐formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time‐lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 μM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 μM) plus external calcium inhibited locomotion.
Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase
Authors: Stemple Derek L. , Sweet Stuart C. , Welsh Michael J. , Richard McIntosh J. 
Published: February 2005

AbstractWe have compared the exchange kinetics of fluorescein‐labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady‐state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells preequilibrated to either 26 ± 2°C or 36 ± 2°C. A pulse of laser light focused to a 5‐μm diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half‐spindles and from the two polar regions were calculated for each image in a post‐bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26°C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 ± 2°C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.
Wave of free calcium at fertilization in the sea urchin egg visualized with fura‐2
Authors: Hafner Mathias , Petzelt Christian , Nobiling Rainer , Pawley James B. , Kramp Douglas , Schatten Gerald 
Published: February 2005

AbstractA wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura‐2 in combination with low‐light‐level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340‐nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band‐like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.
Determination of the average shape of flagellar bends: A gradient curvature model
Authors: Eshel Dan , Brokaw Charles J. 
Published: February 2005

AbstractData obtained by manual digitization of photographs of flagellar bending waves have been analyzed by determining size parameters for the bends by least‐squares fitting of a model waveform. These parameters were then used to normalize the data so that the average shape of the bends could be determined. Best fits were obtained with a model waveform derived from the constant curvature waveforms used previously but with provision for a linear change in curvature across the central region of the bend–the gradient curvature model (GCM). The central regions of the GCM bending waves are separated by transition regions with length determined by a parameter called the truncation factor (FT). Fitting the GCM to sine‐generated bending waves give optimal fit when FT = 0.34. Fitting the GCM to four different samples of flagellar bending waves gave best fits with values of FT ranging from 0.17 for ATP‐reactivated Lytechinus spermatozoa beating at approximately 10 Hz to 0.32 for live spermatozoa of Arbacia. The difference between the Arbacia waveforms and a sine‐generated waveform is therefore very small, but a sine‐generated waveform lacks the degree of freedom represented by FT that is required to fit other waveforms optimally.The residual differences between the waveform data and optimal GCM waveforms were averaged and found to be small. In most cases, the curvature in the central region of the optimal GCM decreased in magnitude towards the tip of the flagellum; however, this slope was highly variable and sometimes positive. Significant variations in both this slope and FT were found in individual bends as they propagated along a flagellum.
The cytoskeletal microtubular system of some naked dinoflagellates
Authors: Brown D. L. , Cachon J. , Cachon M. , Boillot A. 
Published: February 2005

AbstractThe cytoskeletal microtubule system has been studied in six species of unarmoured Dinoflagellates using immunofluorescence and electron microscopy. Several structures have been detected and described: (1) a subpellicular layer of microtubules, constituting the microtubular cytoskeleton, running singly or in bundles from the anterior part of the cell to the posterior; (2) a feeding apparatus, containing a ribbon of microtubules, which corresponds to a small peduncle in some species and is simply represented by a cytostome in some other species; and (3) the longitudinal flagellum that runs in a long intracytoplasmic pocket before becoming free at the extremity of the sulcus. A thorough study of the organization of the microtubular structures in a wide spectrum of Dinoflagellates is a prerequisite for understanding the evolutionary history of the group.
Nanometer‐scale measurements using video light microscopy
Authors: Schnapp B. J. , Gelles J. , Sheetz M. P. 
Published: February 2005

AbstractVideo and digital image processing have been used to amplify the contrast of light microscopic images, making it possible to observe in real time the diffraction images of cell structures 10 times smaller than the Raleigh resolution limit of 0.2 μm. In this paper we discuss how quantitative analysis of diffraction images can be used to extract information about motion or structure at the nanometer level. This issue is considered in the context of a method for tracking the motion of kinesin‐coated beads on microtubules with 1–2 nm precision (Gelles et al.: Nature 331:450–453, 1988).
Actin polymerization and pseudopod extension during amoeboid chemotaxis
Authors: Condeelis J. , Hall Anne , Bresnick Anne , Warren Vivien , Hock Rick , Bennett Holly , Ogihara Satoshi 
Published: February 2005

AbstractAmoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F‐actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F‐actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross‐linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin‐binding proteins that are present in the cell cortex.