摘要:

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

ISSN:0886-1544    

DOI:10.1002/cm.970080302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractThe monoclonal antibody, Tau‐1, which had previously been used to localize tau to the axonal compartment in brain has been reutilized for light and electron microscopic immunohistochemistry following phosphatase treatment of tissue. We report here that a significant quantity of tau in the central nervous system is phosphorylated in situ at or near the Tau‐1 epitope, preventing the binding of the Tau‐1 antibody. Upon removal of this/these phosphate group(s), however, Tau‐1 was observed in the somatodendritic compartment of neurons as well as in axons. Furthermore, intense staining was also observed in astrocytes and in perineuronal glial cells. This immunoreactivity was present along the lengths of microtubules and on ribosomes (polysomes). Treatment of immunoblots of extracts of whole cerebral cortex with phosphatase confirmed the immunohistochemical results in that a 50–65% increase in Tau‐1 binding to the tau region of the blot was noted. Moreover, a novel monoclonal antibody, Tau‐2, was also used in these experiments. This antibody binds only to tau and localizes along microtubulesin axons, somata, dendrites, and astrocytes and on ribosomes (polysomes) without phosphatase

ISSN:0886-1544    

DOI:10.1002/cm.970080303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

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.

ISSN:0886-1544    

DOI:10.1002/cm.970080304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractA procedure adapted from that described by Mitchison and Kirschner [Nature 312:232–237, 1984] was used to isolate centrosomes from human lymphoid cells. High yields of homogeneous centrosomes (60% of the theoretical total, assuming one centrosome per cell) were obtained. Centrosomes were isolated as pairs of centrioles, plus their associated pericentriolar material. Ultrastructural investigation revealed: 1) a link between both centrioles in a centrosome formed by the gathering in of a unique bundle of thin filaments surrounding each centriole; 2) a stereotypic organization of the pericentriolar material, including a rim of constant width at the proximal end of each centriole and a disc of nine satellite arms organized according to a ninefold symmetry at the distal end and; 3) an axial hub in the lumen of each centriole at the distal end surrounded by some ill‐defined material.The total protein content was 2 to 3 × 10−2pg per isolated centrosome, a figure that suggests that the preparations were close to homogeneity. The protein composition was complex but specific, showing proteins ranging from 180 to 300 kD, one prominent band at 130 kD, and a group of proteins between 50 and 65 kD. Actin was also present in centrosome preparations.Functional studies demonstrated that the isolated centrosomes were competent to nucleate microtubules in vitro from purified tubulin in conditions in which spontaneous assembly could not occur. They were also very effective at inducing cleavage when microinjected into unfertilizedXenop

ISSN:0886-1544    

DOI:10.1002/cm.970080305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractWe report a developmental sequence in the type and frequency of behaviours of neurons differentiating in vitro. We characterised these changes with extensive analysis of time‐lapse sequence from both the continuing cell line phenochromocytoma PC12 and primary mixed cell culture of cat and mouse central nervous system. PC12 cells activated by nerve growth factor (NGF) differentiate in a uniform and synchronous manner. This allowed the first quantification of changes in different neuron behaviours during morphogenesis.Shortly after NGF activation, PC12 cells are highly labile in morphology and exhibit a large variety of morphological behaviours. During the first week of differentiation, the frequency of these behaviours declines, and gross morphology becomes more stable. The frequency of neurite initiation after 1 week in NGF is one‐seventh what it was after 2 days in NGF. Over the same period, neurite retraction declines to one‐third, and somal migration ceases altogether. Growthcone activity does not decline during development. These behaviour changes correlate with published data on the differentiation of the neurite cytoskeleton.A qualitatively similar ontogeny was noted in the differentiation of CNS neurons in mixed cell culture. Major differnces occur in the relative timing of changes in behaviours. Mature, stable morphology is not detected in these cultures until 7 weeks in

ISSN:0886-1544    

DOI:10.1002/cm.970080306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

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 s

ISSN:0886-1544    

DOI:10.1002/cm.970080307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractDouble immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell‐substrate focal contacts and intermediate filaments. Most of the vinculin‐containing adhesion plaques coincided with the ends of vimentin‐positive fibrils.This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method.Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid‐induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts.The possible role of the intermediate filaments in the formation and maintenance of focal contacts is di

ISSN:0886-1544    

DOI:10.1002/cm.970080308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

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 i

ISSN:0886-1544    

DOI:10.1002/cm.970080309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970080301

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY