Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

Cytoskeletal organization in locomoting cells of the V2 rabbit carcinoma

The relationship between the organization of cytoskeletal elements and locomotory activity was studied in single cells of the V2 rabbit carcinoma. Like migratory fibroblasts, and unlike colony-forming epithelial cells, these cells show a pronounced horizontal polarization, and develop a large lamella at their leading front. With affinity-purified antibodies and a combination of light and electron microscopic techniques, actin and alpha-actinin (but not myosin and tropomyosin) were found highly concentrated within the marginal region of the leading lamella, both in ruffles and in the underlying zone of contacts with the substratum. Close contacts prevailed in the locomotory cells and small focal contacts developed only in cells detaching from others. Focal contacts always contained small microfilament bundles. Reorganization of actin filaments is suggested as the fundamental event for the dynamic contact formation of the leading lamella. Large microfilament bundles (stress fibers) were absent in all stages of locomotion.Since locomotory behavior and shape changes of V2 cells are the same on glass as on the surface of a natural membrane, the rabbit mesentery, organization and distribution of contractile elements of cultured V2 cells probably reflect the in vivo situation.     

Dorsoventral Differences in Cell-Cell Interactions Modulate the Motile Behaviour of Cells from the Xenopus Gastrula

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum

In this paper I sought to determine how the expression of differentiated traits of chick retinal pigmented epithelial (RPE) cells in vitro can be modulated by varying both the biochemical and the spatial complexity, and the mechanical properties, of the growth substratum. I have used glass derivatized with proteins of a basement membrane extract (nondeformable, two-dimensional substratum) and gels of reconstituted basement membrane extract (viscoelastic, three-dimensional substratum). These two biochemically similar substrata were compared to an inert substratum (untreated glass) and to the native basement membrane of the RPE, i.e., Bruch's Membrane. With immunofluorescence microscopy, I have shown that RPE cells, given space, will spread on their native basement membrane and form stress fibres and focal contacts, analogous to the stress fibres and integrin-, talin-, and vinculin-containing focal contacts of the cells grown on glass. Therefore, the stress fibres and focal contacts present in cultured cells are not artifacts of growth in vitro, but are a natural cellular response to the nondeformability of commonly used tissue culture substrata. The proteins of the basement membrane promote expression of some of the differentiated traits by RPE cells in vitro: however, the fully differentiated phenotype is expressed by RPE cells only when their spreading is prevented by low resilience of a substratum. Basement membrane gels generally are not resilient enough to support RPE cell spreading; however, the cells spread and form stress fibres, and integrin-, talin-, and vinculin-containing focal contacts when they are presented with areas of the gel which locally acquired higher resilience. The extent of cell spreading is determined by the deformability of substratum, hence elastic forces operating within the substratum determine the maximal cell traction allowable and, indirectly, the cytoarchitecture. Therefore, in addition to biochemical composition, the mechanical properties of substrata play important role in regulation of expression of the differentiated phenotype of cells in vitro and, possibly, in vivo.     

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.     

Adhesion to the substratum improves the motility ofAmoeba proteus in the absence of a cell nucleus

Summary Anucleated fragments ofAmoeba proteus obtained by dissection and kept on an untreated glass surface fail to adhere to this substratum, lose motor polarity, and stop moving, at least for several hours. If they are transferred after the operation to a highly adhesive surface (polylysine-coated glass), they adhere to the substratum, although locomotion is not spontaneously restored. However, after exposure to a light-shade difference along their body they start moving towards the shaded area and continue locomotion as long as the photic stimulus is acting. Disorganisation of the F-actin cytoskeleton of anucleated fragments was observed on the untreated glass but reorganization on the polylysine-coated surface. The anucleated fragments can show transient recovery of slight spontaneous motor activity and react promptly to external stimuli after up to several days on untreated glass. These intermittent activity periods are enabled by reconstruction of F-actin cytoskeleton in the anucleated fragments during their temporary adhesion to the glass. It is concluded that the injurious effect of cell nucleus removal on the locomotor capacity of amoebae can be compensated by the simultaneous enhancement of cell adhesion and application of a stimulus restoring the motor polarity of the cell. The compensation is achieved by cytoskeletal reorganization.     

Dorsoventral differences in cell-cell interactions modulate the motile behaviour of cells from the Xenopus gastrula.

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.     

Minipodia, novel structures for extension of the lamella: a high-spatial-resolution video microscopic study.

Extension of leading lamellae has been analyzed by high-spatial- and -time-resolution video microscopy. Many tiny semicircular profiles, hereby named minipodia, have been found at the leading edge of locomoting fibroblasts in culture. These new structures are not "miniruffles" since minipodia seem to originate underneath the leading lamella. Once initiated, minipodia quickly expand and become indistinguishable from what have been known as lamellipodia. The minipodial front becomes the new focal leading edge. So, it appears that leading lamellae do not extend directly themselves, but minipodia protrude underneath them to become new leading edges. These findings are not compatible with the current concept of cell movement, and a new model is proposed.     

3D coupling of fibronectin fibril arrangement with topology of ventral plasma membrane

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from < 25 nm at focal adhesions to 40-50 nm at close contacts and > 80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is > 80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.     

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

Abstract

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from <?25 nm at focal adhesions to 40–50 nm at close contacts and >?80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is >?80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.     

Cell-substratum adhesion of neurite growth cones, and its role in neurite elongation.

The adhesion of chick embryo sensory neurons to glass coverslips was examined with interference reflection optics. On untreated glass, adhesive contacts are common only beneath growth cones and are small. On polylysine-treated glass growth cones are highly spread, microspikes reach treat lengths and extensive adhesive contacts underlie growth cones, microspikes and nerve fibers. Veils, expanded from the growth cone, are adherent to the substratum either centrally or laterally, while the extending edge of the cell margin is non-adherent. Linear adhesions are frequent beneath microspikes and pass centrally beneath the growth cone margin. The distribution of linear adhesions resembles that of microfilament bundles seen within whole mounts of growth cones. Adhesive contacts stabilize extensions of the growth cone margin and may influence the organization of the microfilamentous network within the growth cone. Regulation of microfilament organization by adhesion may influence microfilament functions in growth cone mobility and the assembly of neurite structures.     

Golgi structure in three dimensions: functional insights from the normal rat kidney cell

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.     

Surface movements during the spreading of blood platelets

When human blood platelets spread on a substratum they increase their surface area as much as 4-fold. We investigated the mechanism of spreading by light microscopy and by scanning and transmission electron microscopy. Contact of a platelet with a glass surface induces formation of thin extensions which spread out over the substratum. These extensions resemble the actin-containing microspikes and lammelipodia of tissue cells in culture and appear to be drawn from the peripheral cortical layer associated with the plasma membrane. If platelets are initially labeled on their external surface with cationic ferritin or lentil-conjugated gold particles and then allowed to spread, the labels are retained in the central region, or granulomere. Proteins released by the spreading platelet--fibronectin and fibrinogen--also remain in this central unspread region. Peripheral regions of spread platelet surface (hyalomere) were unlabeled following the above procedures but could be labeled with cationic ferritin or lentil-conjugated gold provided these were applied after spreading was completed. These markers are cleared with time from the periphery, moving centripetally to accumulate at the granulomere. We suggest, on the basis of these observations, that platelets spread onto a substratum by a closely similar mechanism to that used by cells such as fibroblasts. In both cases the spreading involves the peripheral actin cortex and is accompanied by a continual centripetal movement of surface components--a "membrane flow"--which continues even after spreading is completed.     

The ER body,a novel endoplasmic reticulum-derived structure in Arabidopsis

Plant cells develop various endoplasmic reticulum (ER)-derived structures with specific functions. The ER body, a novel ER-derived compartment in Arabidopsis, is a spindle-shaped structure (approximately 10 microm long and approximately 1 microm wide) that is surrounded by ribosomes. Similar structures were found in many Brassicaceae plants in the 1960s and 1970s, but their main components and biological functions have remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein with an ER-retention signal. A large number of ER bodies are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently nai1, a mutant that does not develop ER bodies in whole seedlings, was isolated. Analysis of the nai1 mutant reveals that a beta-glucosidase, called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.     

Effect of calcium on the morphology of human platelets spread on glass substrates

The morphology of human platelets spread on glass substrates is sensitive to the presence of calcium. In the absence of Ca2+, cells spread from buffered salt solution develop radially oriented filopodia and subsequently a broad hyalomere surrounding the central region of the cell from which granules are frequently exocytosed. In the presence of Ca2+ cell rounding and apparent withdrawal from the substrate occurs. Scanning (SEM) and transmission (TEM) electron microscopy of cells rounded in the presence of Ca2+ show fibrous elements connecting the cells to the substratum as well as adherent to the substrate in the vicinity of the rounded cells. Interference reflection microscope (IRM) images of these cells are heterogeneous: some contain small discrete darker regions suggesting the presence of focal specializations at the ventral cell surface. In contrast IRM images of cells spread in the absence of Ca2+ indicate predominantly broad areas of unspecialized contact with the substrate in agreement with TEM observations. These results suggest that Ca2+ may enhance platelet-substrate adhesion by initially promoting the formation of focal specializations which become more pronounced as cell rounding occurs possibly due to Ca2+ activation of an actomyosin-based contractile mechanism.     

Locomotion of sponges and its physical mechanism

Active locomotion by individual marine and freshwater sponges across glass, plastic and rubber substrata has been studied in relation to the behavior of the sponges' component cells. Sequential tracing of sponge outlines on aquarium walls shows that sponges can crawl up to 160 microns/hr (4 mm/day). Time-lapse cinemicrography and scanning electron microscopy reveal that moving sponges possess distinctive leading edges composed of motile cells. Sponge locomotion was found to be mechanically similar to the spreading of cell sheets in tissue culture both with respect to exertion of traction (which causes the wrinkling of rubber substrata) and with respect to the patterns of adhesive contacts formed with the substratum (as observed by interference reflection microscopy). Other similarities include the orientation of sponge locomotion along grooves and the preferential extension onto more adhesive substrata. Neither the patterns of wrinkling produced in rubber substrata nor the distributions of adhesive contacts seen by interference reflection microscopy show evidence of periodic, propagating waves of surface contractions, such as would be expected if the sponges' mechanism of locomotion were by peristalsis or locomotory waves. Our observations suggest that the displacement of sponges is achieved by the cumulative crawling locomotion of the cells that compose the sponge's lower surface. This mode of organismal locomotion suggests new explanations for the plasticity of sponge morphology, seems not to have been reported from other metazoans, and has significant ecological implications.     

Cell-cell contacts alter intracellular pH.

Intracellular pH, an important regulatory factor for many cellular activities, was shown to be modulated by cell adhesion to the solid substratum. In the present work we have shown that cell-cell contacts also affect intracellular pH. pH(i) depends on how many contacts the cell has established with the substratum and the neighboring cells. pH(i) is low in single cells, not contacting each other. It increased with the increase of cell density. pH(i) is again decreased in confluent (topoinhibited) monolayers. pH(i)-shifts triggered by cell-cell contacts seem to be mediated by Na+/H(+)-antiporter. Dependence of pH(i) on cell density could be simulated by different concentration of Arg-Gly-Asp--which is part of the site of extracellular matrix proteins involved in integrin binding. The dependence of pH(i) on cell-cell contacts is discussed in relation to the phenomena of topoinhibition.