摘要:

AbstractPtK1cells were treated with 10 μg/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana‐like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 μg/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed that most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revelaed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosome congression with most chromosomes aligning at the metaphase plate after 10–15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti‐tubulin staining of a monolayer of cells treated with 10 μg/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970320402

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe have analyzed the kinetics of synthesis, phosphorylation, and stability of the soluble and insoluble plakoglobin (PG) and their interactions with Dsg1 and E‐cadherin in Madin‐Darby canine kidney (MDCK) epithelial cells in the absence of cell adhesion and after the induction of cell‐cell contact. Using a combination of biochemical and morphological approaches, we show that newly synthesized PG enters a soluble:insoluble pool of proteins in a 60:40 ratio regardles of cell‐cell contact. Following synthesis, PG is increasingly found in the insoluble pool. Although cell‐cell contact does not effect either the size of each pool or the rate or efficiency of the transfer from the soluble into the insoluble pool, it results in a significant increase in the metabolic stability of the newly synthesized insoluble PG. The soluble PG initially forms separate complexes with E‐cadherin and Dsg1. PG‐Dsg1 complexes become insoluble and localize to the desmosome. PG‐E‐cadherin complexes remain soluble and are distributed intracellularly. The insoluble PG and E‐cadherin detected at the cell periphery remain distinctly separate, as demonstrated previously [Hinck et al., 1994:J. Cell Biol.125:1327–1340; Nathke et al., 1994:J. Cell Biol.125:1341–1352]. In addition, we detected a separate pool of PG which is not associated with either Dsg1 or E‐cadherin and after the induction of cell‐cell contact becomes primarily insoluble and is distributed along the lateral membrane. Phoshorylation analysis showed that there is a significantly greater amount of phosphorylated PG in the soluble pool than in the insoluble pool. In addition the soluble pool is both serine and theronine phosphorylated, whereas the insoluble PG is primarily phosphorylated on serine resid

ISSN:0886-1544    

DOI:10.1002/cm.970320403

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

年代:1995

数据来源: WILEY

摘要:

AbstractMicrotubules oriented in the apicobasal axis of columnar epithelial cells arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF‐B (hepatocyte), Caco‐2 (intestine), and Madin‐Darby canine kidney (MDCK). Caco‐2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole‐induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF‐B and Caco‐2 cells contained two pools of γ‐tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non‐centrosomal γ‐tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal γ‐tubulin in apical cytoplasm suggests multiple mechanisms by which microtubule nucleation might occur in epithelial cells

ISSN:0886-1544    

DOI:10.1002/cm.970320404

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

年代:1995

数据来源: WILEY

摘要:

AbstractSalivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule‐free domains. Additionally, vimentin filaments are absent from large domains of microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules. © 1995 Wiley‐Liss,

ISSN:0886-1544    

DOI:10.1002/cm.970320405

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

年代:1995

数据来源: WILEY

摘要:

Abstract3T3 mouse fibroblasts responded differently to specific near‐infered signals than epithelial CV1 cell. Furthermore, signals with the same wavelength and energy changed the percentages of attracted and repelled 3T3 cells if their intensity modulation was altered. I found this result in a 22 month long study which established a spectrum of motile responses of 781 individual 3T3 cells and 148 CV1 cells to the near‐infrared emissions of microscopic, pulsating light sources using the infrared spot‐irradiation phase‐contrast (IRSIP) microscopic [Albrecht‐Buehler, 1991:J. Cell Biol.114:493–502]. Thus the response of cultured, mammalian cells to near‐infrared light signals is not merely a matter of total energy absorption by cirtain cytoplasmic componets. Since it seems to depend on the cell type and the temporal pattern in which the light energy is emitted, it appears to imply the existence of a new kind of cellular information. © 1995 W

ISSN:0886-1544    

DOI:10.1002/cm.970320406

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

年代:1995

数据来源: WILEY

摘要:

AbstractUsing video‐enhanced differential interference microscopy and digital image processing, we have observed organelle motility inAcanthamoeba castellanii.In amoebae taken from cultures in rapid growth phase, mitochondria and small particles moved over distances of several microns and at an average velocity of ∼2 μ/s. Mitochondrial motility was verified by intensified fluorescence microscopy of cells that were labeled in vivo with the DNA‐binding dye DAPI or the mitochondria‐specific dye Mito Tracker. We further studied the role of microtubules (MTs) in the translocation of cell organelles. Double‐labelling of fixed cells bules with mitochondrial markers (anti‐F1β antibody, Mito Tracker) and cytoskeletal markers (anti‐tubulin antibody, rhodamine‐phalloidin) demonstrate that the mitochondria colocalize with MTs in the subcortical cell area and are excluded from the F‐actin‐rich cell cortex. Colchicine treatment resluted in an almost complete depolymerization of MTs and an inhibition of organelle motility. Moreover, we have directly visualized MTs in vivo in flattened amoebae. Mitochondria and small particles moved along the MTs in a bidirectional mode at an average velocity of ∼1 μm/s. We conclude that the observed movement of mitochondria and small particles inAcanthamoeba castellaniimainly occurs via microtubules and associated motor proteins

ISSN:0886-1544    

DOI:10.1002/cm.970320407

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

年代:1995

数据来源: WILEY

摘要:

AbstractIn epithelial cells interactions between the actin cytoskeleton and cell‐cell junctions regulate paracellular permeability and partcipate in morphogenesis. We have studied the relationship between supracellular morphology and actin‐junction interactions using primary cultures of porcine thyroid cells grown either as three‐dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO‐1 and E‐cadherin, respectively, and showed F‐actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin‐rich zonula adherens and in cell‐substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton‐insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)‐cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 μM) caused actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO‐1 and E‐cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP

ISSN:0886-1544    

DOI:10.1002/cm.970320408

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.970320401

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

年代:1995

数据来源: WILEY