Cell motility and the cytoskeleton最新文献

The different patterns of motility of rat spermatozoa during epididymal transit were studied in vitro using high-speed videomicroscopy. The sperm images were analysed after manual tracing as well as with a computer imaging system. The present work is the first which reports both the swimming path of the sperm head and the characteristics of flagellation in this species. The hook-shaped head of the rat spermatozoa allowed us to demonstrate the two-dimensional (2D) swimming movement compared to the three-dimensional (3D) sperm motion which was mainly related to rotation of the head. Immotile spermatozoa entered the initial segment of the testis and showed rigid flagella. The potential for sperm motility occurred abruptly in the proximal caput region, and different patterns of flagellation were observed: vibrating, motile in place, motile with a static curvature of the midpiece resulting in a spinning motion or a circular path, and forward progressive movement with regular rotation of the head. The pattern of sperm movement became homogeneous in the distal cauda where the whole sperm population swam in a straight line. A static curvature appeared in the midpiece portion when the spermatozoa reached the proximal caput region. The formation of the static curvature was observed on both sides of the rat flagellum which were easily indicated by the head-shaped projection of the head and the axonemal side of the principal wave. As soon as they moved, the spermatozoa successively initiated principal (P) and reverse (R) waves, but the waves were visible only distal to the static curvature. The midpiece stiffness progressively decreased during the epididymal maturation; simultaneously the static curvature showed a larger radius and then disappeared. Consequently, the initiation of waves which was first seen in the distal part of the flagellum of immature cells occurred progressively near the junction with the head of maturing spermatozoa. These changes in sperm motion previously shown in rams and now in rats might be a general phenomenon in mammals. The high resolution of this computer imaging system applied tosperm motion showing a well-characterized "side of the flagellum" should allow sensitive detection of biochemical effects on flagellar beating.

We have used a combination of immunofluorescence microscopy, northern blotting, ELISA, and isoelectric focusing to characterize the expression of neuronal Class III beta-tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway by retinoic acid. Following 48 h differentiation, beta-III tubulin mRNA is evident and beta-III tubulin appears in the mitotic spindle of neuroblasts. Neurite outgrowth is obvious by day 3, and beta-III tubulin protein and mRNA levels increase concurrently until approximately day 7, when beta-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic beta-III tubulin isoforms appear during neuronal differentiation. The expression of these isoelectric variants occurs concomitant with a temporal increase in the levels of beta-III tubulin present in the colchicine-stable microtubules. These results implicate posttranslational modifications of beta-III tubulin in the increased microtubule stability noted in differentiating P19 neurons.

We used fluorescent analogue cytochemistry to study in vivo dynamics of myosin II in Dictyostelium discoideum. We labeled myosin with biotin or tetramethyl-rhodamine iodoacetamide (IATR). The labeled myosin shows normal activities as reversible filament assembly and Ca2+ and actin-activatable Mg(2+)-ATPase. We used the biotin-myosin as a probe examining the effects of microinjection on the amoebae and the ability to associate with endogenous actin cytoskeleton. The biotin-myosin incorporates into certain actin populations and localizes to the cortex with the highest accumulation in the posterior end of polarized amoebae. The dynamics in live amoebae were probed by TR-myosin. We monitored the dynamics for a long period to determined the dynamic reorganization corresponding specific cellular behaviors. The TR-myosin converges into a discrete actin- and myosin-rich structure located at the posterior end ("myosin-organizing center"). The rod-shaped TR-myosin exhibits linear orderly arrays emanating from the organizing center which extend about two-thirds of the cell length. The myosin arrays show a dynamic reorganization when the amoebae move. To examine if the observed myosin dynamics are related to filamentous (F-) actin, we disrupted the F-actin by cytochalasin D. The ratioed image of TR-myosin (vs. FITC-dextran) demonstrates that myosin in these cells accumulates in the cortex but does not form the organizing center. Overall, the results suggest that the filamentous myosin organizes into orderly arrays in the live cytoplasm and its translocation occurs by means of F-actin cables, converging into the organizing center.

The present paper describes in detail the complex movement of the protozoon Tritrichomonas foetus. By the use of analogue and digital video techniques, we were able to analyze frame by frame the beatings of the anterior flagella and discuss their role in the movement of the cell. We also measured the productive displacement of the cell during one flagellar beating cycle. The obtained data were digitally improved and compared to analogue quantifications. It is shown that during 1 s of recorded movement, T. foetus performs 4 complete anterior flagella beating cycles (with active-like and recovery-like beatings). In each cycle the cell swims +/- 6.5 microns forwards, after the recovery of +/- 1.5 microns of receded movement. These observations led us to conclude that the estimated average speed of T. foetus is 25 microns/s, and that all flagella participate in the cell movement. The recurrent flagellum continuously contribute to the forward movement of the protozoon. The cell also performs rotational movements. The obtained results led us to suggest a model for the movement of T.foetus.

CENP-E is a protein of the kinesin superfamily that appears as small paired globules at kinetochores of chromosomes in mammalian cells during prometaphase and metaphase of mitosis [Yen et al., 1992: Nature 359:536-539]. In the present study we found that a significant number of chromosomes during early prometaphase in HeLa cells (approximately 30%) were stained with a CENP-E antibody in the form of large C-shaped "collars" that partially encircled the chromosomes. The C-shaped CENP-E collars were present only transiently and were completely replaced by small paired globular forms prior to metaphase. Most chromosomes had persistent CENP-E collars in cells blocked at mitosis with a vinblastine concentration sufficient to prevent all microtubule formation. Attachment of newly formed microtubules to the kinetochores after removal of vinblastine resulted in loss of the collars and replacement with small paired globules. Similarly, a higher proportion of chromosomes isolated from vinblastine-treated cells contained CENP-E collars (73%), and the "capture" (i.e., attachment) of microtubules by the chromosomes resulted in conversion of the collars into small paired globules in vitro. Thus, the CENP-E collars form prior to microtubule attachment and disappear after attachment of the chromosomes to the spindle. The CENP-E collars may facilitate capture of microtubules by chromosomes during prometaphase.

The dynamic behavior of microtubules has been measured in non-polarized, polarized, and hepatocyte growth factor treated Madin-Darby canine kidney epithelial cells. In a nocodazole disassembly assay, microtubules in polarized cells were more resistant to depolymerization than microtubules in non-polarized cells; microtubules in scattered cells were nearly completely disassembled. Analysis of fluorescent microtubules in living cells further revealed that individual microtubules in polarized cells were kinetically stabilized and microtubules in scattered cells were highly dynamic. Individual microtubule behavior in polarized cells was characterized by a suppression of the average rate of shortening, an increase in the average duration of pause, a decrease in the frequency of catastrophe transitions, and an increase in the frequency of rescue transitions, when compared with microtubules in non-polarized cells. In contrast, microtubule behavior in epithelial cells treated with hepatocyte growth factor was characterized by increase in the average rates of microtubule growth and shortening, a decrease in the frequency of rescue transitions, and an increase in the frequency of catastrophe transitions, when compared with polarized cells. Dynamicity, a measure of the gain and loss of subunits from microtubule plus ends, was 2.7 microns/min in polarized cells and 11.1 microns/min in scattered cells. These results demonstrate that individual microtubule dynamic behavior is markedly suppressed in polarized epithelial cells. Our results further demonstrate that in addition to its previously characterized effects on cell locomotion, hepatocyte growth factor stimulates microtubule dynamic turnover in lamellar regions of living cells.

Adult rat cardiomyocytes were placed in tissue culture to determine the relationships of their vinculin positive costameres, their attachments associated with the costameres, the fate of their myofibrils. The costameric structures were detected using interference contrast microscopy and the visualization of the fluorescently labeled vinculin and alpha-actinin molecules. The cardiomyocytes isolated from the heart retained their myofibrils upon attachment to the cell surfaces. One group of cells then rounded up, only to respread after 6 days in culture. These cells initially demonstrated costameric distributions of attachments and vinculin. These relationships were lost during the rounding-up process only to be regained as the cells respread. The second group of freshly isolated cardiomyocytes did not round up but began to spread on the substratum by sending out lamellipodia from their rectangularly shaped body. These newly cultured cardiomyocytes initially exhibited costameric distributions of close attachments detected by interference microscopy. Over the next 3 days although the cells remain attached to the substratum, the costameric attachments were gradually lost. Nevertheless, when similar cells were injected with fluorescently labeled vinculin, costameric distributions of vinculin could be detected in the absence of costameric attachments. Cardiomyocytes, injected with fluorescent alpha-actinin, revealed that during the first few days in culture the existing myofibrils disassembled from the edges of the cell towards the middle. The center group of myofibrils was retained as the cells began to spread. Our observations of living cells support the hypothesis that proteins in addition to vinculin are needed for cardiomyocytes to generate costameric attachments to the cell surfaces. We speculate that the ability of the vinculin-attached Z-lines of adult cardiomyocytes to dissociate from the extracellular matrix may aid in the remodeling of the adult heart in the repair process after myocardial infarction and also in stress induced hypertrophic growth.

Using several specific monoclonal antibodies, we investigated the occurrence and distribution of different post-translationally modified tubulin during interphase and division of the primitive flagellated protist Trichomonas vaginalis. Immunoblotting and immunofluorescence experiments revealed that interphasic microtubular structures of T. vaginalis contained acetylated and glutamylated but non-tyrosinated and non-glycylated [Brugerolle and Adoutte, 1988: Bio Systems 21: 255-268] tubulin. Immunofluorescence studies performed on dividing cells showed that the extranuclear mitotic spindle (or paradesmosis) was acetylated and glutamylated, which contrast with the ephemeral nature of this structure. Newly formed short axostyles also contained acetylated and glutamylated tubulin suggesting that both post-translational modifications might take place very early after assembly of microtubular structures. Our results indicate that acetylation and glutamylation of tubulin appeared early in the history of eukaryotes and could reflect the occurrence of post-translational modifications of tubulin in the primitive eukaryotic cells. These cells probably had a highly ordered cross-linked microtubular cytoskeleton in which microtubules showed a low level of subunit exchange dynamics.

Microtubules are thought to influence cell shape as structural components of an integrated cytoskeletal matrix. Here we show that microtubules can affect the dynamics of macrophage pseudopodia without being integrated into their structure. Macrophages landing on glass surfaces spread within 15 min into flattened circular cells with radial symmetry, and the radial distribution of microtubules reflected this symmetry. Depolymerization of microtubules using nocodazole, colchicine, or vinblastine did not inhibit macrophage spreading or the early establishment of radial symmetry. Shortly after spreading, however, macrophages without microtubules gradually became asymmetric, assuming irregular, lobed profiles. The asymmetry resulted from exaggerated protrusion and retraction of pseudopodia, with net retraction overall. This loss of radial symmetry could be inhibited by treatment of initially spread cells with cytochalasin D, indicating that the change in cell shape was mediated by the actin cytoskeleton. Intact microtubules suppressed the exaggerated pseudopod movements, even when they were separated by a distance from the cell margin. In cells treated with taxol, microtubules remained clustered near the cell center after spreading, yet the dynamics of pseudopodia at the cell margin were reduced and cells maintained a circular profile. Similarly, in cells treated with low concentrations of nocodazole, a much reduced microtubule cytoskeleton nonetheless suppressed pseudopod dynamics. We propose that microtubules act to stabilize cell shape at a distance from the cell edge via a biochemical intermediate that affects the structure or function of the microfilament system.