摘要:

AbstractWe studied the properties of actinogelin, a Ca2+‐regulated actin cross‐linking protein isolated from Ehrlich tumor cells or rat liver. Chicken gizzard α‐actinin was used as a Ca2+‐insensitive control. Actinogelin, which has very high gelation activity under low Ca2+conditions, was found using electron microscopic or fluorescence studies to induce formation of a characteristic structure in which actin filaments and bundles radiate to (or converge from) all directions from spot‐like core structures. A similar structure was induced with actinogelin, even in the presence of 0 7 saturation of tropomyosin. No such structure was detected with actinogelin under high Ca2+conditions, and only a few were found with gizzard α‐actinin. Because reconstituted structures are similar to those observed intracellularly, actinogelin may be important in the formation of similar microfilament organization in the cells. It seems also important that these structures are reconstituted with only two purified protein components, i.e., actinogelin and actin.Immunocompetition studies showed that actinogelin and gizzard α‐actinin partially shared antigenicity, and their molecular shape and peptide maps were similar. Their amino acid compositions [Kuo et al., 1982], subunit and domain structures, and binding sites on actin [Mimura and Asano, 1987]are also very similar. Therefore, it is concluded that actinogelin belongs to α‐actinin superfamily proteins. Furthermore, the presence of functionally different subfamilies concerned with Ca2+sensitivy, gelation‐efficiency, and others is discussed. Actinogelin, which induces networks of actin filaments, may be classified

ISSN:0886-1544    

DOI:10.1002/cm.970100402

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

年代:1988

数据来源: WILEY

摘要:

AbstractAbfrontal ciliated cells ofMytilus edulisgill beat when mechanically stimulated, a consequence of a Ca++‐based generator potential and regenerative response. In contrast, the lateral ciliated epithelial cells arrest when stimulated, a consequence of a Ca++‐based generator potential and a Na+/Ca++‐based regenerative response. Iontophoretic injection of EGTA in abfrontal cells, followed by mechanical stimulation, results in a large, prolonged depolarization that returns to the resting level stepwise. It has been hypothesized that this phenomenon is caused by successive Ca++‐dependent repolarizations in coupled cells, first in adjacent cells and then in the injected cell, in accord with relative EGTA loading. We have now demonstrated this same stepwise repolarization phenomenon in the Na+/Ca++‐dependent lateral ciliated cells. In this case, each repolarization step is often preceded by a small spike. With either cell type, using two‐electrode recording techniques, we can detect the stepwise repolarization in distant cells, proportionately decremented when the second (KCl) electrode is some distance from the injection (EGTA) electrode and stimulus. When force is applied between the electrodes and nearest the KCl electrode, a greater initial response is recorded from this electrode, presumably resulting from depolarization of its impaled cell, prolonged by EGTA diffusion through the intervening cell junctions. The subsequent repolarization steps are of approximately the same size, suggesting repolarization of cells between the two electrodes. These observations are consistent with the cell coupling/EGTA loading hypothesis and indicate that both cell types mediate repolarization through Ca++and propagate ciliary beat or arrest through intracellul

ISSN:0886-1544    

DOI:10.1002/cm.970100403

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

年代:1988

数据来源: WILEY

摘要:

AbstractDictyosteliummyosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V‐8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V‐8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with32P was analyzed by SDS‐PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp

ISSN:0886-1544    

DOI:10.1002/cm.970100404

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

年代:1988

数据来源: WILEY

摘要:

AbstractDirected movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membranous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract‐coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule‐dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells).Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH8) solutions, high ionic strength ([KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP‐PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 μM), or N‐ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP‐PNP or vanadate. Motility was not affected by cytochalasin D (2 μM) or cAMP (1 mM). Kinesin (Mr= 116,000), an anterograde microtubule‐based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP‐PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organell

ISSN:0886-1544    

DOI:10.1002/cm.970100405

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe 51‐kD protein, a protein component of the mitotic apparatus in sea urchin eggs, is involved in the aster‐forming activity previously shown in vitro [Toriyama et al., 1988]. Postembedding immunofluorescent labelings of eggs from fertilization through first cleavage showed that the 51‐kD protein is localized in sperm asters, centrosomal regions, spindles, basal regions of astral microtubules, and regions surrounding daughter nuclei at telophase in situ. Immunofluorescence and immunoblot analyses detected the 51‐kD protein uniformly in unfertilized eggs, but not in spermatozoa. When unfertilized eggs were treated with taxol, the 51‐kD protein was shown to be associated with taxol‐induced cytasters. Immunoblot analysis revealed that similar protein species are present in the mitotic apparatus of other species of sea urchin. It was suggested that the 51‐kD protein may be involved in microtubule nucleation and microtubule matrix in sea urchin

ISSN:0886-1544    

DOI:10.1002/cm.970100406

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

年代:1988

数据来源: WILEY

摘要:

AbstractA high‐molecular‐weight polypeptide, named B‐band, was partially purified from sea urchin sperm flagella using selective extraction, hydroxylapatite chromatography, and sucrose density gradient centrifugation. The molecular weight of the B‐band was 440,000 by continuous system of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Sedimentation coefficient of the B‐band protein was 10.5 S, and its Stokes radius was 10 nm. When examined by low‐angle rotary shadowing electron microscopy, this molecule appeared to be composed of four globular heads and two curved linkers (“double headphone shape”), which was quite different from the shape of 21 S dynein, the outer arm dynein. Flagellar axonemes were also subjected to several chemical dissections. The B‐band was not extracted with treatments that remove both arm structures but was solubilized with treatments that extract other components such as radial spokes and nexin links. The B‐band protein in the axoneme was also more susceptible to trypsin digestion than the arm structures. These results suggest that the B‐band protein is a “double headphone‐shaped” component of the axonemal structures and makes up the elastic structure that might regulate the active sliding betwee

ISSN:0886-1544    

DOI:10.1002/cm.970100407

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe effects of protease substrates and inhibitor, which have been previously shown to inhibit mammalian sperm motility (de Lamirande, E., and Gagnon, C. [1986] J. Cell Biol. 102:1378–1383), were investigated using reactivated sea urchin and carp spermatozoa as models of “9 + 2” flagella. Aprotinin in the 2 to 20 μM range interfered with sperm motility by reducing both the beat frequency and the percentage of motile spermatozoa. These inhibitory effects of aprotinin were reversible either by dilution or by the addition of high concentrations of MgATP to the incubation medium. Protease substrates with a lys–ester bond, such as N‐α‐benzyloxycarbonyl‐lys thiobenzyl ester (BLT), also affected motility, but in the 0.1 to 0.5 mM range. As with aprotinin, both the flagellar beat frequency and the percentage of motile spermatozoa were partially and completely decreased, respectively. Analysis of the beat frequencies as a function of MgATP concentration in the presence and absence of 6 μM aprotinin indicated that this protease inhibitor affects sperm motility by decreasing the maximal flagellar beat frequency rather than by altering the axoneme's apparent Km for MgATP. Furthermore, aprotinin concentrations that blocked flagellar reactivation completely inhibited the sliding of microtubules from trypsinized axonemes. Basic proteins or polypeptides of pI close to that of aprotinin (10.3) were also potent inhibitors of the reactivation of motility. However, the characteristics of their inhibition of flagellar beat frequencies and reversibility of their effects suggested that they might be acting on sites different from those sensitive to aprotinin. The inhibitory effects of protease inhibitor and substrates, as well as results of experiments showing the absolute requirement of an intact ester bond for the inhibitory action of protease substrates, suggest that the involvement of a protease in the reactivation of 9 + 2 flagellar beating might be considered as a possible mechanism to explain aprotinin

ISSN:0886-1544    

DOI:10.1002/cm.970100408

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe migration of keratocytes isolated fromXenopustadpole epidermis has been investigated in vitro. In saline the cells move with a mean speed of 5–6 μm/min. Migration is slowed down in saline with diminished sodium content and ceases in media containing not more than 4 mM sodium. Inhibition of the Na+/K+‐2Cl−cotransporter by piretanide reduces the speed of migrating cells to about one‐third of the control level, the same accounts to inhibition of the Na+/H+antiport with amiloride at pH 7.2. At pH 6.6, however, amiloride only slightly influences locomotion. Depolarization of the plasma membrane by increased extracellular K+concentration or by inhibition of the Na+/K+pump by ouabain is only of minor influence during more than 1 h. Hyperpolarization of the cells using the sodium ionophore monensin impedes locomotion; this inhibition depends on an active Na+/K+pump. Ionophore‐mediated breakdown of the K+gradient strictly inhibits locomotion. The experiments have shown that a continuous flux of sodium ions is indispensable for the maintenance of cell locomotion. These ions may exert their action primarily by affecting cytosolic free calcium concentrat

ISSN:0886-1544    

DOI:10.1002/cm.970100409

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.970100410

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.970100401

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

年代:1988

数据来源: WILEY