摘要:

AbstractHuman fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti‐α‐actinin, and anti‐myosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types f stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron‐dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber‐membrane association site by using immunocy‐tochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including β1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti‐integrin αvsubunit or anti‐focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber‐membrane contact has some important functions different from those o

ISSN:0886-1544    

DOI:10.1002/cm.970310302

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross‐striated myofibrils were permeabilized with Triton X‐100 and incubated with gelsolin. Immunoflurorescence microcopy localized both endogenous and exogenous gelsolin in the I‐Z‐I‐regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross‐striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin stell bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X‐100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric ac

ISSN:0886-1544    

DOI:10.1002/cm.970310303

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe flageliar axoneme of the mutantpf18lacking the central pair does not beat, but undergoes a nanometer‐scale, high‐frequency oscillation (hyper‐oscillation) in the presence of ATP [Yagi et al., 1994:Cell Motil, Cytoskeleton29:177–185]. The present study demonstrates that the amplitude of the hyper‐oscillation increases significantly in the simultaneous presence of ATP and ADP. In addition, the hyper‐oscillation under these conditions sometimes takes on an exceptionally simple asymmetric pattern, in which the maximal shearing velocity exceeds 50 μm/sec, much higher than the maximal velocity of ordinary dynein‐microtubule sliding. The asymmetric oscillation thus appears to be at least partly driven by an internal elastic force. Its amplitude suggests that the axoneme has an elastic component that can be stretched by as long as 0.1 μm. Analyses of the asymmetric pattern further suggests that the axonemal dyneins have a tendency to attach to and detach from the doublets cooperatively and that the mechanochemical cycle of dynein has an inherent refractory period of about 2 msec, during which dynein cannot interact wi

ISSN:0886-1544    

DOI:10.1002/cm.970310304

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

年代:1995

数据来源: WILEY

摘要:

AbstractAdherence of cells to the extracellular matrix via focal adhesions is known to modulate many cellular functions. However, the role of focal adhesions in the regulation of secretion is unclear. To examine this we have used the RBL‐2H3 rat mast cell line, in which we and others have observed cytoskeletal rearrangements and increased cell spreading during secretion. All activators of secretion examined, whether acting specifically through or bypassing the IgE‐receptor, induced the assembly of focal adhesions, as defined by the localization of vinculin and talin. The extent of focal adhesion formation correlated with the extent of secretion and the time course of secretion also correlated with that of the assembly of focal adhesions. To examine the mechanism by which focal adhesion formation occurred, the protein kinase C inhibitor bisindolylmaleimide was used. Bisin‐dolylmaleimide caused complete inhibition of both secretion and focal adhesion formation induced by antigen or the calcium ionophore A23187. Although PMA did not induce secretion, it induced focal adhesion assembly which was inhibited by bisindolylmaleimide. The inhibitor had no effect on secretion or focal adhesion formation induced by the ATP analogue, ATPγS in permeabilized cells, indicating ATPγS acts after the activation of protein kinase C in the secretory pathway. These data provide novel evidence that the formation of focal adhesions may have a role in the process of secretion from mas

ISSN:0886-1544    

DOI:10.1002/cm.970310305

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

年代:1995

数据来源: WILEY

摘要:

AbstractA means of determining quantitative maps of the tractions exerted by locomoting cells on a substratum has been developed. This method is similar to the Harris silicone substratum assay [Harris et al., 1980:Science208:177–179], but uses an improved non‐wrinkling film that deforms more predictably in response to traction forces. The method also utilizes a mathematical analysis of rubber deformation to produce the final map of the distribution of tractions. The resulting maps consistently showed that fish keratocytes exert a steady‐state “pinching” on the substratum, perpendicular to the cell's direction of locomotion. No significant rearward tractions were detected at or near the front edge of the cell. Likewise, no significant forward tractions associated with peeling of adhesions were found at the back of the cell. A second assay uses deflection of a lightly attached glass microneedle to measure the total force exerted by locomoting cells. Forces of approximately 4.5 × 10−3dyn were required to “stall” locomoting keratocytes. The implications of these findings for cell moveme

ISSN:0886-1544    

DOI:10.1002/cm.970310306

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

年代:1995

数据来源: WILEY

摘要:

AbstractComputer‐assisted methods have been employed to obtain a high resolution description of pseudopod expansion, cellular translocation, and the subcellular dynamics of MSP fiber complexes in the motile sperm of the nematodeAscaris suum. AlthoughAscarissperm translocating in a straight line or along a curved path do not retract their pseudopod or significantly alter pseudopod shape, they move in a cyclic fashion, with an average period between velocity peaks of 0.35 × 0.05 min, which is independent of the forward velocity of sperm translocation. Expansion is confined to a central zone at the distal edge of the pseudopod for sperm translocating in a straight line and to a left‐handed or right‐handed lateral zone in the direction of turning, for sperm translocating along a curved path. For cells translocating in a straight line, the branch points and kinks of MSP fiber complexes move in a retrograde direction in relation to the substratum at an average velocity of 11 μm per min which is independent of the forward velocity of sperm translocation. The distal (anterior) end of a fiber complex, however, moves distally at the speed of sperm translocation when it emanates from the expansion zone, but when it is displaced to a nonexpanding surface of the pseudopod, it stops moving distally. When a cell is anchored to the substratum and is, therefore, nonmotile, the velocity of fiber complexes moving in a retrograde direction doubles. The unique aspects of pseudopod and MSP fiber complex dynamics inAscarisare compared to the dynamics of pseudopod formation and actin filament dynamics in traditional actin‐based amoeboid cells, and the treadmill model for MSP polymerization is reassessed in light of the discovery that fiber complex branch points move proximally (posteriorly) at a f

ISSN:0886-1544    

DOI:10.1002/cm.970310307

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

年代:1995

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970310301

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

年代:1995

数据来源: WILEY