摘要:

AbstractThe vimentin intermediate filament (VIMF) network is more sensitive to heat‐induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1Chinese hamster ovary (CHO) cells (Coss and Wachsberger:Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert:European Journal of Cell Biology37:169–174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1or S phase) or in the well‐spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival.CHO cells, either in the process of spreading or in the well‐spread state, were sensitized to heat‐induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1but not necessarily to the death of cells treated in S phase.The degree of acrylamide sensitization of heat‐induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well‐spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment

ISSN:0886-1544    

DOI:10.1002/cm.970130202

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

年代:1989

数据来源: WILEY

摘要:

AbstractRetinal pigment epithelial (RPE) cells maintained in organ culture on Bruch's membrane and the associated choroid spread and migrate into a linear wound along the exposed basal lamina. Changes in cell shape, in the organization of microfilaments, and in cell‐cell and cell‐substratum interactions during this time were examined by epifluorescence and transmission electron microscopy. In contrast to cuboidal stationary cells distant from the wound edge, which display well‐developed apical circumferential microfilament bundles (CMBs) associated with zonulae adhaerentes junctions, the migrating RPE cells near the wound edge instead are flat, and, in addition to microfilament bundles near junctions between adjacent cells, display prominent stress fibers. Furthermore, monoclonal antibodies to vinculin labeled regions at the terminal ends of these stress fibers indicating that the RPE cells form focal contacts with the basal lamina at these sites. Electron microscopy of these regions of cell‐substratum interaction confirmed the presence of microfilament bundles that terminate on the cell membrane. Folds present in the basal lamina near these sites suggest that tension is being generated by the microfilaments in the stress fibers as the migrating cells pull on the underlying basal lamina through these adhesion

ISSN:0886-1544    

DOI:10.1002/cm.970130203

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

年代:1989

数据来源: WILEY

摘要:

AbstractInterference‐reflection microscopy (IRM) is the only method presently available with which to visualize cell‐substratum adhesions in living tissue culture cells continuously for long periods of time without the use of fluorescent markers (Curtis:J. Cell Biol.20:199–215, 1964; Izzard and Lochner:J. Cell Sci.21:129–159, 1976). This method utilizes approximately 1% of the incident illumination to produce the IRM image (Verschueren:J. Cell Sci.75:279–301, 1985) and so far has required the use of high‐intensity light sources in the visible spectral range (400–800 nm). Unfortunately, visible light of this intensity and spectral range induces marked changes in the behavior and morphology of motile fibroblasts, including cessation of locomotion. In contrast, the present paper reports that continuous observations of live cells in IRM for periods of up to 8 hours are possible if the illuminating light is in the red to near‐infrared range (650–950 nm) and without any observable change in normal cell morphology or behavior. In addition, we describe how the technique of Y‐contrast image processing can be applied to IRM images to create a three‐dimensional image of the ventral ce

ISSN:0886-1544    

DOI:10.1002/cm.970130204

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

年代:1989

数据来源: WILEY

摘要:

AbstractMammalian eggs and embryos contain an extensive detergent‐resistant cytoskeletal network, including many elements which have been referred to as sheets in hamster eggs. In this study we examined the structure of the sheet‐like components by using embedment‐free sections and freeze‐fracture electron microscopy and found that the sheets are composed of both filamentous and particulate components. In addition, exposure to a high salt extraction medium resulted in the disappearance of the sheets at the ultrastructural level. SDS‐polyacrylamide gel electrophoresis of the cell fractions revealed four stainable proteins solubilized by the high salt extraction with one of the proteins being greatly enriched. Because these cytoskeletal sheets undergo an extensive reorganization coincident with key events during early development they serve as internal markers for the establishment of polarity and subsequent differentiation of the first embryonic epithelium, the trop

ISSN:0886-1544    

DOI:10.1002/cm.970130205

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

年代:1989

数据来源: WILEY

摘要:

AbstractFollowing our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses.Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously.Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA‐induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]iand inhibited its subsequent decline. Trypsin‐induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earl

ISSN:0886-1544    

DOI:10.1002/cm.970130206

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

年代:1989

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970130201

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

年代:1989

数据来源: WILEY