摘要:

AbstractIn this study, we follow changes in localization of the centrin‐related 165,000‐Mrprotein of PtK2cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury,J. Cell Biol.107:2669–2678, 1988). By immunofluorescence microscopy, the 165,000‐Mrprotein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half‐cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telo‐phase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000‐Mrprotein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (sp

ISSN:0886-1544    

DOI:10.1002/cm.970180102

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

年代:1991

数据来源: WILEY

摘要:

AbstractActin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross‐linking protein alpha‐actinin. Ultrathin sections of chicken retina were double‐immunolabelled with monospecific antibodies against actin and alpha‐actinin. The highest relative amount of alpha‐actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha‐actinin to actin labelling are where the actin filaments are most highly cross‐linked into bundles and linked to the plasma membrane by alpha‐actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell‐cell or cell‐substrate

ISSN:0886-1544    

DOI:10.1002/cm.970180103

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

年代:1991

数据来源: WILEY

摘要:

AbstractCalyculin‐A, an inhibitor of type 1 and 2A phosphatases, was applied extracel‐lularly to 3T3 fibroblasts. At 0.1 μM, calyculin‐A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phos‐phorylation following treatment with calyculin‐A was observed in vimentin, the 20‐kD myosin light chain, and an unidentified 440‐kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells.Calyculin‐A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin‐A (0.1 μM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin‐A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin‐A an unusual “ball‐like” structure was observed with transmission electron microscopy. This unique structure was 2–3 μM in diameter and was located close to the nucleus.The use of calyculin‐A adds further support to the idea that cell shape is controlled, at least in part, by concerted acti

ISSN:0886-1544    

DOI:10.1002/cm.970180104

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

年代:1991

数据来源: WILEY

摘要:

AbstractThis study focuses on the dynamic reorganization of actin and myosin (“conventional” myosin, or myosin‐II) during cytokinesis inD. discoideum.This is the first study identifying the birefringence of the spindle microtubules as well as three sets of microfilamentous structure inDictyostelium.The change of organization in these fibrillar structures was followed in real‐time with video microscopy, using a Universal Polarizing Microscope equipped with polarized‐light (POL) and differential interference contrast (DIC) optics combined with digital image processing. High‐frequency mitotic cells were obtained by semi‐synchronous culture, and high‐resolution observations were made by utilizing the agar‐overlay method (Yumura et al.:Journal of Cell Biology99:894–899, 1984). The molecular identity of the birefringent structures was determined by fluorescence microscopy. Through‐focus observations were performed with an axial resolution of 0.3 μm depth of field.The actomyosin fibrils show a dramatic reorganization throughout mitosis. The fibrils at the leading lamellipodia disappear, and there is a striking assembly of the cortical actomyosin in pro‐metaphase, which is accompanied by a decrease in cell volume. The cortical actomyosin gradually increases through anaphase. After late anaphase, very active polar lamellipodia, with an average life of less than 1 minute, are formed. We confirmed that the polar lamellipodia include actin, but not myosin‐II. At the cleavage furrow, the microfilaments form two distinctive structures: circular contractile ring at the equator, and a cortical filament array parallel to the polar axis. Myosin is localized in the contractile ring, but not associated with the axial array of F‐actin. Actomyosin in the contractile ring gradually transforms into cortical network at the posterior region of daughter cells. The constriction of the furrow is accompanied by a drastic efflux of water as evidenced by highly active contractile vacuole formation and turbulent motion of minute vesicles connected to the furrow. This study demonstrates the presence of a new microfilament structure, as well as the dynamic property of the contractile ring, and sheds new light on the contractile mechan

ISSN:0886-1544    

DOI:10.1002/cm.970180105

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

年代:1991

数据来源: WILEY

摘要:

AbstractMicrotubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15‐fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.:Journal of Cell Biology107:1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP‐tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respec

ISSN:0886-1544    

DOI:10.1002/cm.970180106

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

年代:1991

数据来源: WILEY

摘要:

AbstractMotion of nucleoli within interphase nuclei, known as nuclear rotation, may be used as a measure of motion of chromatin domains within the global confines of the nucleus. Mechanisms by which chromatin domains are transposed remain enigmatic. It has been established that nuclei are anchored by a network of intermediate filaments, structural proteins which share epitopes with nuclear lamins and possibly representing a constraint on nuclear rotation. It is postulated that selective removal of this constraint, by acrylamide, would result in increased chromatin motion. Mean rates of nucleolar displacement were quantified in neurons, in vitro. Nuclear rotation increased from a mean control rate of 0.102 ± 0.002 μm/min (n = 52) to a maximum mean rate of 0.207 ± 0.026 μm/min (n = 11), after 23 hr of exposure to 4 mM acrylamide. Despite this significant increase in motion of intranuclear domains, cytoplasmic structures in the immediate juxtanuclear area did not exhibit increases in rates of motion. Immunocy‐tochemistry was used to visualize cytoskeletal structures and to assay selective disruption of neurofilaments by acrylamide. Increased rates of chromatin motion coincided with breakdown of the intermediate filament network. Ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was also associated with a significant(P<0.005) change in the thickness of the nuclear lamina, decreasing from 20.9 ± 5.10 nm (n = 159) in controls to 18.9 ± 3.1 nm (n = 148), to 19.5 ± 3.6 nm (n = 240) and to 16.1 ± 4.4 nm (n = 103) at 4, 8 and 22 hr exposure, respectively. Moreover, the number of mito‐chondria per unit area changed significantly(P<0.0001) with exposure to acrylamide, increasing from 9.1 ± 2.2 mitochondrial profiles in controls to 16.5 ± 5.3 profiles after 22 hr exposure to acrylamide. Distribution of other cytoskel‐etal components, actin and microtubules, was not altered and does not appear to play a significant role in the observed increase in rates of nuclear rotation. We conclude that the removal of the damping effects on chromatin motion normally imposed by the nuclear lamina and by intermediate filaments results in increased c

ISSN:0886-1544    

DOI:10.1002/cm.970180107

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

年代:1991

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970180108

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

年代:1991

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970180101

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

年代:1991

数据来源: WILEY