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Migrating cells advance by first protruding a lamellipodium, a thin sheet of cytoplasm that consists mostly of filamentous actin (F-actin). The region immediately behind the lamellipodium is also relatively flat and has been termed the lamella ([Heath and Holifield, 1991][1]). Adhesion to the matrix

To quantitatively characterize the mechanical processes that drive phagocytosis, we observed the FcgammaR-driven engulfment of antibody-coated beads of diameters 3 mum to 11 mum by initially spherical neutrophils. In particular, the time course of cell morphology, of bead motion and of cortical tension were determined. Here, we introduce a number of mechanistic models for phagocytosis and test their validity by comparing the experimental data with finite element computations for multiple bead sizes. We find that the optimal models involve two key mechanical interactions: a repulsion or pressure between cytoskeleton and free membrane that drives protrusion, and an attraction between cytoskeleton and membrane newly adherent to the bead that flattens the cell into a thin lamella. Other models such as cytoskeletal expansion or swelling appear to be ruled out as main drivers of phagocytosis because of the characteristics of bead motion during engulfment. We finally show that the protrusive force necessary for the engulfment of large beads points towards storage of strain energy in the cytoskeleton over a large distance from the leading edge ( approximately 0.5 microm), and that the flattening force can plausibly be generated by the known concentrations of unconventional myosins at the leading edge.

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

We reported earlier that isolated plasma membranes trigger a number of responses comprising contact inhibition of motility, including loss of oriented detyrosinated microtubules (Glu MTs) from the lamella of motile fibroblasts. In this study, we show that the membranes trigger this loss of Glu MTs, not by binding to cells, but by removing an essential component from the medium necessary to maintain oriented Glu MTs. Preincubation of membranes with medium containing serum followed by removal of the membranes by sedimentation rendered the membrane-treated medium capable of triggering the loss of oriented Glu MTs. Membrane activity was inhibited by high concentrations of serum and removal of serum from medium triggered the loss of oriented Glu MTs similar to that triggered by membranes. These results suggest that the membranes trigger the loss of Glu MTs by inactivating factors in serum that are required for the maintenance of oriented Glu MTs. By fractionating serum, we have identified lysophosphatidic acid (LPA) as the principal serum factor that is responsible for supporting oriented Glu MTs. The activity of LPA to maintain oriented Glu MTs upon serum withdrawal was half maximal at 100 nM and no activity was observed with structurally related phospholipids. Serum LPA levels were sufficient to account for the ability of serum to support oriented Glu MTs. Enzymatic degradation of serum LPA strongly reduced the ability of serum to support oriented Glu MTs. That membranes degrade LPA was shown by the ability of membranes to block LPA's ability to maintain oriented Glu MTs, and by direct measurement of the loss of radiolabeled LPA after incubation with membranes in vitro. These results show that isolated plasma membranes trigger the loss of Glu MTs from the lamella of motile cells by degrading serum LPA. Coupled with earlier results showing that membranes trigger a number of contact inhibition responses, our data suggest a new model for contact inhibition of motility in which local degradation of LPA and/or interference with LPA-stimulated signalling pathways initiates a contact inhibition response in colliding cells.

Patches of aggregated membrane proteins on motile fibroblasts are transported from the surfaces of the leading lamella to a site just ahead of the nucleus in the phenomenon known as capping. A major cell surface glycoprotein, Pgp-1 (GP80), was tagged with a monoclonal IgG and then aggregated with fluorescent secondary antibodies. Correlated digitized fluorescence and phase-contrast microscopy were used to map the trajectories of fluorescent Pgp-1 patches located in various regions of the cell surface. The response of patches located in lamellar and nonlamellar regions to spontaneous retraction of the trailing cell margin, or tail was examined in detail. During capping, Pgp-1 patches accumulated at a morphologically distinct site on the cell surface, the ‘null border’, corresponding to the boundary between lamelloplasm and endoplasm and the posterior edge of the dorsal cortical F-actin sheath. Posterior to this site, gradual forward movement of patches accompanied the gradual narrowing phase of the trailing edge retraction that occurs prior to abrupt detachment of the tail, but patches did not actually accumulate at the null border. The rate of forward patch movement was generally greater at positions further behind the boundary. Patch movement correlated approximately with forward organelle movement in the trailing region of the cell. The boundary was also apparent during simultaneous capping and retraction when forward patch transport on the trailing edge and rearward transport of patches across the lamellar surface appeared to converge on the null border. Forward patch transport was strictly confined to regions behind the boundary while retrograde patch transport was confined to the lamellar region ahead of the boundary. Patches are thought to be linked to the cortical cytoskeleton and their transport is discussed in terms of the very different cortical cytoskeletal dynamics occurring in the leading and trailing edges of locomoting cells.

We have previously shown that detyrosinated microtubules (Glu MTs), which are oriented toward the direction of locomotion in motile fibroblasts, disappear from the area adjacent to cell-cell contact soon after a cell-cell collision. To identify cell surface molecules that trigger this phenomenon, we have established a system in which this and other cellular reactions to cell-cell contact can be reproduced by the addition of isolated plasma membranes. Experimental wounds were made in confluent monolayers of NRK cells, and cells at the wound margin were allowed to develop oriented Glu MTs. Test samples were added to these cells and after a 1 hour incubation the distributions of Glu MTs, tyrosinated MTs (Tyr MTs) and microfilaments were determined by immunofluorescence. When plasma membranes isolated from NRK cells were added, oriented Glu MTs disappeared from the leading lamella of target cells and instead a small number of Glu MTs were found clustered around the nucleus. As observed for cell-cell contact, plasma membranes did not significantly affect the distribution of Tyr MTs. We also found that both cell-cell contact and membrane treatment caused the collapse of lamellipodia and loss of associated staining with antiactin antibody. Time-lapse recordings of directed locomotion of NRK cells showed that membranes suppressed the forward movement of cells. The loss of Glu MTs from the leading lamella was the most amenable response for quantification and we used it to examine the biochemical properties of the membrane activity. The ability of membranes to induce the loss of oriented Glu MTs was observed at as low as 4 micrograms/ml of membrane protein and was detectable 10 minutes after membrane addition. The loss of oriented Glu MTs was reversible upon removal of membranes, demonstrating that the membranes were not toxic to the cells. The oriented Glu MT reducing activity could be solubilized from the membranes by detergent, was enriched in a plasma membrane fraction, and was labile to heat and acid treatment. In summary, we have successfully reconstituted a number of responses of contact inhibition using solubilized preparations of membranes. Our preliminary results suggest that there is a specific factor in plasma membranes that is capable of triggering contact inhibition. With the assay we have developed, it should now be possible to dissect contact inhibition of motility at the molecular level.

Limpet blood cells are suitable for the study of a number of interdependent features of cell behaviour; microspike formation, spreading and locomotion on a solid substrate, and aggregation. The effects of cytochalasin B (CCB) and colchicine on each of these activities is studied with a view (a) to determining with which functions these 2 reagents interfere; and (b) to gaining information on the relationships between these functions in normal cell behaviour. Rapid spike formation and fast aggregation in a shaker system, which are normal features of the behaviour of amoebocytes in limpet blood or in seawater, are totally but reversibly inhibited by the presence of low concentrations of CCB (e.g. 0.5 µg/cm3). Similarly, the spreading of amoebocytes on to a solid substrate is greatly inhibited by CCB, both in rate and extent, and the rapid locomotion on a solid substrate of normal amoebocytes is completely abolished by CCB. The first observable effects of CCB on spread amoebocytes are loss of optical integrity of the spikes and retraction of parts of the anterior cell margin. The loss of distinctive cell shape, the inhibition of spreading on to a glass surface and the lack of motility on such a surface may all be immediate consequences of the disruption of spike structure. CCB inhibits aggregation but does not disrupt preformed contacts between cells, which suggests that it acts on an early stage in the formation of stable contact rather than on the adhesiveness per unit area of contacting cell surface. A possible link between the effects of CCB on spikes and on aggregation is the proposal that low-diameter projections are needed to establish initial contact between cells. Alternatively, the spikes may be required for the rapid spreading of cells, not only on to glass but also over the surfaces of other cells, enabling them to increase their mutual contact area very rapidly and thus stabilize an adhesion. Amoebocytes maintained in the presence of 50 µg/cm3 colchicine for a number of hours gradually lose their bipolar form and the entire cell margin becomes occupied by the spike-supported lamella which normally constitutes the leading edge of the cell. Thus microtubules are probably not necessary for the skeletal functions of spikes or for their roles in spreading and aggregation, but they do appear to play a part in the control of spike orientation. Macrophages, cells lacking obvious spikes and showing little sign of bipolarity, appear unaffected by CCB or colchicine and spread normally on to a glass slide in the presence of either reagent. This, together with the limited spreading of amoebocytes in CCB, suggests that at least 2 distinct mechanisms may operate in the spreading of cells on to a solid substrate.

The accessibility of the nervous system of Manduca sexta to horseradish peroxidase and lanthanum during metamorphosis has been studied. In the fifth instar larva, peroxidase penetrates the thick neural lamella but does not reach the perineurium. In the first week after larval-pupal ecdysis, when the intercellular channels between hypertrophied perineurial I cells are the least tortuous, the enzymic tracer bypasses the perineurial I tight and gap junctions and penetrates as far as the perineurial II bracelet layer. During this time, there is active uptake of peroxidase by the perineurial I cells but no reaction product is found in the underlying perineurial II cell bracelet. In the second week after larval-pupal ecdysis, when the perineurium is a flattened interdigitating cell layer, peroxidase does not penetrate as far as the perineurial I and II interspace and there is little or no uptake of the protein by the perineurial I cells. Lanthanum, in contrast, passes the perineurial I tight and gap junctions, which are presumed to be macular, and travels within the entire perineurial I and II interspace throughout metamorphosis. As it travels within this interspace, it gains access to the perineurial II bracelet connexions, which differ in their ability to restrict lanthanum. Lanthanum is able to penetrate the entire overlapping connexion with its predominant gap junctions before being stopped, but penetrates the more complex interdigitating connexion with its extensive tight, gap and septate junctions for only a short distance. The relevance of the perineurial II bracelet layer and its intercellular connexions to the maintenance of a blood-brain barrier in this developing nervous system is discussed.

Haemocytes suspended in the blood of the limpet Patella vulgata rapidly form microspikes and aggregate in response to withdrawal from the haemocoel. It has previously been suggested that the contacts between colliding cells in shaken suspension are stabilized by the rapid spreading of participant cells over each other's surfaces by a microspike-dependent process. In this communication we report that an electron-microscopic study of the contacts formed between cells in the early stages of aggregation suggests that intercellular contact elicits a rapid response from the organelles concerned with cell motility. This response leads to the rapid progression of each initial collision through 3 major categories of contact morphology, namely cell body/lamella, lamella/lamella and cell body/cell body. There is a concomitant increase in the area of mutually contacting surface between participating cells. On the basis of these results, a model is constructed for the participation of sub-surface components in the process of cellular aggregation. The sequence of development of contact found between aggregating cells is envisaged to be a consequence of cellular locomotory activities resembling those by which these cells spread on to a solid substrate. Included in this process is an element of intercellular recognition. It is suggested that the involvement of motile elements in cell aggregation is a general feature of aggregating cells. This would permit a re-interpretation of aggregation experiments which show specificity of cell adhesion, for total adhesive interactions between cells would depend not only on the adhesiveness per unit area of contacting cell surface but also on the extent or rate of formation of flat contact areas. The latter element could be regulated by a recognition system between contacting cells independently of the force per unit area which holds the 2 surfaces together.

The fine structure of the metamorphosing abdominal nerve cord of Manduca sexta has been studied. In fifth instar larvae, the connectives are ensheathed by a complex, thickened neural lamella. The underlying perineurium at this stage consists of 2 layers. The outer layer consists of interdigitating type I cells which are attached to the overlying neural lamella by hemidesmosomes, and to each other by occasional gap and tight junctions which persist throughout development. They are attached by desmosomes to a thin underlying type II cell layer, which is joined by gap and tight junctions and which has desmosomal attachments with the underlying glial membranes. The larval axons are surrounded by multiple glial wrappings containing bundles of microtubules. During the first week after larval-pupal ecdysis, the neural lamella degenerates and is phagocytosed by invading haemocytes. The underlying perineurial I cells gradually become hypertrophied and vacuolated. At the same time the type II layer, which does not increase in size, appears to be composed of either one or two cells which form a continuous ‘bracelet’ around each connective. The cellular bracelet is joined at one or two places by extensive gap, tight and septate junctions, and gap junctions are also seen along its perineurial I and glial borders. The underlying axons are embedded in vast amounts of glial cytoplasm containing relatively few microtubules. During the second week after larval-pupal ecdysis, the neural lamella is reformed and the perineurium flattens again. Type I and II cell junctions remain as described in earlier stages. Before adult emergence, the axons are again wrapped by glial cells rich in microtubules.

The connective tissue sheath surrounding the nervous system of Locusta migratoria has been studied histochemically. It consists of an outer non-cellular layer, the neural lamella, and an inner layer of cells, the sheath-cells. The neural lamella has been identified as being composed of a collagen-type protein and neutral mucopolysaccharide on the evidence of its histochemical reactions and the identification of hydroxyproline by paper chromatography in a hydrolysate of the neural lamella. The sheath-cells possess large numbers of lipochondria composed of phospholipids and cerebrosides, and small spherical mitochondria. The cytoplasm also contains lipids (some of which may be cerebrosides), glycogen, and RNA.

The embryological development of the connective tissue sheath around the nervous system has been investigated in Schistocerca gregaria. The sheath cells appear to be derived from outlying ganglion cells. The neural lamella is first visible when the embryo is 9 days old and it increases in thickness until hatching occurs on the twelfth day. It is produced entirely by the sheath cells. The sheath cells have numerous lipid droplets in their cytoplasm. Some neutral mucopolysaccharide and proteins are also present. The histochemical reactions of the neural lamella after its formation suggest that it is composed of collagenous proteins embedded in neutral mucopolysaccharides. The sheath cells are typical fibroblasts during the formation of the neural lamella. The cisternae of the endoplasmic reticulum are dilated into vesicles which contain a somewhat electron-dense material. No intracellular fibrils were observed. Collagen fibrils with banding of periodicity between 55 and 60 mµ. are seen in the neural lamella from 11 days onwards.

The body-wall of adult Euchromadora vulgaris is composed of the 3 layers common to all nematodes, the cuticle, epidermis, and muscle cells. The cuticle is composed of 4 layers, a thin membrane resolvable only by the electron microscope, and 3 layers which can be observed in the light microscope. Histochemical tests show that the cuticle is predominantly protein and contains collagen. Of the 3 main layers of the cuticle, the outermost is about 0.4µ, thick and it is penetrated at regular intervals by grooves which divide the cuticle into annuli. This layer has several features in common with the external cortical layer of the cuticle in Ascaris lumbricoides; it is hardened by disulphide bonds and possibly quinone tanning, and is resistant to collagenase. The middle layer is about 1 to 1.5µ thick and is formed from a series of overlapping plates. The rod-like bodies of de Man are located in this layer and are hollow. Internally, the cuticle is bounded by a basal lamella about 0.2µ thick. The epidermis is thickened to form 4 chords and is composed of a large number of cells, which contain filamentous mitochondria with many cristae, granules of glycogen, and, in the pharyngeal region, pigment spots. The fibrillar zone of the muscle cell contains myofilaments of two types, large filaments 20 to 25 mµ in diameter, which are surrounded by smaller filaments 5 to 7 mµ in diameter. There are filamentous mitochondria, glycogen and a nucleus in the protoplasmic bulb. Covering the muscle cell is a thin membrane, the sarcolemma, which is infolded at regular intervals between groups of myofilaments. The sarcolemma is fused with the basal cuticular layer at both ends of each muscle cell.

The connective-tissue sheath surrounding the nervous system of Periplaneta americana consists of two layers, the neural lamella and the sheath cells beneath it. The neural lamella is composed of a collagen-type protein and neutral muco-polysaccharide. The sheath cells possess numerous lipochondria and mitochondria; the former consist of phospholipid and some cerebroside. The cytoplasm of the sheath cells contains some RNA, glycogen, and lipid.

The sheath is composed of an outer non-cellular layer, the neural lamella, and an inner layer of sheath cells. The neural lamella possesses a large number of collagen fibrils arranged in layers with differing orientations. The sheath cells are flattened on the inner surface of the lamella and the cytoplasm contains lipochondria, mitochondria, and small amounts of endoplasmic reticulum.

Several aspects of the behaviour of polyoma virus-transformed BHK cells in culture have suggested that they are more deformable than BHK cells. This possibility was tested by applying negative pressure at the cell surface by means of a micropipette. It was found that PyBHK cells in early mitosis are twice as deformable as BHK cells in the same stage. In addition, the taut, non-ruffling margins of both cell types when fully spread are much less deformable than the extending, ruffling leading lamella. The degree of deformability of these cells is correlated with the distribution and organization of microfilaments and, consistent with this, deform-ability increases greatly in the presence of cytochalasin B. The significance of deformability studies such as these is discussed.

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 α-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.

Volume/osmotic pressure relationships were compared in Xenopus epidermal cells treated with the calcium antagonist lanthanum and in control cells. In lanthanum-treated cells the volume was found to be proportional to the reciprocal of the osmotic pressure in the hypotonic range, but not in the control cells. This difference is interpreted as being caused by contractions of the actomyosin system in control cells, which require external calcium. On the basis of the different slopes of the measurements under these two conditions the contractile force of the cells in isotonic conditions was calculated to be 0.96 × 10(5)Nm-2. The observation that in hypotonic media the cell body is enlarged in the direction of movement provides evidence that the cell body/lamella transition region is the part of the cell that contracts most during locomotion. By analysing cell speed in media with varying osmotic pressures, the relationship between internal pressure and cell locomotion was revealed.