摘要:

AbstractRelationships among the endoplasmic reticulum (ER), microtubules, and bead movements on the cell surface were investigated in the thin peripheral region of A6 cells, a frog kidney cell line. ER tubules were often aligned with microtubules, as shown by double‐labeling with DiOC6(3) and anti‐tubulin in fixed cells. In living cells stained with DiOC6(3) and observed in time lapse, there were frequent extensions, but few retractions, of ER tubules. In addition, there was a steady retrograde (towards the cell center) movement of all of the ER at ∼0.3 μm/min. Since microtubules are often aligned with the ER, microtubules must also be moving retrogradely. By simultaneous imaging, it was found that the ER moves retrogradely at the same rate as aminated latex beads on the cell surface. This indicates that the mechanisms for ER and bead movement are closely related. Cytochalasin B stopped bead and ER movement in most of the cells, providing evidence that actin is involved in both retrograde movements. The ER retracted towards the cell center in nocodazole while both ER and microtubules retracted in taxol. Time lapse observations showed that for both drugs, the retraction of the ER is the result of retrograde movement in the absence of new ER extensions. Presumably, ER extensions do not occur in nocodazole because of the absence of microtubules, and do not occur in taxol because taxol‐stabilized microtubules move retrogradely and there is no polymerization of new microtubule tracks for ER elongation. © 1994 Wiley‐Liss, Inc.This Article is a US Government work and, as such, is in the public domain in the United States

ISSN:0886-1544    

DOI:10.1002/cm.970290402

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

年代:1994

数据来源: WILEY

摘要:

AbstractIn the large eggs (∼1 mm) of the ctenophoreBeroe ovata, female pronuclei migrate long distances to join stationary male pronuclei in the peripheral cytoplasm that surrounds the yolky interior. We have investigated the mechanism of nuclear migration using time lapse video recording, automated image analysis, visualization of microtubules by immunofluorescence and rhodamine‐tubulin injection, and electron microscopy. Female pronuclei migrated at average speeds of 0.2 μm/sec, and were found to show periodic oscillations in velocity. Alternating phases of acceleration and deceleration occurred with an average periodicity of 235 seconds covering distances of 47 μm (about 3 times the nuclear diameter). Migration velocities and velocity oscillations were similar in fertilized and unfertilized eggs; however, changes in migration direction were much more frequent in unfertilized eggs. Characteristic deformations of the pronuclear membrane and occasional rotation of the nuclear contents were observed during migration. Inhibitor studies indicated that microtubules are required for nuclear migration. In fertilized eggs the top of the nucleus was found to move through the dense layer of aligned sperm aster microtubules. The frequent changes in direction of pronuclear migration in unfertilized eggs reflect the random organization of the microtubule layer in the absence of sperm derived centrosomes. Densely packed endoplasmic reticulum was found intermeshed with sperm aster microtubules and connected extensively with the nuclear membrane during migration. Most nuclear pores were grouped in an infolding of the nuclear membrane. We suggest that in fertilized eggs the female pronucleus is transported to the minus ends of sperm aster microtubules using motor molecules attached either to the outer nuclear membrane and/or to the network of connecting ER. © 1994 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970290403

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

年代:1994

数据来源: WILEY

摘要:

AbstractDuring cytokinesis, daughter cells are cleaved in two by the constriction of an actin‐rich contractile ring which encircles the equator of the dividing cell. Filamentous myosin II is present in the contractile ring and necessary for constriction of the furrow, as shown in several cell types [Satterwhite and Pollard, 1992: Curr. Opin. Cell Biol. 4:43–52]. However, no functional role nor distinctive localization has been previously identified for non‐filamentous “unconventional” myosins, such as myosin I, during cytokinesis. Using antibodies to adrenal medullary myosin I, we report that myosin I is localized in 3T3 fibroblasts to the mid‐equatorial plane during late‐cytokinesis, as well as to the polar edges as previously described in ameboid cells [Fukui et al., 1989:Nature341:328–331]. Confocal microscopy revealed that myosin I is concentrated at the midbody region in a nearly continuous transverse disk, extending from the cortical region of the furrow through the midbody itself. These findings suggest that, in addition to the accepted role of filamentous myosin II in constriction of the contractile ring, non‐filamentous myosin I might contribute to motile events occurring late in cytokinesis. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970290404

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

年代:1994

数据来源: WILEY

摘要:

AbstractAddition of protein kinase inhibitor H‐7 leads to major changes in cell structure and dynamics. In previous studies [Citi, 1992:J. Cell Biol.117:169–178] it was demonstrated that intercellular junctions in H‐7‐treated epithelial cells become calcium independent. To elucidate the mechanism responsible for this effect we have examined the morphology, dynamics, and cytoskeletal organization of various cultured cells following H‐7‐treatment. We show here that drug treated cells display an enhanced protrusive activity. Focal contact‐attached stress fibers and the associated myosin, vinculin, and talin deteriorated in such cells while actin, vinculin, and N‐cadherin associated with cell‐cell junctions were retained. Furthermore, we demonstrate that even before these cytoskeletal changes become apparent, H‐7 suppresses cellular contractility. Thus, short pretreatment with H‐7 leads to strong inhibition of the ATP‐induced contraction of saponin permeabilized cells. Comparison of H‐7 effects with those of other kinase inhibitors revealed that H‐7‐induced changes in cell shape, protrusional activity, and actin cytoskeleton structure are very similar to those induced by selective inhibitor of myosin light chain kinase, KT5926. Specific inhibitors of protein kinase C (Ro31‐8220 and GF109203X), on the other hand, did not induce similar alterations. These results suggest that the primary effect of H‐7 on cell morphology, motility, and junctional interactions may be attributed to the inhibition of actomyosin contraction. This effect may have multiple effects on cell behavior, including general reduction in cellular contractility, destruction of stress fibers, and an increase in lamellipodial activity. It is proposed that this reduction in tension also leads to the apparent stability of cell‐cell junctions in low‐c

ISSN:0886-1544    

DOI:10.1002/cm.970290405

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

年代:1994

数据来源: WILEY

摘要:

AbstractAnti‐actin monoclonal antibodies were prepared using phalloidin‐stabilized actin that was purified from pea roots by DNase I affinity chromatography. One monoclonal antibody, designated mAb3H11, bound plant actin in preliminary screenings and was further analyzed. Immunoblot analysis showed that this antibody had a high affinity for plant actin in crude and purified preparations but a low affinity for rabbit muscle actin. In immunoblots of plant extracts separated on two‐dimensional gels it appeared to bind all actin isoforms recognized by the JLA20 anti‐chicken actin antibody. Using immunofluorescent cytochemistry, the antibody was used to observe actin filaments in aldehyde‐fixed and methanol‐treated tobacco protoplasts. These results indicate that mAb3H11 should be a useful reagent for the study of plant actins. © 1994 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970290406

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

年代:1994

数据来源: WILEY

摘要:

AbstractRat basophilic leukemia cells secrete histamine and serotonin in response to cross‐linking of the IgE receptor by multivalent antigen [Metzger et al., 1986:Ann. Rev. Immunol. 4:419–470]. Receptor crosslinking also induces phosphorylation of the light and heavy chains of myosin II with kinetics similar to that of secretion [Ludowyke et al., 1989:J. Biol. Chem.264:12492–12501]. Here we show that myosin II localization changes after activation with similar kinetics. Furthermore, these changes are coincident with changes in cell shape and increase in motile activity induced by activation. Within 2 min, activated cells begin to flatten, spread on their substratum, and extend lamellipodia which show active ruffling. Quantitation of the extent of cell spreading from video micrographs shows that 48% of the cells increase significantly in surface area by 5 min and 71% by 15 min. Myosin II is uniformly distributed in unactivated cells but is deficient in newly formed lamellipodia that start to appear at 2 min after activation. In contrast these lamellipodia show strong staining for actin. Further changes in myosin organization are detected by 15 min after activation when myosin reappears in the cell periphery, is concentrated in the perinuclear area, and is also organized in punctate linear arrays that extend from the nucleus to the cell periphery. The kinetics of the early cell shape changes and formation of the myosin‐deficient lamellipodia correlate well with, and may relate to, the increase in the level of myosin II phosphorylation reported by Ludowyke et al. [1989:J. Biol. Chem.264:12492–12501]. Changes in the distribution of cell surface‐bound IgE also occur upon antigen activation, and they correlate with the myosin distribution in a manner that suggests that they may be driven by myosin II. © 1994 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970290407

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

年代:1994

数据来源: WILEY

摘要:

AbstractRat basophilic leukemia (RBL‐2H3) cells undergo morphological and cytoskeletal changes during antigen‐induced secretion of allergic mediators. The exact role these changes play in the process of secretion is unclear. Using confocal microscopy we now show that PMA + A23187 causes extensive F‐actin rearrangements during secretion of [3H] 5‐HT. We also describe for the first time the association of myosin with F‐actin during this secretory process. In unstimulated cells, myosin and F‐actin are concentrated at the plasma membrane with no evidence of stress fibres. Upon addition of PMA or A23187, both F‐actin and myosin are rearranged into membrane ruffles and discrete aggregations (foci), followed by the formation of parallel stress fibres located on the ventral membrane. This is in contrast to reports in other cell types in which PMA has been described as causing the disruption of F‐actin stress fibres. The time course of secretion coincides with the formation of the foci and ruffles whilst the stress fibres form after the majority of secretion has occurred. These changes are accompanied by a 40% decrease in cell height and a two‐fold increase in cell spreading and they occur in the absence of extracellular calcium but are inhibited by the protein kinase C inhibitor, Bisindolylmaleimide, which also inhibits secretion. The formation of myosin‐decorated stress fibres, foci, and ruffles is not sufficient to cause secretion, as PMA alone induces these changes without any secretion. The relevance of actin and myosin rearrangements for the regulation of secretion is discussed. ©

ISSN:0886-1544    

DOI:10.1002/cm.970290408

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

年代:1994

数据来源: WILEY

摘要:

AbstractApical cells of protonemata of the mossCeratodon purpureusare unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos‐methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30–60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load‐bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity. © 1994 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970290409

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

年代:1994

数据来源: WILEY

摘要:

AbstractCyclic AMP (cAMP) has been implicated in the regulation of movement of certain cultured cell types. We have studied the effects of cAMP on epithelial cell motility using serum‐free NBT‐II cells, derived from a rat bladder carcinoma. The random movement of these cells on type I collagen was reduced upon elevation of intracellular cAMP by several means and this effect was reversible. Alterations in the organization of the cytoskeletal proteins F‐actin and α‐actinin occurred concurrently with the reduction in motility, and the arrangement of these proteins resembled that seen in non‐motile cells on glass. In addition, pretreatment of cells with KT5720, a cAMP‐dependent protein kinase (PKA)‐specific inhibitor, prevented the dibutyryl cAMP‐induced reduction in cell movement as well as the associated cytoskeletal changes. These results suggest that elevation of PKA is responsible for the observed effects on cell motility and cytoskeletal reorganization and demonstrate a role for PKA in the regulation of cell motility in this system. © 199

ISSN:0886-1544    

DOI:10.1002/cm.970290410

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

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970290411

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

年代:1994

数据来源: WILEY