摘要:

AbstractThe dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video‐enhanced, differential‐interference contrast (VE‐DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus‐ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut‐off<30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus‐ended growth was observed, and self‐assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non‐buffer factors which stimulate elongation velocity by 6.5‐fold without self‐assembly, increase catastrophe frequency by 20‐fold,

ISSN:0886-1544    

DOI:10.1002/cm.970210102

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have previously described actin edge‐bundles (AEBs) as cables of microfil‐aments lining the webbed edges of 3T3 cells (Zand and Albrecht‐Buehler:Cell Motil. Cytoskeleton13:195–211, 1989). We have suggested that AEBs, along with their cell‐substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re‐formation induced by the following micro‐manipulations(1)Scoring of the webbed edge of a 3T3 cells with a microneedle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20–40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20–80 minutes, the new web decreased in length and depth, until it regained its approximate original shape.(2)Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by a

ISSN:0886-1544    

DOI:10.1002/cm.970210103

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

年代:1992

数据来源: WILEY

摘要:

AbstractFilamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non‐muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F‐actin and activates motile responses. In vitro, all purified F‐actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F‐actin within cells may not be identical. Typically, F‐actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton‐extracted cells. To determine whether the two measures of F‐actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F‐actin in basal, non‐adherent endo‐toxin‐free PMNs measured by both techniques was performed. F‐actin as NBD‐phallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F‐actin in PreFix cells is higher than in PostFix cells (54.25 ± 3.77 vs. 23.5 ± 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 ± 3.5% vs. 47.2 ± 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F‐actin pool from basal cells while a stable F‐actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F‐actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F‐actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with antigelsolin. Our studies show that at least two distinct F‐actin pools coexist in endotoxin‐free, basal PMNs in suspension: (1) a stable F‐actin pool which is a minority of total cellular F‐actin, Triton insoluble, resistant to depolymerization at 4°C, gelsolin‐poor, and localized to submembranous areas of the cell; and (2) a labile F‐actin pool which is the majority of total cellular F‐actin, Triton soluble, depolymerizes at 4°C, is gelsolin‐rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate acti

ISSN:0886-1544    

DOI:10.1002/cm.970210104

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe B‐CK isozyme of cytoplasme creatine kinase is localized distinctly in the terminal web region of the intestinal epithelial cell brush border (Keller and Gordon:Cell Motil. Cytoskeletoa19:169–179, 1991). Experiments were performed to determine whether this CK is energetically coupled to the myosin II that is present in the circumferential ring and interrootlet structural domains of the brush border terminal web. In isolated brush borders, ATP‐dependent circumferential ring contraction and interrootlet myosin solubilization were supported either by an exogenous PEP‐pyruvate kinase‐based ATP‐regeneration system (PEP‐PK) or by the addition of phosphocreatine to the endogenous B‐CK‐based ATP‐regeneration system (PCr‐B‐CK). Addition of an exogenous hexokinase‐glucose ATP‐hydrolysis system (HK‐G) effectively blocked both contraction and myosin solubilization in the PEP‐PK assay. In contrast, HK‐G had no significant effect on PCr‐B‐CK‐supported brush border contraction, although it did inhibit interrootlet myosin solubilization. Thus, when high‐energy phosphate is supplied as phos‐phocreatine, brush border B‐CK imparts to the circumferential ring myosin a selective energetic advantage over other ATPases. These results suggest that myosin and B‐CK are functionally coupled in the brush border circumferential ring, where they might comprise one end of an energy circuit that supplies energy for contraction, but that colocalizaton of CK with myosin in the brush border interrootlet domain i

ISSN:0886-1544    

DOI:10.1002/cm.970210105

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have previously described stable mouse C127 cell lines in which a CaM mini‐gene has been expressed in a bovine papilloma virus‐based expression vector (Rasmussen and Means:EMBO J.6:3961–3968. 1987). Elevation of CaM to levels five‐fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV‐transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell‐cycle‐dependent changes in the levels of mRNAs for histone H4, glyceraldehyde‐3‐phosphate dehydrogenase, β‐actin, vimentin, and β‐tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell‐cycle‐dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of β‐tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the moiecular basis of CaM‐dependent

ISSN:0886-1544    

DOI:10.1002/cm.970210106

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe studied the effect of cytochalasins (B, D, and E) on the F‐actin content in human neutrophils and lymphocytes using NBD‐phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration‐ and time‐dependent increase in the F‐actin content in both cell types. The order of potency was cytochalasin D>E>B. The increase in F‐actin content was accompanied by a decrease in the G‐actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin‐binding or sequestering proteins. 2‐deoxyglucose (20 mM) caused a decrease in the basal F‐actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than cur

ISSN:0886-1544    

DOI:10.1002/cm.970210107

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position‐specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N‐cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N‐cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N‐cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate comp

ISSN:0886-1544    

DOI:10.1002/cm.970210108

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe investigated the swimming patterns of trout sperm using computer‐assisted analyses of video microscopy. Under full activation conditions, in which 80–100% of sperm activate their motility, sperm swim in circular paths for 2–5 sec, followed by 30–60 sec of a more linear swimming, and, finally, cessation of movement, with a straightening of the flagella. Threshold activation, in which 50% of the sperm activate, is characterized by circular patterns of swimming for less than 20 sec, with straightened flagella upon cessation. Full activation and threshold activation are observed in low‐K+solution or in an Mg++‐supplemented K+solution. Similarities in swimming patterns in low‐K+solution and in a Mg++‐supplemented K+solution suggest a common underlying mechanism of activation. Initiation of movement in solutions with high Ca++to K+ratio is similar to activation in K+‐free solution. However, sperm in Ca++‐supplemented media resume circular swimming within 20–25 sec after activation, and, upon cessation of movement, the flagella are frequently cane shaped or bent. Differences in swimming patterns upon activation by high Ca++concentration suggest additional effects of Ca++on regulating swimming patterns. We used the fluorescent Ca++indicator Fluo‐3 to measure changes in intracellular Ca++concentration upon activation. Intracellular Ca++concentration transiently increases upon activation, with peak Ca++concentration coinciding with the period of circular swimming. This transient increase in Ca++concentration is seen in the absence of external Ca++, providing strong evidence for the released of Ca++from intracellular

ISSN:0886-1544    

DOI:10.1002/cm.970210109

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

年代:1992

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970210101

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

年代:1992

数据来源: WILEY