摘要:

AbstractIn this report, we describe an in vitro system for analyzing microtubule‐based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol‐stabilized microtubules assembled in low speed supernatants ofLytechinusegg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (–) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (−) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin‐like. The implications of these molecular interactions for mitosis and other motile events are

ISSN:0886-1544    

DOI:10.1002/cm.970060602

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

年代:1986

数据来源: WILEY

摘要:

AbstractRedistribution of alpha‐actinin during fertilization was investigated by means of the microinjection of fluorescently labeled egg alpha‐actinin in the sea urchin,Hemicentrotus pulcherrimus. Upon fertilization, labeled alpha‐actinin accumulated locally around the sperm binding site, where the fertilization cone formed soon afterwards. The accumulation propagated all over the cortex within 10 sec after a latent period of 10–20 sec. When an egg in Na‐free seawater was injected with both alpha‐actinin and calcium buffer (intracellular free Ca2+concentration = 9 μM), the accumulation of alpha‐actinin was similar to that in normal seawater, which suggests that the accumulation did not depend on the increase in intracellular pH but only on the increase in the intracellular free Ca2+concentration. In immature oocytes the accumulation was detected in the cortical region, including the huge protruding cytoplasm where the sperm entered. When labeled egg alpha‐actinin was injected into starfish (Asterias amurensis) oocytes followed by insemination, it accumulated in the cortical layer in a manner similar to the case of sea urchin, except that the accumulation in fertilization cones of maturing oocytcs or reception cones of immature oocytes appeared ringlike and rodlike, respectively. Moreover, just after the arrival of the meiotic apparatus, egg alpha‐actinin accumulated in the cortical region, where the formation of the polar body was expected. This suggests that the meiotic apparatus somehow induced the differentiation of the cortex so as to form a polar body. It is concluded that the cortical region where alpha‐actinin accumulated coincided with the microfilament‐rich region. This suggests that alpha‐actinin plays a role in forming the cortical mes

ISSN:0886-1544    

DOI:10.1002/cm.970060603

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

年代:1986

数据来源: WILEY

摘要:

AbstractAlthough the mammalian epidermal basal cell hemidesmosome bears some superficial resemblance to one half of a desmosome at the ultrastructural level, examination of the structure of the electron‐dense submembranous plaques of the hemidesmosome and desmosome reveals that they differ with respect to their overall morphology and dimensions. Based on these findings, we wondered whether components of the desmosome are present in the hemidesmosome. In order to determine this we prepared a number of stratified squamous epithelial tissues for indirect immunofluorescence using antibody preparations directed against known desmosome components including desmoplakin and certain glycoproteins. These antibody preparations do not show reaction with hemidesmosomes by indirect immunofluorescence criteria. We have also utilized bullous pemphigoid (BP) autoantibodies that have been shown to recognize hemidesmosomes in mammalian skin cells [Mutasim et al., J. Invest. Derm., 84:47–53, 1985]. Double label indirect immunofluorescence observations of neonatal mouse skin prepared using desmoplakin antibodies and BP autoantibodies reveal that hemidesmosomes that are stained by the BP autoantibodies are not recognized by the desmoplakin antibodies. We confirmed these findings at the ultrastructural level by indirect immunogold localization of desmoplakin antibodies and BP autoantibodies. Therefore, the hemidesmosome does not appear to be one half of a desmosome and may possess a very different molecular organization relative to the desmosome. We raise the possibility that the variability between the hemidesmosome and desmosome that we detect at the morphological and immunological level may reflect the functional differences of these two types of juncti

ISSN:0886-1544    

DOI:10.1002/cm.970060604

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

年代:1986

数据来源: WILEY

摘要:

AbstractMicrotubule‐associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells.In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cell

ISSN:0886-1544    

DOI:10.1002/cm.970060605

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

年代:1986

数据来源: WILEY

摘要:

AbstractWhen detergent‐extracted cell models ofChlamydomonas reinhardtiiwere left in the presence of 1 mM Mg‐ATP for more than 30 minutes flagellar axonemes tended to become frayed into fine bundles of microtubules. Under such conditions, bundles made up of a pair of outer‐doublet microtubules displayed oscillatory bending movements of low (<2 Hz) frequencies. The two doublet microtubules underwent association‐dissociation cycles coupled with gross bending movement. A model is presented to explain this phenomenon by unidirectional sliding interaction between the two micro

ISSN:0886-1544    

DOI:10.1002/cm.970060606

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

年代:1986

数据来源: WILEY

摘要:

AbstractEnergy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP‐dependence of ATPase activity was investigated for ATP concentrations ranging from 4 μM to 600 μM ATP. Using Eadie‐Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 μM and 161 μM ATP, and they were referred to, respectively, as the high‐affinity dynein ATPase (HADA) and the low‐affinity dynein ATPase (LADA). Investigation of movement‐coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement‐coupled. The apparent Michaelis constants of movement‐coupled HADA and LADA, 12 μM and 271 μM ATP, respectively, were two‐ to four‐fold greater than the apparent Michaelis constants of movement‐uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 μM and 290 μM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement‐coupled HADA, and that flagellar beat frequency is controlled primarily by movement‐coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distin

ISSN:0886-1544    

DOI:10.1002/cm.970060607

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

年代:1986

数据来源: WILEY

摘要:

AbstractSlow pulsatile movements of Schwann cells in vitro were studied quantitatively by using time‐lapse cinemicrography. Schwann cells from peripheral nerves of 3‐day‐old rats were cultured in serum‐free medium. Most Schwann cells showed intermittent episodes of pulsatile movement; each episode consisted of one or several contractile pulses. About half of the episodes consisted of a single pulse, and episodes with more than four pulses were rare. The average episode of activity lasted 2.6 min, while the average duration of a single pulse was 1.5 min. The mean quiescent interval between episodes of activity was 3.7 min. Some cells showed no pulsatile activity. Active cells averaged 6.6 episodes/h. The fraction of time which a Schwann cell spent in pulsatile activity varied widely, with an average of 28%. Behavior of Schwann cells in HEPES‐buffered Hanks saline was generally similar to that in the complete medium. Raising K+to 40 mM or Ca++to 10 mM did not markedly affect the time course of the pulsatile motility, although the contractions were more vigorous in the high Ca++. Pulsatile movement was reversibly inhibited by cytochalasin B and appeared to be potentiated by drugs that disrupt mic

ISSN:0886-1544    

DOI:10.1002/cm.970060608

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

年代:1986

数据来源: WILEY

摘要:

AbstractSea urchin coelomocytes contain an actin‐based cytoskeleton that undergoes major organizational changes as the cells transform from one morphology (petaloid) to another (filopodial). The molecular mechanisms directing and regulating this cytoskeletal reorganization are not well understood; Ca2+has been implicated, but the specific targets of its action have not been identified. Since the effect of Ca2+on a variety of cellular processes has been shown to be mediated by the Ca2+‐binding protein calmodulin, we investigated the role of this protein in coelomocyte morphological transformation. The calmodulin inhibitory drugs trifluoperazine, chlorpromazine, and calmidazolium were shown to inhibit coelomocyte morphological transformation in response to hypotonic shock in a dosedependent fashion and at concentrations consistent with their reported potencies as anti‐calmodulin agents. Calmodulin isolated from coelomocytes using trifluorophenothiazine affinity chromatography co‐migrates with bovine brain calmodulin on 15% SDS‐polyacrylamide gels and demonstrates a characteristic shift in electrophoretic mobility in the presence of Ca2+. Another diagnostic for calmodulin, Ca2+‐dependent activation of exogenous 3':5' cyclic AMP phosphodiesterase, was demonstrated by whole coelomocyte homogenates, heat‐treated homogenates, and the affinity purified coelomocyte protein. Localization of calmodulin in coelomocytes by indirect immunofluorescence reveals an association of calmodulin, at least in part, with the actin‐based cytoskeleton. Calmodulin‐binding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using125I‐calmodulin overlay procedures. Ca2+‐dependent calmodulin‐binding in these preparations was demonstrated for all but the Mr30,000 and 20,000 coelomocyte polypeptides. The majority of the calmodulin‐binding proteins identified in whole petaloid coelomocyte preparations are also found in Triton X‐100 insoluble cytoskeletal fractions. Immunoblotting with antiserum raised against chicken erythrocyte alpha‐spectrin suggests that the 240,000 Mrcalmodulin‐binding polypeptide corresponds to coelomocyte alpha‐spectrin. This protein was enriched in isolated coelomocyte filopodia where, we propose, it serves an analogous function to its counterpart in erythrocytes, in linking the actin‐cytoskeleton to the plasma membrane. Thus, calmodulin is present in coelomocytes and possibly participates in the morphological transformation of these cells through regulation of cytoskeletal and/or

ISSN:0886-1544    

DOI:10.1002/cm.970060609

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

年代:1986

数据来源: WILEY

摘要:

AbstractIn a preceding study [Blum, J.J., and Reed, M.C. (1985): Cell Motil. 5:507–527], factors responsible for the shape and velocity of the leading edge of the radiolabeled organelle profile were analyzed, but processes that might influence the shape of the plateau‐like region behind the advancing wave were ignored. It is now shown that deposition of material from the fast transport system into membrane‐associated structures, degradation of such deposited material and its return to the soma by the retrograde transport system, or leakage of radiolabeled material from the axon can account for the shape of the plateau. Furthermore, these processes are compatible with the maintenance of such structural inhomogeneities as the nodes of Ra

ISSN:0886-1544    

DOI:10.1002/cm.970060610

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

年代:1986

数据来源: WILEY

摘要:

AbstractWe describe here antigenic cross‐reactivity between the human 40‐kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF‐7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40‐kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes.In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40‐kD cy

ISSN:0886-1544    

DOI:10.1002/cm.970060611

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

年代:1986

数据来源: WILEY