摘要:

AbstractTaxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calciumlabile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 μM Ca2+was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane‐free mitotic spindles, isolated with a calcium‐chelating, nonionic detergent buffer, were stored in an EGTA‐gylcerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA‐buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60–120 min; but in isolated spindles perfused with buffer that contained 100 μM Ca2+, BR decreased by 90% within 2–5 sec. In contrast, spindles that were pretreated for 3 min with 1 μM taxol, or for about 30 sec with 10 μM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 μM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 μM or 10 μM taxol then washout, stability of spindle BR to 100 μM Ca2+decreased exponentially with a time constant of 30–60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol‐stabilized micro

ISSN:0271-6585    

DOI:10.1002/cm.970040302

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

年代:1984

数据来源: WILEY

摘要:

AbstractReorganization of intermediate filaments during cell spreading is examined by immunofluorescence, electron microscopy, and time‐lapse video microscopy. A juxtanuclear cap, believed to correspond to the intermediate filament distribution center, was observed to be spatially related to the organization of the intermediate filament network as cells spread. A keratin cap was observed, which appeared spontaneously in motile PtK1 cells. Cap formation may be a consequence of retraction of intermediate filaments from the cytoplasm as cells move. The position of this juxtanuclear cap is related to the direction of movement, located on the side of the nucleus near the advancing edge of the cell. As the cell spreads, the cap disappears as the keratin filament network returns to the cytoplasm. Evidence presented here is consistent with the hypothesis that the distribution center mediates keratin filament organization during cell shape chang

ISSN:0271-6585    

DOI:10.1002/cm.970040303

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

年代:1984

数据来源: WILEY

摘要:

AbstractFluorescent derivatives of cellular proteins that retain their native characteristics have become useful probes to investigate the dynamics of specific cytoskeletal proteins. In the experiments reported here, a previously characterized fluorescent derivative of tubulin, bimane‐tubulin [Wadsworth and Sloboda, 1982a], was used to investigate microtubule assembly in vitro. The results demonstrate that bimanetubulin was competent to assemble onto a variety of organizing centers in vitro, including microtubule organizing centers (MTOCs) present in homogenates of sea urchin eggs, isolated mitotic apparatuses (MAs), and lysed mitotic cells. When homogenates of fertilized sea urchin eggs containing MTOCs were incubated with bimane‐tubulin at 37°C, discrete areas of linear fluorescence were observed. Only diffuse fluorescence was observed when calcium or colchicine was added to the homogenate or if the temperature was maintained at 0°C. Negative‐stain electron microscopy of the fluorescent arrays revealed morphologically normal microtubules radiating from electron dense regions. When mitotic spindles, isolated in glycerol containing buffers and therefore cold stable, were incubated with bimane‐tubulin, linear fluorescence was observed emanating from the spindle poles but not from the region occupied by the kinetochores. MAs incubated with bimane‐labeled bovine serum albumin or bimane‐labeled microtubule‐associated proteins showed only diffuse fluorescence. However, when mitotic cells which were hypotonically lysed in the absence of detergents or microtubule stabilizing solvents, were perfused with bimane‐tubulin intense fluorescence was observed in the asters and throughout the spindle. Two experiments suggested that the fluorescence observed in the results outlined above was due to the assembly of normal microtubules from the fluorescent subunits. First, the observed fluorescence was sensitive to cold temperataure, which is known to disassemble microtubules. Second, when the isolated, fluorescent MAs were examined by thin section electron microscopy, microtubules of normal diameter were seen. No aggregated material appeared associated with the walls of the microtubules, which might have been expected if the fluorescent protein was nonspecifically adsorbed to the microtubules. The results of these experiments demonstrate that isolated, stabilized MAs support the growth of new microtubules from the spindle poles while labile spindles, present in lysed cells, incorporate fluorescent tubulin throughout the spindle and asters. The significance of these results for hypotheses concerning microtubule assembly and disassembly during mit

ISSN:0271-6585    

DOI:10.1002/cm.970040304

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

年代:1984

数据来源: WILEY

摘要:

AbstractWe have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin‐based systems using mixtures of actin and the actin‐binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelat

ISSN:0271-6585    

DOI:10.1002/cm.970040305

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

年代:1984

数据来源: WILEY

摘要:

AbstractIontophoretic application of ATP to the flagellum of the demembranated hamster spermatozoon produced a planar pair of bends at the two ends of the stimulated site. During bend propagation, torsion appeared in the vicinity of the interbend in some responses such that the distal bend was twisted clockwise when viewed from the base of the flagellum. This pattern of propagation is consistent with the instantaneous configurations of free‐swimming cells previously described. The technique used here establishes that the three dimensionality arises from propagation per se, and does not depend on forces developed during swimming. The rolling of both free‐swimming intact and demembranated spermatozoa was examined by two‐color darkground videomicroscopy and the direction of rotation was, as predicted, always anticlockwise. A hypothetical mechanism, involving differential speeds of propagation of active sliding within the active microtubule subset, is proposed to account for the observed wave

ISSN:0271-6585    

DOI:10.1002/cm.970040306

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

年代:1984

数据来源: WILEY

ISSN:0271-6585    

DOI:10.1002/cm.970040307

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

年代:1984

数据来源: WILEY

ISSN:0271-6585    

DOI:10.1002/cm.970040301

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

年代:1984

数据来源: WILEY