摘要:

AbstractSeveral characteristics of the sliding disintegration ofTetrahymenaciliary axonemes were found by turbidimetric assay, the ATP‐regenerating system, and quantitative determination of the ATP concentration. At ATP concentrations exceeding 40 μM, the response in terms of turbidity was biphasic and could be divided into three phases. The dependence of each phase on ATP concentration was examined. The time duration of phase 2 increased with ATP concentration. When the ATP concentration was kept constant by the ATP‐regenerating system, consisting of pyruvate kinase and phosphoenol pyruvate, the time duration of phase 2 increased with the concentration of phosphoenol pyruvate. On examining the change in turbidity with decreasing ATP concentration, the transition from phase 2 to phase 3 was found to occur at an ATP concentration of 40 μM.Dark‐field and electron microscopy indicated the sliding disintegration to be closely correlated with the degree of tubidity. At phase 1, one or two doublets extruded from most of the axonemes, and disintegration failed to progress during phase 2. At the transition point from phase 2 to 3, at about 40 μM, ATP, other doublets were noted to extrude from the axonemes one after the other, causing turbidity to be minimal by the end of phase 3.The ATP concentration dependence of stepwise sliding disintegration suggests that each axoneme may possess the ability to regulate doublet microtubule sliding at lower or higher concentrations of ATP. In response to local differences or gradients of ATP concentration along the axoneme, the axonemes may cause localized sliding of doublets, thus subsequently generating active bending

ISSN:0886-1544    

DOI:10.1002/cm.970090302

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe Ca+2‐sensitive actin‐binding protein isolated fromDictyostelium discoideum, 30,000‐D protein (Fechheimer and Taylor:J. Biol. Chem.259:4514–4520, 1984;) has recently been localized in filipodia of substrate‐adhered amoebae (Fechheimer:J. Cell Biol.104:1539–1551, 1987). We have determined that this protein has a Mrof 34,000 daltons and is strictly colocalized with actin filaments in both substrate‐attachedDictyosteliumamoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34‐kilodalton (kD) protein that cross‐reacts specifically with antibody to theDictyosteliumbundling protein. Mammalian 34‐kD protein is colocalized with F‐actin in stress fibers and the cortical cytoskeleton in substratadhered fibroblasts. In substrate‐adhered vegetativeDictyostelium, F‐actin and 34‐kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34‐kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetativeDictyostelium, F‐actin and 34‐kD protein were not colocalized in cells that had progressed through the development cycle. In fruiting bodies, 34‐kD protein was detected by immunofluorescence microscopy only in prespore cells, while F‐act

ISSN:0886-1544    

DOI:10.1002/cm.970090303

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, andHalocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg ofM. occidentalishad a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic rescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.

ISSN:0886-1544    

DOI:10.1002/cm.970090304

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

年代:1988

数据来源: WILEY

摘要:

AbstractWe have compared the exchange kinetics of fluorescein‐labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady‐state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells preequilibrated to either 26 ± 2°C or 36 ± 2°C. A pulse of laser light focused to a 5‐μm diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half‐spindles and from the two polar regions were calculated for each image in a post‐bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26°C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 ± 2°C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to

ISSN:0886-1544    

DOI:10.1002/cm.970090305

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

年代:1988

数据来源: WILEY

摘要:

AbstractWe have used the mouse monoclonal antibody 6‐11B‐1, specific for acetylated α‐tubulin, to determine the distribution of acetylated α‐tubulin in in vitro–assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated α‐tubulin. Immunofluorescence, using 6‐11B‐1 and antitubulin B‐5‐1‐2, a monoclonal antibody specific for α‐tubulin, demonstrated the colocalization of both α‐tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated α‐tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod‐inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerizati

ISSN:0886-1544    

DOI:10.1002/cm.970090306

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe peroxidase‐antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)‐mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be s

ISSN:0886-1544    

DOI:10.1002/cm.970090307

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

年代:1988

数据来源: WILEY

摘要:

AbstractAmerican horseshoe crab sperm flagella have the typical 9+2 structure whereas Asian horseshoe crab sperm flagella have a 9+0 axoneme lacking central pair and central sheaths. Beat patterns of the American and the Asian horseshoe crab sperm were recorded by means of a high‐speed video system (200 fields/second) and were compared in order to study the role of the central pair of the axoneme in ciliary and flagellar movement.The American horseshoe crab sperm beat with relatively planar waves, whereas the Asian horseshoe crab sperm beat with right‐handed helical waves. These results suggest that the central complex plays an important role in forming planar waves, whereas it is not essential for the conversion of microtubule sliding into axonemal be

ISSN:0886-1544    

DOI:10.1002/cm.970090308

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

年代:1988

数据来源: WILEY

摘要:

AbstractA wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchinLytechinus pictus. The use of the fluorescent calcium chelator fura‐2 in combination with low‐light‐level TV microscopy and image processing allows the visualization of the Ca2+wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340‐nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band‐like appearance of the wave resembles the Ca2+wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+originating from the respective points of sp

ISSN:0886-1544    

DOI:10.1002/cm.970090309

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

年代:1988

数据来源: WILEY

摘要:

AbstractAs the species name indicates, the large heterotrichous ciliateSpirostomum ambiguumis characterized by a twisting contraction of the cell body that is easily triggered by various kinds of external stimuli. On the basis of morphological studies, contraction and extension of this organism have been considered to result from antagonistic actions of myoneme and microtubular ribbons. After many trials, we have succeeded in preparing cell models to examine induced contractions and extensions of the cell body. The contraction of this model was induced by increasing the free Ca2+concentrations even in the absence of Mg‐ATP and was reversed by adding Mg‐ATP without Ca2+. Using dynein ATPase inhibitors such as vanadate and ATP analogs, furthermore, the experiments revealed that the ATPase that generated the force between the two neighboring microtubular ribbons might be a dynein‐like A

ISSN:0886-1544    

DOI:10.1002/cm.970090310

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

年代:1988

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970090301

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

年代:1988

数据来源: WILEY