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PubMed1991

Inhibition of spindle elongation by taxol.

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole-pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273-281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 microM taxol, the rate and extent of pole-pole separation, measured from time-lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtubules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.

Alkaloids↗

PubMed1992

Distribution of detyrosinated microtubules in motile NRK fibroblasts is rapidly altered upon cell-cell contact: implications for contact inhibition of locomotion.

Fibroblasts migrating into an experimental wound contain an extensive array of detyrosinated microtubules (Glu MTs) oriented in the direction of migration, whereas nonmotile cells in the interior of a monolayer contain Glu MTs that are primarily coiled around the nucleus. To determine the role of cell-cell contact in the formation of these distinct arrays of Glu MTs, we studied the distribution of Glu MTs by immunofluorescence in NRK fibroblasts that had been fixed at different intervals after they had established contact with other cells. Time-lapse video recordings were made of the contacting cells to provide a record of cellular behavior. In motile cells that became completely surrounded by virtue of contact with other cells, Glu MTs were found mostly coiled around the nucleus. The proportion of cells whose Glu MTs extended to the original leading edge decreased dramatically after the cells had been surrounded for 10 min or more. At earlier times, when the contact was confined to a portion of the cell margin, Glu MTs were absent from the area behind the contact site, yet were still oriented toward the noncontacting and ruffling margins. The contact-induced alteration of Glu MTs was not due to the cessation of forward locomotion of cells per se, since immobilization of cells with cytochalasin D did not cause a dramatic change in Glu MTs. That cell-cell contact also specifies the type of Glu MTs formed in cells was shown by experiments in which MTs were regrown following complete depolymerization with nocodazole. The remodeling of Glu MTs during cell-cell contact may be involved in cellular repolarization during contact inhibition of locomotion and will be a useful marker for further dissecting the molecular events of contact inhibition of motility.

Cell Movement↗

PubMed1983

Timing of dispersal in juvenile jewel fish during development is unaffected by available space.

The spatial distributions of 2 groups of sibling juvenile jewel fish were examined between 58 and 99 days of age to determine the effects of spatial experience on the timing of dispersal from a more aggregative behavioral mode. One group was provided a seminatural 1000-liter aquarium, the other with 70% less space per fish, but both groups were able to observe the same number of fish. Using a computer and digitizing tablet, distances between 1st-6th nearest neighbors were measured from time-sampled video recordings of spatial distributions in either overhead or underneath plan views. Both groups exhibited changes in aggregative behavior that were remarkably similar. Between 65 and 92 days of age, both groups showed gradual dispersal, which accelerated abruptly at 93-99 days of age. These results suggest that the timing of dispersal is regulated by developmental processes operating independently of spatial experience.

Animals↗

PubMed1989

Nutritive and nonnutritive sucking and the temporal organization of the suckling behavior of domestic piglets.

Detailed video recordings of the suckling behavior of one piglet from each of four litters were analyzed to determine how the components of piglet suckling behavior are organized in relation to the time of milk ejection and the temporal pattern of grunting by the sow. Early in the suckling episode, most piglets massaged the udder with their snouts, and then changed gradually to sucking the teats with slow mouth movements (1-2/sec). The piglets then had a distinct phase of sucking with rapid mouth movements (4-5/sec) which began suddenly and lasted about 5 to 15 sec. The weight gains of piglets removed at different times showed that piglets consumed milk during the fast sucking but not during the preceding slow sucking, regardless of how much slow sucking had occurred. Three of the four sows showed a characteristic increase in rate of grunting about 20 to 25 sec before fast sucking began. The piglets' change from massaging to slow sucking often coincided with the increase in grunt rate, but the timing of the transition varied greatly. This suggests that the change in grunting is one but not the only cue used by the piglets to time their suckling behavior. During the slow, nonnutritive sucking, mouth movements were highly variable in duration, with occasional short bursts of rapid mouth movements. This contrasted with the more uniform duration of fast sucking movements. Evidently, in piglets, nutritive and nonnutritive sucking differ in both rate and temporal patterning.

Animals↗

PubMed1990

The excitatory neurotransmitter glutamate causes filopodia formation in cultured hippocampal astrocytes.

Can neurons induce surrounding glia to provide a more favorable microenvironment? Synapses and nerve growth cones have been shown to release neurotransmitters (Hume et al. Nature 1983;305:632-634; Kater et al. Trends Neurosci. 1988;11:315-321; Young and Poo Nature 1983;305:634-637) providing a possible mechanism for this type of control. The excitatory neurotransmitter glutamate induces an increase in the number of filopodia on the surface of astrocytes cultured from the neonatal rat hippocampus. This seems to be associated with a receptor-mediated event that is activated to a lesser degree by the quisqualate and kainate, but not NMDA receptors. In addition, time-lapse video recordings have revealed a rapid extension of filopodia from the apical margins of cells treated with glutamate. The apical margins of glutamate-treated cells studied with electron microscopy contained dense cortical actin networks that are devoid of microtubules. Coated pits are often seen to invaginate from the apical membrane in the vicinity of filopodia. A receptor-binding step may be followed by a rapid reorganization of cortical actin resulting in actin-containing filopodia. This process may be mediated by inositol lipid hydrolysis. Pyramidal neurons settled on glial cultures induced filopodia to form around the entire margin of growth cones and neurite tips suggesting that these events might occur in situ.

Animals↗

PubMed1994

"Short-stops" in rats with fimbria-fornix lesions: evidence for change in the mobility gradient.

Rats with damage to the hippocampal formation and allied structures are hyperactive in many test situations but the cause of this hyperactivity is not known. Here the activity of control rats and rats with fimbria-fornix lesions is documented in tests of overnight activity. Details of activity are then characterized from video recordings of behavior in an open field. Rats with fimbria-fornix lesions make significantly more stops of shorter duration and thus more individual trips than control rats but they do not differ in the distance traveled on individual trips or in travel speed. It is suggested that the main difference between fimbria-fornix rats and control rats is that when fimbria-fornix rats stop they remain "still" for shorter durations than do control rats. This finding is discussed in relation to a theory of locomotor/exploratory behavior, and in relation to its implications with respect to the performance of fimbria-fornix rats in studies of learning and memory.

Animals↗

PubMed2004

Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility.

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor receptor (EGFR) in many cancers is associated with progression of superficial to invasive forms of the disease. The naturally occuring type III mutant epidermal growth factor receptor (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. EGFRvIII is expressed frequently by a number of human solid tumours including those of the lung, breast, prostate, brain and ovary. Our study was designed to investigate the effect of EGFRvIII expression on cell motility and compare it to that of ligand-activated EGFR using transfected fibroblasts. We show here using time-lapse video recording that expression of EGFRvIII greatly enhances the motility of fibroblasts independently of ligand stimulation. In addition, expression of EGFRvIII caused a marked increase in the number of cellular protrusions (lamellipodia) and a reduction in the number of stress fibers and focal adhesions. The EGFR tyrosine kinase inhibitor, AG1478, and the MEK inhibitor, U0126, blocked these cellular effects of EGFRvIII. Two cell lines expressing different levels of EGFR were used for comparison. The low-expressing cell line responded to EGF treatment by increasing motility in a manner very similar to the motility induced by EGFRvIII. In contrast, the high-expressing cell line responded to EGF by detachment from the extracellular matrix and decreased motility. Cellular detachment was correlated to a high phosphorylation of PLC-gamma, whereas increased motility was correlated to a high level of ERK phosphorylation. Overall these results indicate that tumor-associated EGFR mutations might be critical for tumor cell motility, invasion and thus progression of disease.

3T3 Cells↗

PubMed1991

Invasion of Rous sarcoma virus-transformed retinal cells: role of cell motility.

Transformation of retinal neuro-epithelial cells by Rous sarcoma virus (RSV) leads to many alterations in cell phenotype, including changes in cell movement, cell-cell adhesion and protease secretion. To define and quantitate the alterations in cell movement, we analyzed video recordings of cultured cells using the computer-assisted Dynamic Morphology System (DMS). Control neuro-epithelial cells showed very low levels of translocation and membrane activity. After transformation, neuro-epithelial cells exhibited increased membrane activity, although directed cell translocation remained low. Developing retinas also contain a small proportion of Müller glial cells, which were purified by repeated passaging of control cultures. In contrast to neuro-epithelial cells, both control and RSV-transformed glial cells showed high levels of translocation and membrane activity. To analyze how different kinds of cell movement affect invasive behavior, we compared the ability of control and RSV-transformed cells to invade the chorio-allantoic membrane of developing chicken embryos. Control neuro-epithelial cells were not invasive. RSV-transformed neuro-epithelial cells, which showed low levels of translocation as revealed by DMS, were invasive. Similarly, RSV-transformed glial cells were invasive while control glial cells, which translocated, were not invasive. These results suggest that high levels of cell translocation are not necessary for invasion. In addition, the results suggest that elevated membrane activity in neuro-epithelial cells may be important for their invasion.

Allantois↗

PubMed1987

Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts.

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 microgram/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.

Animals↗

PubMed1994

Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes.

This study examined the effects of cryopreservation on cellular organization, chromosomal complement, and developmental potential of immature and mature mouse and human oocytes. Chromosomal analyses were performed by DNA fluorescence microscopy and karyotyping on the same metaphase II-stage oocytes before and after freezing. Cellular analyses involved electron microscopy, time-lapse video recording, and fluorescent-probe microscopy of cortical granules. The findings demonstrate that while profound cytoplasmic, nuclear, and nucleolar alterations occur in the immature oocyte during cryopreservation, an apparently normal nucleus and cytoplasm is re-established progressively after thawing and culture. The resulting oocytes mature at high frequency and for the mouse, are fertilizable and capable of normal preimplantation of embryogenesis. Cryopreservation of mature mouse and human oocytes is not accompanied by a significant increase in the frequency of aneuploidy. However, cryopreserved human oocytes, while fertilizable, arrest development during the early cleavage stages and display aberrant patterns of cytokinesis. The possible etiologies of developmental failure in the human embryo that may be related to oocyte cryopreservation, as well as the potential benefits of cryopreservation of the immature oocyte, are discussed with respect to clinical and commercial applications.

Aneuploidy↗

PubMed1987

Pacemaker region in a rhythmically contracting embryonic epithelium, the enveloping layer of Oryzias latipes, a teleost.

The primary objectives of this study were to determine the embryonic stage at which the Oryzias latipes enveloping layer (EVL) begins to contract rhythmically, and to determine where these contractions arise within the EVL. Using time-lapse video recording, we showed that the contractions begin at stage 14 (the stage of the embryonic shield) and arise in the ventral region of the EVL, which is centered at 180 degrees longitude from the embryonic shield. We have called this the pacemaker region for the contractions. Using fluorescein diacetate as a vital stain, we showed that the ventral region of the EVL continues to act as a pacemaker even after the EVL is detached from the rest of the egg. Rhythmic contractile activity ceased when we removed a group of about 130 cells--10% of the total EVL--from the pacemaker region; comparably large wounds elsewhere had no effect on the contractions. When we cut detached EVLs into ten pieces, only 2.4 +/- 1.8 (mean +/- SD, N = 11) of them contracted rhythmically, even though a considerably larger proportion of the EVL cells participate in the contractions in undisturbed blastoderms. We conclude that the pacemaker cells are necessary for rhythmic contractile activity and that cells outside this region do not contract spontaneously. The contractile waves are propagated at a velocity of 14-54 microns sec-1. This value, which is two to three orders of magnitude slower than the propagation of epithelial action potentials, is similar to the rate of propagation of waves of increased cytosolic Ca2+ in other systems. We propose that the medaka EVL is a good system in which to study certain aspects of epithelial morphogenesis.

Animals↗

PubMed1989

Ascidian sperm penetration and the translocation of a cell surface glycosidase.

Sperm bind to vitelline coat (VC) glycosides of ascidian eggs by means of a sperm surface glycosidase (Hoshi et al.: Zool Sci 2:65, 1985). In the genus Ascidia, N-acetylglucosamine (NAG) is the VC ligand. After initial binding by the tip of the head, sperm pass through the VC and perivitelline space leaving the single mitochondrion outside. This process can also be followed in vitro on a coverslip. Analysis of recorded video images shows that the sperm moves away from the anchored mitochondrion. Our model for sperm penetration suggests that mitochondrial translocation is responsible for driving the sperm into the egg. In the work presented here, we have demonstrated that ascidian sperm have N-acetyl-beta-D-glucosaminidase (NAGase) activity with an acidic pH optimum. This enzyme, which can be removed from the sperm with Triton X-100, binds to concanavalin A, demonstrating that it is glycosylated. Histochemical methods disclose that the enzyme is originally located at the tip of the head but subsequently remains with the surface overlying the mitochondrion during translocation. Fluorescent Con A was used as a second label for localization of the enzyme on the cell surface during translocation. Colocalization of both probes of the enzyme support a crucial facet of our model; the sperm surface VC binding site remains over the mitochondrion during translocation. This would couple mitochondrial translocation with sperm penetration and drive the sperm into the egg.

Acetylglucosaminidase↗

PubMed1997

Chemotaxis by human neutrophils and their cytokineplasts treated with inhibitors of nitric oxide synthase: no suppression of orientation or trajectory.

Inhibitors of nitric oxide (NO) synthase are reported to inhibit both the adherence of polymorphonuclear leukocytes (PMN) to substrate and chemotaxis (directed locomotion) of PMN as determined in Boyden chamber assays. In the current study, we examined both human blood PMN and granule-poor motile cytoplasts derived from them (cytokineplasts, CKP), under direct microscopic observation with concomitant time-lapse video recording, for their ability to respond chemotactically to an erythrocyte destroyed by laser microirradiation. In this system we can observe directly and continuously the orientation and trajectory of PMN before, during, and after establishment of a chemotactic gradient. For both PMN and CKP we employed three different inhibitors of NO synthase (N(omega)-methyl-L-arginine, N-iminoethyl-L-ornithine, and diphenyleneiodonium) in at least twice the concentrations employed to inhibit chemotaxis of PMN in Boyden chambers or killing of bacteria in CKP. Although small differences in adhesion might not have been appreciated, treated PMN and CKP were each indistinguishable from untreated controls in their ability to orient in a newly created chemotactic gradient and in their trajectories toward the chemotactic target.

Arginine↗

PubMed2004

Mechanical functions of setae from the mouth apparatus of seven species of decapod crustaceans.

The mouthpart setae of seven species of decapods were examined with macro-video recordings and scanning electron microscopy. The general mechanical (nonsensory) functions of the different mouthparts are described and an account of their setation is given. This offers the possibility to determine the mechanical functions of the different types of setae. Pappose setae do not participate in food handling but in general make setal barriers. Plumose setae likewise do not contact food objects but assist in current generation. Papposerrate setae are rare but they were seen to assist in pushing food particles into the mouth. Serrulate setae are very common and mainly participate in gentle food handling and grooming. Serrate setae are used for more rough food manipulation and grooming. The roughest shredding, tearing, and manipulation of prey items are handled by the cuspidate setae. Simple setae seem to be divided into two populations with very different functions. On the maxillipeds of Panulirus argus they are used for shredding, tearing, and holding the food objects, but on the basis of maxilla 2 of three other species they appear to have very little mechanical influence and only when handling small prey items. The functional scheme seems to be consistent within the Decapoda.

Animals↗

PubMed2006

Ontogeny of form and function: locomotor morphology and drag in zebrafish (Danio rerio).

Many fish species transform in body shape during growth, but it remains unclear how this influences the mechanics of locomotion. Therefore, the present study focused on understanding how drag generation during coasting is affected by ontogenetic changes in the morphology of zebrafish (Danio rerio). The shapes of the body and fins were measured from photographs of fish ranging in size from small larvae to mature adults and these morphometrics were compared to drag coefficients calculated from high-speed video recordings of routine swimming. We found that the viscous drag coefficient of larval and juvenile fish increased by more than an order of magnitude during growth and the inertial drag coefficient decreased at a comparable rate in adults. These hydrodynamic changes occurred as zebrafish disproportionately increased the span of their fins and their body changed shape from elongated to streamlined, as reflected by the logistic growth of a newly defined streamlining index, SL. These results suggest that morphological changes incur a performance cost by generating greater drag when larvae and juveniles operate in the viscous regime, but later provide a performance benefit by reducing pressure drag in the inertial regime of the adult stage.

Animals↗

PubMed2002

Spermatophore transfer in the hermit crab Clibanarius vittatus (Crustacea, Anomura, Diogenidae).

Although mating has been described in several hermit crab species, the mechanics of spermatophore transfer have not previously been demonstrated. Evidence from pleopod and gonopore morphology, video observations, and inseminated females indicates that in Clibanarius vittatus the male applies a spermatophoric mass directly onto the female via the gonopores rather than with modified pleopods 1-2 (gonopods) and/or genital papillae as in many other decapods. The single second pleopod of males of C. vittatus has a simple endopod with no apparent modifications for sperm transfer. There are no genital papillae extending from the male gonopores. The globular spermatophores are aligned in rows surrounded by a seminal secretion in the male ducts (vasa deferentia that terminate in ejaculatory ducts opening to the exterior via the gonopores). During copulation, described from time-lapse video recordings, the ventral surface of the last thoracic segment of the male, bearing the gonopores, was apposed to the ventral cephalothorax of the female. A massive amount of seminal secretion containing spermatophore ribbons, termed here the spermatophoric mass and described for the first time in a hermit crab species, was observed covering the sternites and coxae of pereopods 1-5 of a recently copulated female. It is suggested that during copulation the male emits the contents of the ejaculatory ducts directly onto the female without the aid of gonopods or genital papillae. Although spermatophore transfer is simple in C. vittatus, the presence of modified anterior pleopods or elongate genital papillae (sexual tubes) in other paguroidean species suggests the possibility of a more complex insemination process in these other hermit crabs.

Animals↗

PubMed1991

Characterization of ramified microglia in tissue culture: pinocytosis and motility.

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: gamma-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time-lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.

Animals↗

PubMed1990

Mechanism of failure of the ligamentum flavum of the spine during in vitro tensile tests.

Thirty ligamenta flava were dissected from pig lumbar spines with their attachments to the bone of the laminae still intact. These specimens were tested to failure, in tension, at strain rates of 0.03 and 0.2 s-1. Video recordings were made of the specimens during testing. Specimens failed because the ligaments became detached from the bone. The maximum stress that the ligaments were able to transmit was approximately 3 MPa. However, the corresponding strain was higher when testing at the slower strain rate than at the faster rate; these strains were 1.6 +/- 0.5 and 1.0 +/- 0.3, respectively. All specimens were able to transmit stress after the maximum value had been attained because threads of ligament continued to remain attached to bone. Complete failure did not occur until the last of these threads had pulled away. Scanning electron micrographs showed no ligament, other than a few isolated pieces of fibre, adhering to bone after failure had occurred.

Animals↗