Increased Perviousness on CT for Acute Ischemic Stroke is Associated with Fibrin/Platelet-Rich Clots.

BACKGROUND AND PURPOSE: Clot perviousness in acute ischemic stroke is a potential CT imaging biomarker for mechanical thrombectomy efficacy. We investigated the association among perviousness, clot cellular composition, and first-pass effect. MATERIALS AND METHODS: In 40 mechanical thrombectomy-treated cases of acute ischemic stroke, we calculated perviousness as the difference in clot density on CT angiography and noncontrast CT. We assessed the proportion of fibrin/platelet aggregates, red blood cells, and white blood cells on clot histopathology. We tested for linear correlation between histologic components and perviousness, differences in components between "high" and "low" pervious clots defined by median perviousness, and differences in perviousness/composition between cases that did and did not achieve a first-pass effect. RESULTS: Perviousness significantly positively and negatively correlated with the percentage of fibrin/platelet aggregates (P = .001) and the percentage of red blood cells (P = .001), respectively. Higher pervious clots had significantly greater fibrin/platelet aggregate content (P = .042). Cases that achieved a first-pass effect (n = 14) had lower perviousness, though not significantly (P = .055). The percentage of red blood cells was significantly higher (P = .028) and the percentage of fibrin/platelet aggregates was significantly lower (P = .016) in cases with a first-pass effect. There was no association between clot density on NCCT and clot composition or first-pass effect. Receiver operating characteristic analysis indicated that clot composition was the best predictor of first-pass effect (area under receiver operating characteristic curve: percentage of fibrin/platelet aggregates = 0.731, percentage of red blood cells = 0.706, perviousness = 0.668). CONCLUSIONS: Clot perviousness on CT is associated with a higher percentage of fibrin/platelet aggregate content. Histologic data and, to a lesser degree, perviousness may have value in predicting first-pass outcome. Imaging metrics that more strongly reflect clot biology than perviousness may be needed to predict a first-pass effect with high accuracy.

Phototoxicity and photoinactivation of blebbistatin in UV and visible light.

Blebbistatin was recently identified as a selective, cell-permeant inhibitor of myosin II. Because blebbistatin is likely to be used extensively with fluorescence imaging in studies of cytoskeletal dynamics, its compatibility with common excitation wavelengths was examined. Illumination of blebbistatin-treated bovine aortic endothelial cells at 365 and 450-490 nm, but not 510-560 or 590-650 nm, caused dose-dependent cell death. Illumination of blebbistatin alone at 365 and 450-490 nm changed its absorption and emission spectra, but the resultant compounds were not toxic. In addition, photoreacted blebbistatin no longer disrupted myosin distribution in cells, indicating loss of pharmacological activity. Fluorescence microscopy showed that upon illumination, blebbistatin became bound to cells and to protein-coated glass, suggesting that toxicity may arise from light-induced reaction of blebbistatin with cell proteins. Blebbistatin should be used only with careful consideration of these photochemical effects.

Turnover rates at regulatory phosphorylation sites on myosin II in endothelial cells.

Assembly and motor activity of non-muscle myosin II can be regulated by phosphorylation. Because myosin II-containing structures undergo continuous assembly, disassembly, and remodeling in living cells, especially during cell migration, myosin II should undergo frequent phosphorylation and dephosphorylation. This study examines the turnover of phosphate on myosin II in stationary and migrating endothelial cells. Cultured bovine aortic endothelial cells were metabolically labeled with (32)P-phosphate, and the incorporation of phosphate into myosin II was assessed by quantitative phosphor imaging of electrophoretic gels of myosin II immunoadsorbed from cell lysates. Likewise, phosphate turnover was measured upon chasing the (32)P with unlabeled phosphate. Phosphate incorporated very slowly into heavy chains, taking >8 h to plateau, and turned over at

Regulatory light chain phosphorylation and the assembly of myosin II into the cytoskeleton of microcapillary endothelial cells.

During the crawling movements of non-muscle cells, myosin II-containing structures assemble and disassemble with a high degree of spatial and temporal heterogeneity. In order to understand how this is controlled, we examined factors that influence the association of myosin II with detergent-resistant cytoskeletons of cultured endothelial cells. Treatment of cells with 0.05% Triton X-100 in an actin-stabilizing buffer released approximately 42% of the myosin II from the cytoplasm. Most remaining myosin II was dissociated from the cytoskeleton by treatment with ATP or AMPPNP, but not ADP, suggesting that myosin II is retained as ATP-sensitive filaments or via rigor-like binding to F-actin. Disruption of actin filaments with cytochalasin or latrunculin prior to detergent permeabilization sharply decreased the amount of myosin II retained, suggesting the latter type of association. Because phosphorylation of myosin II affects filament assembly and actin binding in vitro, phosphorylation levels in soluble and cytoskeletal myosin II were measured. Phosphorylation of myosin heavy chains was not significantly different between the two fractions, but regulatory light chains of cytoskeletal myosin II were 5 times more phosphorylated than in soluble myosin II. Tryptic-peptide mapping showed that cytoskeletal light chains were phosphorylated predominantly at serine 19/threonine 18, which regulates myosin II assembly in vitro, whereas soluble light chains were not phosphorylated or were phosphorylated at threonine 9. Treating cells with the kinase inhibitor, staurosporine, prior to permeabilization decreased light-chain phosphorylation with concomitant reduction in myosin retention. These observations suggest that assembly of myosin II into cytoskeletal structures, where it can generate and resist forces, is regulated in vivo by phosphorylation of myosin light chains at serine 19/threonine 18.

Cytoplasmic dynamics of myosin IIA and IIB: spatial 'sorting' of isoforms in locomoting cells.

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to 'sort' within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells.

Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells.

Asymmetry in the distribution of free versus cytoskeletal myosin II in locomoting microcapillary endothelial cells.

Myosin II is required for normal amoeboid locomotion. In order to understand how myosin II elicits its effects on locomotive behavior, we have mapped myosin II-cytoskeleton interactions in locomoting endothelial cells. Bovine microcapillary endothelial cells were microinjected with fluorescently labeled myosin II, and the distribution of myosin II was imaged in the living cells by fluorescence microscopy. The same cells were then permeabilized with Triton X-100 and imaged again. The second set of images showed only myosin II that was associated with detergent-insoluble cytoskeleton. Dividing the image of retained myosin II by that of total myosin II produced a map of the extent to which myosin II was associated with the detergent-resistant cytoskeleton at any point in the cell. In cells migrating at the edge of a scrape wound, myosin II was preferentially retained in a region approximately 10 microm wide located just behind the cells' leading lamellipodia. Relatively little myosin II was retained in perinuclear cytoplasm. A vector representation of the distribution of total versus retained myosin II demonstrated that myosin II retention was sharply polarized with respect to locomotion, favoring the front of migrating cells. Myosin II-enriched cytoskeleton in this region may help polarize protrusive activity and/or move cytoplasmic bulk forward. Patches of myosin II retention were also observed in adherent tails of many cells, consistent with a role in pulling the rear of the cell forward.

Potential of machine-vision light microscopy in toxicologic pathology.

Major developments in machine-vision light microscopy and in reagent chemistry have led to a renaissance and revolution in the use of the light microscope in biology, biotechnology, and medicine. The potential use of this technology in the field of toxicologic pathology is discussed. It is suggested that a combination of investigating living cells and tissues and fixed samples using the new technologies will lead to understanding mechanisms of toxicity. Examples of the use of the methods in basic cell biology and medicine are presented.

Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport.

Assembly and motor activity of myosin II affect shape, contractility, and locomotion of nonmuscle cells. We used fluorescent analogues and imaging techniques to elucidate the state of assembly and three-dimensional distribution of myosin II in living Swiss 3T3 fibroblasts. An analogue of myosin II that was covalently cross-linked in the 10S conformation and unable to assemble served as an indicator of the cytoplasmic volume accessible to 10S myosin II. Ratio-imaging of an analogue that can undergo 10S-->6S conversion versus the volume indicator revealed localized concentration of assembly-competent myosin II. In stationary serum-deprived cells and in cells locomoting at the edge of a wound, it was most concentrated in the peripheral cytoplasm, where fibers containing myosin II assemble, and least concentrated in the perinuclear cytoplasm, where they disassemble. Furthermore, fluorescence photobleaching recovery showed myosin II to be less mobile in the periphery than in perinuclear cytoplasm. These results indicate a gradient in the assembly of myosin II. Three-dimensional microscopy of living cells revealed that fibers containing myosin II were localized in the cortical cytoplasm, whereas myosin II was diffusely distributed in the deeper cytoplasm, suggesting that myosin II is assembled preferentially near the cell surface. Localized protein phosphorylation may play a role, because a kinase inhibitor, staurosporine, abolished the gradient of myosin II assembly.

Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.

Characterization of a keratinocyte-specific extracellular epitope of desmoglein. Implications for desmoglein heterogeneity and function.

Despite the presumed importance of desmoglein, a 160-kDa glycoprotein, in desmosome formation and its possible involvement in certain blistering skin diseases, the precise location and function of this protein have not yet been firmly established. We describe here the characterization of a new monoclonal antibody, AE23, against an extracellular epitope of desmoglein. Both the AE23 epitope and another epitope, defined by the previously characterized DG3.4 antibody, reside on a 160-kDa human epidermal desmoglein as evidenced by their identical solubility profile, their coexistence in a 130-kDa desmoglein degradative product, their coadsorption by an AE23 immunoaffinity column, and the identical changes in the two antigens' electrophoretic mobility after air oxidation and deglycosylation. The AE23 epitope is resistant to various endoglycosidases, suggesting that sugar moieties are not involved. Characterization of several proteolytic fragments of this epidermal desmoglein enabled us to map the DG3.4 epitope to a 96-kDa intracellular domain and the AE23 epitope to an extracellular domain flanked by the plasma membrane and the distal N-glycosylation site(s). However, these two epitopes do not always coexist on the same desmoglein molecule. For example, tissue surveys showed that although the DG3.4 epitope is present in the desmogleins of all epithelial cell types, the AE23 epitope is limited to normal keratinocytes. Moreover, electron microscopic localization data indicate that whereas the DG3.4 epitope is detected in the submembranous plaques of desmosomes, the AE23 epitope is present in the intercellular space of both desmosomal and nondesmosomal areas. These results raise the possibility that there exist several biochemically closely related isoforms of desmoglein, one (AE23+/DG3.4+) restricted to epidermal desmosomes, one (AE23+/DG3.4-) uniformly distributed along the keratinocyte cell surface, and another (AE23-/DG3.4+) present in desmosomes of simple epithelia and basal cells of cultured keratinocytes. The uniform distribution of at least one desmoglein-related antigen in the intercellular space of keratinocytes coupled with the realization that different isoforms of desmogleins form a subfamily of cadherins suggest that desmoglein(s) may play a more general role in keratinocyte adhesion than previously appreciated.

The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells.

Serum-deprived Swiss 3T3 fibroblasts constitutively form stress fibers at their edges. These fibers move centripetally towards the perinuclear region where they disassemble. Serum stimulation causes shortening of fibers in a manner suggesting active actin-myosin-based contraction (Giuliano, K.A. and D.L. Taylor. 1990. Cell Motil. and Cytoskeleton. 16:14-21). To elucidated the role of actin-based gel structure in these movements, we examined the effects of disrupting actin organization with cytochalasin. Serum-deprived fibroblasts were microinjected with rhodamine analogs of actin or myosin II and fiber dynamics were monitored with a multimode light microscope workstation using video-enhanced contrast and fluorescence modes. When cells were perfused with greater than or equal to 3 microM cytochalasin B or 0.5 microM cytochalasin D, formation and transport of stress fibers were reversibly inhibited, and rapid and immediate shortening of existing fibers was induced. Quantification of actin and myosin II fluorescence associated with individual shortening fibers demonstrated that fluorescence per length of fiber increased for both components, suggesting sliding filament contraction. However, there was also a net loss of both actin and myosin II from fibers as they shortened, indicating a self-destructive process. Loss of material from fibers coupled with increased overlap of actin and myosin II remaining in the fibers suggested that contraction could be induced not only by increasing the force exerted by contractile motors, but also by decreasing gel structure through partial solation. Finally, cytochalasin accelerated contraction of actin-myosin-based gels reconstituted from purified proteins in the absence of myosin-based regulation, further supporting solation-contraction coupling as a possible mechanism for modulating cytoplasmic contractility (Taylor, D.L. and M. Fechheimer. 1982. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299:185-197).

Modulation of contraction by gelation/solation in a reconstituted motile model.

The actin-based cytoskeleton is a dynamic component of living cells with major structural and contractile properties involved in fundamental cellular processes. The action of actin-binding proteins can decrease or increase the gel structure. Changes in the actin-based cytoskeleton have long been thought to modulate the myosin II-based contractions involved in these cellular processes, but there has been some debate concerning whether maximal gelation increases or decreases contractile activity. To address this question, we have examined how contractile activity is modulated by the extent of actin gelation. The model system consists of physiologically relevant concentrations and molar ratios of actin filaments (whose lengths are controlled by gelsolin), the actin-cross-linking protein filamin, and smooth muscle myosin II. This system has been studied at the macroscopic and light microscopic levels to relate the gel structure to the rate of contraction. We present results which show that while a minimal amount of structure is necessary to transmit the contractile force, increasing the gel structure inhibits the rate of contraction, despite an increase in the actin-activated Mg(2+)-ATPase activity of myosin. Decreasing the total myosin concentration also inhibits the rate of contraction. Application of cytochalasin D to one side of the contractile network increases the rate of contraction and also induces movement comparable to flare streaming observed in isolated amoeba cytoplasm. These results are interpreted relative to current models of the relationship between the state of gelation and contraction and to the potential effects of such a relationship in the living cell.

Tissue-specific distribution of a novel component of epithelial basement membranes.

Monoclonal antibodies against basement membrane (BM) were generated using the matrix deposited by cultured rabbit corneal epithelial cells as immunogen. BM antibodies were identified by immunofluorescent staining of frozen tissue sections and of extracellular matrix of living cultured cells. BM localization was confirmed by immunoelectron microscopy. Antibody AE26 immunoprecipitates a 140,000 Mr component from radiolabeled corneal epithelial cells and recognizes this component plus a 95,000 Mr band on Western blots. The antigen resists extraction by high and low salt and by nonionic detergents, but is solubilized in 4 M urea/1% mercaptoethanol. On isoelectric focusing and nonequilibrium pH gradient gels, AE26 antigen migrates to the acidic region (pI less than 3). The molecule is destroyed by trypsin, but is insensitive to bacterial collagenase. In frozen tissue sections, AE26 stains only BM of stratified epithelia plus trachea, ureter, lung, and intestine, but no other epithelial or nonepithelial BM. AE26 antigen is detected on Western blots of cornea, skin, and lung extracts, but not liver, kidney, or muscle, indicating that this is not due to masking of the epitope. This tissue distribution is different from any previously described BM molecule. Although we have not ruled out the possibility that AE26 recognizes a modification or fragment of a known BM component (particularly entactin), the acidic pI, collagenase resistance, and unusual tissue specificity suggest that AE26 recognizes a new BM protein. The BM heterogeneity demonstrated by AE26 may play a structural role or provide positional signals to the overlying epithelium.

Basement membrane heterogeneity and variation in corneal epithelial differentiation.

We have previously shown that the expression of a major 64-Kda keratin (K3) in corneal epithelium is site-related. It is found suprabasally in limbal epithelium, but uniformly (basal cells included) in central corneal epithelium. In the present study, we used a panel of antibodies against various components of corneal epithelial basement membrane to investigate a possible correlation between basement membrane heterogeneity and differential (basal vs. suprabasal) K3 keratin expression. One of these antibodies, AE27, stains human conjunctival basement membrane weakly, limbal basement membrane heterogeneously, and central corneal basement membrane strongly. Basal cells resting on basement membrane that stains strongly with AE27 tend to stain with monoclonal antibody AE5, which recognizes keratin K3. Basal cells on basement membrane staining weakly with AE27 tend not to stain with AE5. No such correlation exists between AE5 staining and type IV collagen, which is detectable immunohistochemically in conjunctival and limbal basement membrane, but not in corneal basement membrane overlying Bowman's layer. These results suggest that basement membrane of human corneal/conjunctival epithelium can be divided into at least three domains: the conjunctival basement membrane (type IV collagen-positive, AE27-weak), the limbal basement membrane (type IV collagen-positive, AE27-strong), and corneal basement membrane (type IV collagen-negative, AE27-strong). The results also raise the possibility that basement membrane heterogeneity may play a functional role in regulating keratin expression and other aspects of differentiation of corneal epithelium; more experiments are needed to test this hypothesis.

Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture.

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.

The cellular basis of epithelial morphogenesis.

Epithelial tissues are ubiquitous in metazoan organisms, performing many different functions and assuming a variety of shapes. This diversity of form and function is ultimately dependent on the behavior of the cells within the epithelia. For example, it is intercellular adhesion and the control of paracellular permeability by cell junctions that permit epithelia to form barriers and act as selective filters. It is cellular polarity that enables absorptive epithelia to extract materials from a particular side of the sheet; it is the collective contributions of cell proliferation, cellular translocation, and changes in cell shape that sculpt epithelia from simple sheets into folds, pouches and tubes. Clearly, a complete understanding of epithelial morphogenesis is inextricably entwined with questions of cell behavior in general, such as how any cell adheres, moves, and maintains its shape. The study of epithelial systems has lent considerable insight into these problems and should continue to do so, just as examination of the behavior and architecture of nonepithelial cells will undoubtedly clarify many aspects of the cellular events underlying epithelial morphogenesis. Although the action of individual cells ultimately shapes epithelial, coordination of that action is necessary for the development of a coherent tissue. Attention must therefore be given to integrative mechanisms in epithelial morphogenesis. How do the many cells in an epithelial sheet act in virtual unison during folding? What defines the boundaries of epithelial invaginations? How does an individual cell detect its position within, and thereby know its role in the morphogenesis of, the epithelial whole of which it is a part? At the most elementary level, epithelial cells interact via their physical attachments to one other. Even such rudimentary communication affects cell shape, movement, and possibly proliferation and also plays a part in the maintenance of epithelial polarity. Additional signals pass among epithelial cells by a number of other mechanisms as well, most notably electrical coupling. However, many questions remain regarding the quality and quantity of what is communicated between epithelial cells. Accordingly, elucidating the means by which supracellular order is maintained in epithelial tissues may still be regarded as the major problem in the study of epithelial morphogenesis.

Rapid cellular translocation is related to close contacts formed between various cultured cells and their substrata.

Interference-reflection microscopy combined with time-lapse cinemicrography was used to examine the relationship between cell-to-substratum contact patterns and the speeds of translocation for a variety of cell types. Rapid translocation of amphibian leukocytes (average speed = 9.0 micron/min), amphibian epidermal cells (7 micron/min) and teleost epidermal cells (7 micron/min) was found to correlate with patterns of broad grey close contacts. Similar contact patterns were found under freshly seeded (2 h) chick heart fibroblasts (moving 1-3 micron/min), the rapidly advancing (1-5 micron/min) margin of spreading human WI-38 fibroblasts, and isolated MDCK canine epithelial cells (0.5-1.0 micron/min). Conversely, numerous dark streaks of focal contact were found associated with the slow rate of translocation displayed by older cultures (72 h) of chick fibroblasts (less than 0.1 micron/min), well-spread WI-38 cells (less than or equal to 0.3 micron/min) and confluent MDCK cells (less than 0.01 micron/min). It is concluded that close contacts, but not focal contacts, are associated with rapid cellular translocation, and that the build-up of focal contacts is associated with reduced cellular translocation and maintenance of the spread cell shape.

The movement of cell clusters in vitro: morphology and directionality.

The movement of cells in small groups, or clusters, was studied in vitro using epithelioid cells from Gordon-Kosswig melanomas (from poecelid fish) and time-lapse cinemicrography. Tumour explants cultured on glass yield cell sheets from which groups of cells separate and become independently motile clusters. These clusters typically contain 3-30 cells, but may have as many as 50. They propel themselves at speeds of 0.2-4.0 micrometer/min by means of broad hyaline lamellae. The distribution of lamellae around the perimeter of each cluster correlates with both direction and speed of cluster movement, i.e. a cluster moves with its most lamellar region at its leading edge, and the greater the extent of the leading lamellar region the greater the speed. Also, a cluster tends to keep moving in the same direction. This persistence is due to a relatively constant distribution of lamellae. Cells on the trailing edge usually lack lamellae and most are very elongate and oriented perpendicular to the direction of cluster movement. In general, whenever a cell elongates, there is a loss of lamellar activity along its taut edges, parallel to the axis of elongation. Thus, any region with less lamellar activity would tend to be elongated by the outward pull of the more active regions to either side and would, in consequence, suffer a further reduction in lamellar activity. In this way, the distribution of regions of lamellar activity is self-reinforcing and the result is persistence of movement in a particular direction. This phenomenon could play an important role in giving directionality to certain morphogenetic movements, such as neural crest cell migration.