摘要:

AbstractEmploying a newly developed computer‐assisted system for visualizing and quantitating cell motility in three dimensions, we have examined the 3‐dimensional changes in cell shape and the dynamics of pseuodopod extension during translocation ofDictyosteliumamoebae. Amoebae exhibit a 3‐dimensional behavior cycle with an average period of 1.5 min. The cycle includes a transient pseudopod extension phase in the x, y axis followed by a z‐axis expansion phase. Anterior pseudopod extension in the x, y axis is accompanied by a decrease in height, not by uropod retraction. The increase in height is accompanied by uropod retraction. In the pseudopod extension phase in the x, y axes, pseudopods form either anteriorly or laterally, and either on or above the substratum. Pseudopods which initially form on the substratum in almost all cases continue to expand as the anterior end of the cell. In the case of lateral pseuodopods, anteriorization leads to a turn. Approximately half of anterior pseudopod and two‐thirds of lateral pseudopods which initially form above the substratum are retracted. These results suggest that pseudopod‐substratum interaction plays a fundamental role in the regulation of directionality and turning in the translocation phase of the 3‐dimensional behavior cycle. © 1994 W

ISSN:0886-1544    

DOI:10.1002/cm.970270102

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe β‐thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β‐thymosins, we have used isoform‐specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β‐thymosins are uniformly distributed within the cytoplasm. cDNA‐mediated overexpression of β‐thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β‐thymosin were observed to divide. In these cells, β‐thymosin was excluded from the midbody which contains an actin filament‐rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in livin

ISSN:0886-1544    

DOI:10.1002/cm.970270103

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

年代:1994

数据来源: WILEY

摘要:

AbstractPtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows. © 1994 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970270104

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have recently purified and characterized from rat liver, polypeptides of 110‐kDa and 130‐kDa which possess several characteristics of myosin‐1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189‐199, 1993]. What roles these myosin‐1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellular transport or locomotion. As a first step in establishing their function, we have investigated whether these molecules are capable of supporting motility in vitro. Our results clearly demonstrate that the isolated 130‐kDa‐calmodulin complex will translocate filaments at a rate of 0.03‐0.05 μ/sec; motility is inhibited in free calcium ion concentrations above 0.1 μM. This inhibition is reversed with the addition of exogenous calmodulin. These results provide supporting evidence of a motile role for the 130‐kDa‐calmodulin complex in vivo. This is the first demonstration that in higher eukaryotes, myosin‐1 from a tissue other than intestine will support motility. Partial peptide sequence analysis indicates that the 130‐kDa polypeptide resembles the recently described myr 1 [Ruppert et al.: J. Cell Biol. 120:1393‐1403, 1993]or MM1α [Sherr et al.: J. Cell Biol. 1405‐1416, 1993] gene p

ISSN:0886-1544    

DOI:10.1002/cm.970270105

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti‐acetylated tubulin antibody (6‐11B‐1), confirming previous results of Hermo et al. [Anat. Rec. 229:31‐50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2‐D PAGE of mouse and hamster sperm extracts showed a high level of α and β‐tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous α as well as β‐tubulin isoforms. This suggests that the major testis‐specific tubulin isotypes (mα3/7 and mβ3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures.

ISSN:0886-1544    

DOI:10.1002/cm.970270106

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

年代:1994

数据来源: WILEY

摘要:

AbstractDithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule‐based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT‐treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg‐derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division. © 1994 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970270107

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

年代:1994

数据来源: WILEY

摘要:

AbstractBundles of native microtubules isolated from the ovarioles of hemipteran insects are seen to shimmer when observed using dark‐field microscopy. This novel form of microtubule motility becomes even more obvious when the isolated bundles are detergent‐extracted and reactivated. We have studied the nucleotide‐specificity and the drug‐sensitivity of microtubule shimmering in order to obtain information regarding the nature of the motor protein responsible, and to compare its properties with those of previously characterised microtubule motors. The involvement of structural MAPs in the shimmering and in maintenance of microtubule bundles in this system has also been investigated. © 1994 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970270108

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

年代:1994

数据来源: WILEY

摘要:

AbstractMicrotubules treated with the γ‐diketone 2,5‐hexanedione (2,5‐HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5‐HD‐induced microtubule modification. 2,5‐HD‐treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin‐based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5‐HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3‐Acetyl‐2,5‐hexanedione (AcHD), a pyrrole forming, non‐crosslinking congener of 2,5‐HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5‐HD. However, glutaraldehyde, a pyrrole‐independent crosslinking agent which alters microtubule assembly in the same way as 2,5‐HD, slowed microtubule transport. These results indicate that a 2,5‐HD‐induced microtubule modification, possibly a crosslink‐related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule

ISSN:0886-1544    

DOI:10.1002/cm.970270109

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe role of microtubules in random cell migration was investigated using time‐lapse videomicroscopy to record in vitro the shape and motile behavior of guinea pig alveolar macrophages before and after disrupting microtubules with colcemid. Cell migration was quantified in terms of directional persistence time and speed. Motility was also correlated with morphological polarity: cells having a single lamellipodal region (monopolar cells) migrated, whereas those lacking a lamellipod (apolar cells) or with opposing lamellipodal regions (bipolar cells) did not migrate. Within 2 hours, colcemid caused a shift in polarity from 80% monopolar cells to 40% monopolar and 40% bipolar cells and a corresponding decrease from 80% to 40% in the fraction of migrating cells. Mean persistence time and speed decreased only slightly (approximately 20%) for those cells (still monopolar) which continued to migrate in the presence of colcemid. Persistence time and speed actually increased for many individual cells, indicating that random migration did not require intact microtubules. We conclude that colcemid treatment destabilizes monopolarity, leading to the gradual loss of monopolarity and consequent inhibition of migration. While a cell remains monopolar, it will continue to migrate even in the absence of intact microtubules, but microtubules are required for the long‐term maintenance of cellular monopolarity and, thus, for continued motility. © 1994 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970270110

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.970270111

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

年代:1994

数据来源: WILEY