摘要:

AbstractExposure of porcine vascular smooth muscle cells to platelet‐derived growth factor (PDGF; 18–180 ng/ml) but not epidermal growth factor (EGF; 30ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 μM), results in a rapid, reversible, time‐ and concentration‐dependent disapperance of vinculin staining in adhesion plaques; actin‐containing stress fibers also become disrupted following exposure of cells to PDGF. Disapperance of vinculin staining from adhesion plaques is also caused by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA; 200–400 nM), though the time course of the disapperance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF‐induced removal of vinculin from adhesion plaques was inhibited in a concentration‐dependent fashion by 8‐(N, N‐diethylamin) octy1–3,4,5‐trimethoxybenzoate (TMA‐8; 0.25–4 μM) and leupepetin (2–300 μM), and by n‐α‐rosyl‐L‐lysine chloromethylketone (TLCK; 100 μM) and trifluoperazine (TFP; 2.5 μM). Addition of PDGF to vascular smooth muscle cells caused a rapid, tranient increase in cytosolic free calcium, from a basal resting level of 146 ± 6.9 nM (SEM, n=62) to 414 ± 34 nM (SEM, n=22) as determined using the calcium‐sensitive indicator Fura‐2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB‐8 but not leupeption. This rise in cytosolic free calcium was found to occur in ∼ 80% of the sample population and dispalyed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF‐induced disruption of vinculin from adhesion plaques, as determined using the pH‐sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose‐dependent fashion. Both could be inhibited by leupeptin or TMB‐8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF‐induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium‐independent mechanism, and 4) the cellular response to PDGF‐stimulated increases in cytosolic free calcium is heterogeneous. Our data also suggest that cytosolic vinculin distribution is

ISSN:0886-1544    

DOI:10.1002/cm.970080202

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe applied the “agar‐overaly” immunofluorescence techinque (Yumura, S., H. Mori, and Y. Fukui, J. Cell Biol. 99:894–899, 1984) to a semisynchronous culture ofDictyostelium discoideumfor studying the organization changes in the microtubule system during mitosis. Using a flurescent DNA dye DAPI (4′,6′ ‐diamidino‐2‐phenylindole), chromatin fibers and individual chromosomes were visible in cells prepared by this method, whereby the mitotic phase could be critically evaluated.We found that a rapid shortening of the cytoplasmic microtubules was preceded by a structural dislocation from their organizing centers (MTOCs) in the midprophase, resulting in the transient occurrence of free microtubules in the cytoplasm. Statistic analyses showed that microtubule disassembly in prophase was diphasic. Initially long, wavy microtubules shortened from their distal ends. Following dissociation of their proximal ends from the MTOC, all microtubules initiated rapid disassembly, probably from both ends. During this process, microtubule assembly from the now duplicated spindle pole body (SPB) resumed.This study also revealed novel information on the dynamics of theDictyosteliummitotic spindle: 1) Half spindles interdigitate in the spindle center, and the extent of interdigition increases coincidentally with the spindle elongation, and 2) during the anaphase to telophase, a subpopulation of spindle microtubules elongates while the rest of the microtubules disasemble very rapidly.Overall this study indicates the presence of elaborate mechanisms responsible for the selective assembly/disassembly of particular microtubule subpo

ISSN:0886-1544    

DOI:10.1002/cm.970080203

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

年代:1987

数据来源: WILEY

摘要:

AbstractFlurescently labeled heavy mermoyosin, alpha‐actinin, and vinculin were used to localize actin, and vinculin, respectively, in permeabilized and living cells during the process of stress fiber reassembly, which occurred when cells were removed from ATP‐depleting medium (20 mM sodium azide and 10 mM 2‐deoxyglucose). In 80% of the cells recovering from ATP depletion, small, scattered plaques containing actin, alpha‐actinin, and vinculin were replaced by long, thin, periodic fibers within 5 minutes of removal of the inhibitors. These nascent stress fibers grew broader as recovery progressed, until they attained the thickness of stress fibers in control cells. In the other 20% of the cells, the scattered plaques aggregated within 5 minutes of reversal, and almost all the actin, alpha‐actinin, and vinculin in the cell became localized in one perinuclear aggregate, with a diameter of approximaterly 15–25 μm. As recovery progressed, all aggregates resembled rings, with diameters that increased at about 0.5 μm/minute and grew to as large as 70 μm in some giant cells. As the size of the rings increased, fibers radiated outward from them and sometimes spanned the diamater of te rings. The shape of the cells did not change during this time. By 1 hour after reversal, the rings were no longer present and all cells had networks of stress fibers. Indirect immunofluorescence techniques used to localize tubulin and vimentin indicated that microtubules and intermediate filaments were not constituents of the rings, and the rings were not closely apposed to the substrate, judging from reflection contrast optics. The rapid rearrangement of attachment plaques into a perinuclear aggregate that spreads radially in the cytoplasm occurs at the same speed as fibroblast and chromosomal movement, but is unlike other types of intracytopl

ISSN:0886-1544    

DOI:10.1002/cm.970080204

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

年代:1987

数据来源: WILEY

摘要:

AbstractOver the past 30 years filaments 2–5 nm in diameter have been foun in a number of different types of eukaryotic cells. As a group, these fine filaments lack the similarity of composition and function that characterize the three major classes of cytoskeleta elements—microfilaments, microtubules, and intermediate filaments. Six different proteins that form fine filaments have been identified; proposed functions for these fibers range from cell motility to cytoarchitecture. Recent studies, however, have revealed filaments with similar compositions and/or functions in otherwise different cells, sugesting that the fine filaments may eventually fit into a limited number of subgro

ISSN:0886-1544    

DOI:10.1002/cm.970080205

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

年代:1987

数据来源: WILEY

摘要:

AbstractThe amphibian egg undergoes a 30° rotation of its subcortical contents relative to its surface during the first cell cycle, a displacement of 350 μm in 50 min. This is directly visualized by following the movement of an array of Nile blue (a subcortical stain) spots applied to the egg periphery (Vincent, Oster, and Gerhart:Dev Bio 113:484–500, '86). We have investigated the mechanochemical basis of this unusual cell motility. Subcortical rotation depends on microtubule integrity during its entire course and is insensitive to inhibitors of microfilament assembly. It does not depend on newly synthesized proteins for its operation or timing, and it does not involve calcium‐dependent processes. Finally, we show that vegetal fragments of the egg can complete rotation on their own, indicating that mechanochemical components can operate locally in this hemis

ISSN:0886-1544    

DOI:10.1002/cm.970080206

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

年代:1987

数据来源: WILEY

摘要:

AbstractBiochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochmical composition [Brady et al: J. Cell Biol. 99:1716–1724]. To determine the morphologic correlate of cold‐insoluble tubulin, segemnts of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axond exposed to Cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment‐length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold‐ and PT‐exposed axons were significantly decreased, indicating that the presence of short segments of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment lenght and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold‐insoluble tubulin functions as a transportable MT‐organizing comple

ISSN:0886-1544    

DOI:10.1002/cm.970080207

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside‐out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such association may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal element

ISSN:0886-1544    

DOI:10.1002/cm.970080208

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

年代:1987

数据来源: WILEY

摘要:

AbstractIn this study we have applied microtubule‐associated proteins (MAPs) from mammalian brain to both native and reassembled insect ovarian microtubules. Such microtubules, which are normally smooth walled, become decorated with projections similar to those observed when mammalian brain MAPs are added back to assembling or assembled mammalian brain microtubules. The mammalian MAPs were also detected as components of insect microtubules when analyzed by polyacrylamide gel electrophoresis. Our observations suggest that mammalian brain MAPs have common binding sites on microtubules from two widely different sources and indicate the degree of evolutionary conservation of such site

ISSN:0886-1544    

DOI:10.1002/cm.970080209

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

年代:1987

数据来源: WILEY

摘要:

AbstractThis paper is concerned with the proposition that the insertion of membrane mass into the leading edge of a motile cell plays a critical role in directed cell migration. We show by immunofluorescence, with cells transfected with a cloned cDNA encoding the G‐protein of a temperature‐sensitive mutant of vesicular stomatitis virus, that the first cell surface appearance of the G‐protein is indeed at the leading edge of the motile cell. Two drugs capable of inhibiting directed cell migration, cytochalasin D and monensin, appear to function independently, the former by affecting the actin cytoskeleton without affecting the polarized insertion of membrane mass into the cell surface and the latter by abrogating membrane mass insertion without affecting the actin cytoske

ISSN:0886-1544    

DOI:10.1002/cm.970080210

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

年代:1987

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970080211

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

年代:1987

数据来源: WILEY