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PubMed1995

A mechanical function of myosin II in cell motility.

Myosin II mutant Dictyostelium amoebae crawl more slowly than wild-type cells. Thus, myosin II must contribute to amoeboid locomotion. We propose that contractile forces generated by myosin II help the cell's rear edge to detach from the substratum and retract, allowing the cell to continue forward. To test this hypothesis, we measured the speed of wild-type and myosin II null mutant Dictyostelium cells on surfaces of varying adhesivity. As substratum adhesivity increased, the speed of myosin II null mutant cells decreased substantially compared to wild-type cells, suggesting that the mutant is less able to retract from sticky surfaces. Furthermore, interference reflection microscopy revealed a myosin-II-dependent contraction in wild-type but not null mutant cells that is consistent with a balance of adhesive and contractile forces in retraction. Although myosin II null mutant cells have a defect in retraction, pseudopod extension does not cause the cells to become elongated on sticky surfaces. This suggests a mechanism, based possibly on cytoskeletal tension, for regulating cell shape in locomotion. The tension would result from the transmission of tractional forces through the cytoskeletal network, providing the myosin II null mutant with a limited means of retraction and cell division on a surface.

Animals↗
PubMed1995

Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis.

To investigate the dynamics of cell-substratum adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substratum contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from cells at earlier stages of development by small areas of contact to a substratum. Three questions have been addressed in analysing the locomotion of aggregation-competent cells. (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins? During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes the necessity for gradients of adhesion for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role for the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin crosslinking proteins, alpha-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica.

Animals↗
PubMed1998

A cytoplasmic dynein required for mitotic aster formation in vivo.

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

Anaphase↗
PubMed1999

TGFbeta1 induces a cell-cycle-dependent increase in motility of epithelial cells.

We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.

Animals↗
PubMed1975

An ultrastructural study of the spermatozoid of the fern, Marsilea vestita.

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.

Cell Nucleus↗
PubMed1978

An experimental study of the interaction between the soil amoeba Naegleria gruberi and a glass substrate during amoeboid locomotion.

The amoeboid locomotion of the soil protozoon Naegleria gruberi has been studied using reflexion-interference microscopy. Two types of contact are made with a planar glass substrate. One, formed at a considerable distance from the substrate in deionized water (congruent to 100 nm) has been termed 'associated contact' and usually involves a considerable surface area (of the order of 100 micrometer2), i.e. about a third of the cell profile. From this broad platform filopodia are produced which form close contacts ('focal contacts'). In locomotion the area of associated contact is very mobile, in contrast to the focal contacts which, once established, are stable. Focal contact sites are left behind on the glass surface ('footprints') when the amoeba moves away. The cell-substrate gap in the associated contact is greatly affected by the ionic strength of the medium and particularly the valency of the cation component. This suggests that long-range forces of attraction play an important role in keeping the amoeba close to a substrate and thus allow the production of filopodia from the ventral surface to form focal contacts.

Amoeba↗
PubMed1979

Granulocyte migration through endothelium in culture.

Using a model system with which we have previously investigated the adhesion of granulocytes to cultured endothelium, we have now shown that adherent granulocytes migrate through cultured endothelium in a manner closely resembling that found during the acute inflammatory response in vivo. The migration of granulocytes was markedly enhanced in the presence of erythrocytes, whereas blood platelets did not affect migration. Several test agents, including Paroven and some prostaglandins, had different effects upon migration and adhesion. We conclude that the adhesion of granulocytes to endothelium and their migration through it are responses that are, at least in part, separately controlled, and that migration does not depend exclusively upon extravascular stimuli.

Animals↗
PubMed1979

Effect of temperature on dry mass of polytene nuclei in Drosophila.

Nuclei were isolated by an aqueous detergent method from Drosophila prepupal salivary glands, and measured by integrating interference microscopy. There was a highly significant correlation between nuclear volume and dry mass. Dry masses fell into 2, 3 or 4 distinct groups corresponding to polytene replication classes; the mean of a given dry mass group was between 8 and 30% less than twice that of the group below, indicating that the ratio of DNA:dry mass increases during polytenic growth. The proportion of nuclei in the higher mass groups, the mean dry mass of nuclei within a given mass group, and the percentage loss of nuclear dry mass in the first hour after isolation were all higher when animals were reared at 15 degrees instead of 25 degrees C. Nuclear dry mass in prepupae was affected by the temperature during both the embryonic and larval periods, and also to some extent by the nutrition and degree of crowding of the cultures.

Animals↗
PubMed1980

Red blood cell adhesion. II. Interferometric examination of the interaction with hydrocarbon oil and glass.

Using both living and glutaraldehyde-fixed red cells, we have examined adhesion to both oil/saline and glass/saline interfaces by interference reflection microscopy. At low ionic strength, 0.4 mM NaCl, fixed cells adherent to the oil/saline interface show first order whitish yellow zones of closest approach which indicate a separation of similar to or approximately 100 nm. Quantitative interferometry in monochromatic light supports this conclusion. As the salt concentration is increased the separation decreases and the final image shows zero-order black which probably indicates molecular contact with the interface. Similar but less reproducible results were obtained with fixed and unfixed cells on glass. Thes observations show that physical interactions alone can be responsible for adhesion in dilute and concentrated salt solutions. It is not, however, believed that the results necessarily imply the existence of adhesion with a gap in physiological concentrations of salt.

Alkanes↗
PubMed1981

Locomotion of Xenopus epidermis cells in primary culture.

The locomotion of single epidermis cells, grown out from Xenopus laevis tadpole tails has been investigated by time-lapse cinemicrography using phase-contrast and reflection-contrast optics. The cells develop a large, mostly 200-250 nm thick, lamella, which adheres homogeneously to the supporting coverglass and exceeds the projection area of the cell body. From the comparison of RIC-pictures taken at high (1.06) and low (0.62) numerical aperture of illumination (I.N.A.) we deduce that at low I.N.A. the embossment of the medium-facing side of the lamella is visualized. By this method microcolliculi are demonstrated, which form at the edge of the lamellipodium and move backward. They resemble ruffles, but are flatter and no membrane flow towards the perinuclear region is observed. Indirect immunofluorescence reveals an enhanced staining for actin and alpha-actinin in the lamellipodium and in the transition region of cell body and lamella. Tonofilaments do not participate in lamella formation, the relatively few microtubules seem to be oriented in the direction of cytoplasmic flow. Electron micrographs demonstrate the course of fibrils in the cell body and a meshwork of actin filaments and membranous tubules in the lamella. Based on these findings a model for cell locomotion is presented: the motive force is generated by the cell body causing a flow of cytoplasm towards the periphery and extension of the lamella at its edge. The activity of the lamellipodium has to ensure the flat form of the advanced edge; microcolliculi are assumed to represent a small membrane store for the extension of the lamella. The lamellipodium is not involved in the production of motive force. The cell body is anchored to the lamella by radiating fibrils and the fibrillar meshwork is inserted at the 'dorsal' membrane of the lamella and the basal filament cortex of the cell body. This anchorage provides the structural basis for the uptake of lamella material into the cell body in the transition region.

Animals↗
PubMed1982

Cell-substrate contacts in cultured chick embryonic cells: an interference reflection study.

Cell-substrate contacts in explants of different regions of early chick tissues were investigated using the technique of interference reflection microscopy. All the explants spread as epithelial sheets. During initial spreading a peripheral zone of 2-3 cells formed broad contacts with the substrate. In spread explants some cells in the centre made broad substrate contacts. A mat of extracellular material containing fibronectin was found under the explants. Focal contacts and focal adhesions increased in number during culture, and stress fibres were associated with them. These changes in cell contacts appeared more quickly in some tissues than in others. After 24 h, explants of hypoblast and definitive endoblast could easily be distinguished but by 7 days they were very similar. In the absence of serum, specialized cell contacts developed more quickly; in higher concentrations of serum, more slowly. Confrontations between explants were also examined. The most conspicuous feature was that cells in invading explants normally underlapped invaded cells. Invasion from above by an unspread explant could occur even if the invaded explant had formed many focal adhesions.

Animals↗
PubMed1984

An analysis of in vivo cell migration during teleost fin morphogenesis.

In the teleost embryo the pectoral fin bud initially displays an apical ectodermal ridge along its entire distal margin. The ridge subsequently becomes transformed into an apical fold as the distal ectodermal epithelium grows and folds to enclose an extracellular space between the apposed basal surfaces of the epithelium. Collagen fibrils up to 2 micron in diameter, termed 'actinotrichia', are deposited along the proximo-distal axis in two (dorsal and ventral) arrays. The actinotrichia are aligned parallel to one another with a regular spacing along the greater part of their length. Mesenchymal cells migrating distally from the base of the fin bud encounter the dorsal and ventral arrays of actinotrichia and move between them apparently using the fibrils as a substratum. The entire structure is transparent and, using the killifish Aphyosemion scheeli, we have investigated the migration of the mesenchymal cells between 135 and 220 h of development, using Nomarski interference contrast microscopy and time-lapse video recording. The number of cellular processes per cell increased significantly during the period of observation. These processes could be graded according to their diameters. Processes of diameter greater than 2 micron were not usually aligned along actinotrichia and arose at any aspect of the cell body. In contrast, processes with diameters less than 2 micron appeared to be confined to the distal aspects of the migrating cells and showed an increasing tendency to become aligned as development progressed. Time-lapse video recordings revealed that such aligned processes move faster (mean speed 17.98 (+/- 2.25) micron/h) than non-aligned processes (mean speed 4.66 (+/- 0.67) micron/h). Whole cell translocation was generally slower than rates of process movement: the lowest mean value (1.52(+/- 0.36) micron/h) was recorded between 135 and 160 h of development rising to a maximum mean rate (4.72(+/- 0.42) micron/h) between 195 and 220 h; the period of the fastest rate of cell translocation correlated with maximum process alignment along actinotrichia. Thin 1 micron plastic sections revealed that, generally, aligned processes were in close association with the surface of the actinotrichial fibrils and not the spaces between them.

Animals↗
PubMed1984

The mitotic spindle of Chinese hamster ovary cells isolated in taxol-containing medium.

Mitotic spindles from Chinese hamster ovary (CHO) cells were isolated and purified by a one-step procedure in an isolation medium containing the microtubule-stabilizing drug, taxol. Released mitotic spindles were examined by phase-contrast, polarizing and differential-interference microscopy. They were also stained with monoclonal antibody raised against yeast tubulin and examined by epifluorescence microscopy. The spindles were free from visible cytoplasmic contaminants and the chromosomes were generally lost from the preparations. Electron microscopy showed that microtubules were the dominant structural component and sodium dodecyl sulphate/gel electrophoresis showed that tubulin was the major molecular species present, although a number of minor components, possibly representing microtubule-associated proteins (MAPs), were present. The taxol procedure was also useful in obtaining other microtubule-containing structures such as the midbody or the cytoplasmic microtubule complex in interphase cells. The taxol procedure was also used to isolate mitotic spindles from HeLa cells. The HeLa spindles stained positively with an antibody specific for the 210 X 10(3) Mr microtubule-associated protein, indicating that the MAP was retained by the taxol procedure. The taxol procedure appears to be of great advantage in large-scale preparations of spindles for biochemical analysis.

Alkaloids↗
PubMed1984

Membrane-substrate contact under the spermatozoon of Caenorhabditis elegans, a crawling cell that lacks filamentous actin.

Caenorhabditis elegans spermatozoa use a single, persistent pseudopod to crawl at about 20 micrometers/min but, unlike other types of crawling cells, sperm lack both filamentous actin and myosin. Interference reflection microscopy has revealed that sperm form broad grey areas of contact, analogous to the close contacts that have been described underneath other crawling eukaryotic cells, between their pseudopods and their substrate. Individual sperm change the size, shape and pattern of their substrate attachments as they crawl but we found no correlation between the extent of underside of the cell in contact with the substrate and the velocity of locomotion. Two predominant attachment patterns were observed: (1) a single broad contact extending from the front of the pseudopod nearly to the rear of the cell; and (2) two separate contact sites, one under the front of the pseudopod and one under the cell body. Occasionally, under cells exhibiting the second type of attachment pattern, portions of the anterior contact separated and remained stationary relative to the substrate while the cell moved forward. This observation, as well as the continuous change in shape of the contact areas, suggests that sperm continually form new contacts near the tip of the pseudopod and release these contacts backwards. In extreme cases, sperm were able to crawl with only the front of the pseudopod in contact with the substrate. Therefore, we propose that sperm locomotion depends on the interaction of several key events (traction, propulsion, membrane insertion) occurring at the leading edge of the pseudopod.

Animals↗
PubMed1985

Behaviour of neutrophil leucocytes in uniform concentrations of chemotactic factors: contraction waves, cell polarity and persistence.

The essential component of any hypothesis of random or directed cell movement is the mechanism of cell polarity. In this paper we describe the polar behaviour of human neutrophil leucocytes in uniform concentrations of chemotactic factors both in suspension and while moving across surfaces. Neutrophils exposed to uniform concentrations of chemotactic factors in suspension around the dissociation constant (Kd) for the receptor rapidly become distinctly bipolar; neutrophils exposed to supraoptimal uniform concentrations (100-fold greater than Kd) of chemotactic factors in suspension, although morphologically active, never reached the same degree of polarity as cells in optimal concentrations. These differences in polarity were shown to be the direct result of equatorial contraction waves stimulated on the cell surface by interaction with chemotactic factors. In optimal concentrations of chemotactic factors, contraction waves were initiated from one region of the cell, whereas in supraoptimal concentrations of chemotactic factors contraction waves emanated from all areas of the cell surface. Asymmetry in the distribution of surface receptors for Fc and C3b were observed in neutrophils polarized in uniform concentrations of chemotactic factor. In neutrophils, motile but not well polarized (in 10(-6) M-N-formylmethionyl-leucyl-phenylalanine (fMLP), receptors were uniformly distributed. In neutrophils polarized in concentrations of fMLP near the Kd for the receptor (10(-8) M) receptors for C3b and Fc were localized in the anterior region of the moving cell. The link between contraction waves, cell polarity and receptor redistribution and their initiation by chemotactic peptides is discussed in the context of neutrophil locomotion and response to chemical signals.

Cell Movement↗
PubMed1986

Conditions for fibroblast adhesion without fibronectin.

Conditions that permit the adhesion of BHK fibroblasts to a variety of surfaces after inhibition of protein synthesis and competition of any adsorbed fibronectin or vitronectin with the fibronectin cell-binding tetrapeptide, Arg-Gly-Asp-Ser (RGDS), are defined. Exposure of the cells to serum components at any stage in the preparation prevents cell attachment if cycloheximide or fibronectin tetrapeptide is present. If leupeptin is used cell adhesion and spreading occur even when all fibronectin synthesis is suppressed by cycloheximide inhibition, or fibronectin binding by tetrapeptide competition. The adhesions formed under these conditions appear by interference-reflection microscopy and by general properties to be identical to those formed by cells under normal culture conditions. The cell suspensions produced in the presence of leupeptin rather than other trypsin inhibitors show good adhesion at low temperatures, though the cells hardly spread at all. The results suggest that the role of fibronectin in cell adhesion should be reinterpreted in terms of its possible action as an activator rather than as a bonding molecule.

Animals↗