Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell-cell adhesion molecule, platelet-endothelial cell adhesion molecule-1, (PECAM-1), a member of the lg superfamily, in the modulation of cell migration and cell-cell adhesion. PECAM-1 is a 120-130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell-cell contact. Since endothelial cells express PECAM-1 constitutively, we studied the effects of PECAM-1 on cell-cell adhesion and migration in a null-cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM-1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM-1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM-1 transfectants showed peripheral staining at sites of cell-cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full-length PECAM-1 transfectants migrated more slowly than control cells. Thus, PECAM-1 transfected into a null cell appears to localize to sites of cell-cell contact, promote cell-cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell-cell adhesion molecule in the process of endothelial cell migration.     

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.     

Sequential steps in synaptic targeting of sensory afferents are mediated by constitutive and developmentally regulated glycosylations of CAMs.

Sensory afferents in the leech are labeled with both constitutive and developmentally regulated glycosylations (markers) of their cell adhesion molecules (CAMs). Their constitutive mannose marker, recognized by Lan3-2 monoclonal antibody (mAb), mediates the formation of their diffuse central arbors. We show that, at the ultrastructural level, these arbors consist of large, loosely organized axons rich with filopodia and synaptic vesicles. Perturbing the mannose-specific adhesion of this first targeting step leads to a gain in cell-cell contact but a loss of filopodia and synaptic vesicles. During the second targeting step, galactose markers divide afferents into different subsets. We focus on the subset labeled by the marker recognized by Laz2-369 mAb. Initially, the galactose marker appears where afferents contact central neurons. Subsequently it spreads proximally and distally, covering the entire afferent surface. Afferents now gain cell-cell contact, with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant synaptic vesicles prevail where afferents are apposed to central neurons. These neurons develop postsynaptic densities and en passant synapses are forming. Perturbing the galactose-specific adhesion of this second targeting step causes a loss of cell-cell contact but a gain in filopodia and synaptic vesicles, essentially returning afferents to the first targeting step. The transformation of afferent growth, progressing from mannose- to galactose-specific adhesion, is consistent with a change from cell-matrix to cell-cell adhesion. By performing opposing functions in a temporal sequence, constitutive and developmentally regulated glycosylations of CAMs collaborate in the synaptogenesis of afferents and the consolidation of self-similar afferents.     

Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.

Cadherins, a family of transmembrane cell-cell adhesion receptors, require interactions with the cytoskeleton for normal function. To assess the mechanisms of these interactions, we studied the effect of exogenous expression of a mutant N-cadherin, cN390 delta; on epithelial cell-cell adhesion. The intracellular domain of cN390 delta was intact but its extracellular domain was largely deleted so that this molecule was not functional for cell adhesion. cDNA of cN390 delta was attached to the metallothionein promoter, and introduced into the keratinocyte line PAM212 expressing endogenous E- and P-cadherin. When the expression of cN390 delta was induced by Zn2+, cadherin-dependent adhesion of the transfected cells was inhibited, resulting in the dispersion of cell colonies, although their contacts were maintained under high cell density conditions. In these cultures, cN390 delta was expressed not only on the free surfaces of the cells but also at cell-cell junctions. The endogenous cadherins were concentrated at cell-cell junctions under normal conditions. As a result of cN390 delta expression, however, the endogenous cadherins localizing at the cell-cell junctions were largely diminished, suggesting that these molecules were replaced by the mutant molecules at these sites. As a control, we transfected the same cell line with cDNA of a truncated form of N-cadherin cadherin whose intracellular C terminus had been deleted leaving the extracellular domain intact. This molecule had no effect on cell-cell adhesion, nor did it localize to cell-cell contact sites. We also found that the association of the endogenous cadherins with alpha- and beta-catenins and plakoglobin was not affected by the expression of cN390 delta, which also formed a complex with these molecules, suggesting that no competition occurred between the endogenous and exogenous cadherins for these cytoplasmic proteins. These and other additional results suggest that the nonfunctional cadherins whose intracellular domain is intact occupy the sites where the endogenous cadherins should localize, through interactions with the cytoskeleton, and inhibit the cadherin adhesion system.     

Exosomes: A Bubble Ride for Prions?

In certain cell types, endosomal multivesicular bodies may fuse with the cell surface in an exocytic manner. During this process, the small 50-90-nm-diameter vesicles contained in their lumen are released into the extracellular environment. The released vesicles are called exosomes. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types exploit exosomes as intercellular communication devices for transfer of proteins and lipids between cells. The molecular composition of exosomes is determined by sorting events within endosomes that occur concomitantly with the generation of intraluminal vesicles. As other raft-associated components, the glycosylphosphatidylinositol-linked prion protein transits through multivesicular bodies. Recent findings in non-neuronal cell models indicate prion protein association with secreted exosomes. Thus, exosomes could constitute vehicles for transmission of the infectious prion protein, bypassing cell-cell contact in the dissemination of prions.     

Biochemical characterization and functional analysis of two type II classic cadherins, cadherin-6 and -14, and comparison with E-cadherin

Classic cadherins can be grouped based on their deduced primary structures. Among them the type I cadherins have been well characterized; however, little is known about non-type I cadherins. In this study we characterized two human type II cadherins, cadherin-6 and cadherin-14, using a cDNA transfection system. They were each detected as two bands electrophoretically, were expressed on the external cell surface at cell-cell contact sites, and were associated with caten- ins. Direct sequencing of the N-terminal amino acids showed that the two bands of cadherin-14 corresponded to precursor and mature forms, whereas the two bands of cadherin-6 both had the N-terminal sequence of the mature form. Unlike type I cadherins, both cadherin-6 and -14 were not protected from trypsin degradation by Ca2+. We evaluated their adhesive functions by a long term cell aggregation method. The results suggest that both cadherin-6 and -14 have cell-cell binding strengths virtually equivalent to that of E-cadherin and that their binding specificities are distinct from that of E-cadherin. Cadherin-6 and -14 interacted with each other in an incomplete manner. They have a QAI tripeptide in the first extracellular subdomain instead of the HAV motif that is characteristic of type I cadherins and is intimately involved in the adhesive function. The QAI tripeptide, however, appeared not to be involved in the adhesive functions of cadherin-6 and -14.     

Cortical tension initiates the positive feedback loop between cadherin and F-actin

Adherens junctions physically link two cells at their contact interface via extracellular binding between cadherin molecules and intracellular interactions between cadherins and the actin cytoskeleton. Cadherin and actomyosin cytoskeletal dynamics are regulated reciprocally by mechanical and chemical signals, which subsequently determine the strength of cell-cell adhesions and the emergent organization and stiffness of the tissues they form. However, an understanding of the integrated system is lacking. We present a new mechanistic computational model of intercellular junction maturation in a cell doublet to investigate the mechanochemical cross talk that regulates adherens junction formation and homeostasis. The model couples a two-dimensional lattice-based simulation of cadherin dynamics with a reaction-diffusion representation of the reorganising actomyosin network through its regulation by Rho signalling at the intracellular junction. We demonstrate that local immobilization of cadherin induces cluster formation in a cis-less-dependent manner. We then recapitulate the process of cell-cell contact formation. Our model suggests that cortical tension applied on the contact rim can explain the ring distribution of cadherin and actin filaments (F-actin) on the cell-cell contact of the cell doublet. Furthermore, we propose and test the hypothesis that cadherin and F-actin interact like a positive feedback loop, which is necessary for formation of the ring structure. Different patterns of cadherin distribution were observed as an emergent property of disturbances of this positive feedback loop. We discuss these findings in light of available experimental observations on underlying mechanisms related to cadherin/F-actin binding and the mechanical environment.     

Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes)

The morphological features of pinched-off presynaptic nerve terminals (synaptosomes) from rat brain were examined with electron microscope techniques; in many experiments, an extracellular marked (horseradish peroxidase or colloidal thorium dioxide) was included in the incubation media. When incubated in physiological saline, most terminals appeared approximately spherical, and were filled with small (approximately 400- A diameter) "synaptic vesicles"; mitochondria were also present in many of the terminals. In a number of instances the region of synaptic contact, with adhering portions of the postsynaptic cell membrane and postsynaptic density, could be readily discerned. Approximately 20--30% of the terminals in our preparations exhibited clear evidence of damage, as indicated by diffuse distribution of extracellular markers in the cytoplasm; the markers appeared to be excluded from the intraterminal vesicles under these circumstances. The markers were excluded from the cytoplasm in approximately 70--80% of the terminals, which may imply that these terminals have intact plasma membranes. When the terminals were treated with depolarizing agents (veratridine or K- rich media), in the presence of Ca, many new, large (600--900-A diameter) vesicles and some coated vesicles and new vacuoles appeared. When the media contained an extracellular marker, the newly formed structures frequently were labeled with the marker. If the veratridine- depolarized terminals were subsequently treated with tetrodotoxin (to repolarize the terminals) and allowed to "recover" for 60--90 min, most of the large marker-containing vesicles disappeared, and numerous small (approximately 400-A diameter) marker-containing vesicles appeared. These observations are consistent with the idea that pinched-off presynaptic terminals contain all of the machinery necessary for vesicular exocytosis and for the retrieval and recycling of synaptic vesicle membrane. The vesicle membrane appears to be retrieval primarily in the form of large diameter vesicles which are subsequently reprocessed to form new "typical" small-diameter synaptic vesicles.     

The intracellular movement and cycling of ricin

The binding, internalization and recycling of the plant toxin ricin, was studied using electron microscopy and biochemical techniques. For the electron microscope study, ricin was visualized using a gold-labeled second antibody, in the cells of the EJ human bladder carcinoma line growing in monolayer culture. The labeled antibody/toxin complex was found to enter the cell in coated pits and to accumulate in endosomes and to a lesser extent in vesicles associated with the Golgi system. The complex recycled to the cell surface partly in uncoated vesicles, but largely in multivesicular bodies which appeared to exocytose their contents to the extracellular space. Twenty hours after the initial contact with ricin as much as 50% of the cellular label was found on the cell surface mainly associated with shed vesicles. When cells were treated with unlabeled ricin holotoxin and then after 20 h stained post-fixation, ricin molecules, partly associated with vesicles, were present on the cell surface. Biochemical studies showed that ricin was internalized by cells and then released in an intact form to the extracellular space. It was found that less than 10% of the released material had been degraded during its passage through the cells, which is in accord with the low level of label found in the lysosomal system during the morphological study.     

Evidence against involvement of aquaporin-4 in cell-cell adhesion

Aquaporin-4 (AQP4) water channels are expressed strongly in glial cells, where they play a role in brain water balance, neuroexcitation, and glial cell migration. Here, we investigated a proposed new role of AQP4 in facilitating cell-cell adhesion. Measurements were made in differentiated primary glial cell cultures from wild-type versus AQP4 knockout mice as well as in null versus AQP4-transfected L-cells, a cell type lacking endogenous adhesion molecules, and in null versus AQP4-transfected Chinese hamster ovary (CHO)-K1 cells and Fisher rat thyroid cells. Using established assays of cell-cell adhesion, we found no significant effect of AQP4 expression on adhesion in each of the cell types. As a positive control, transfection with E-cadherin greatly increased cell-cell adhesion. High-level AQP4 expression also did not affect aggregation of plasma membrane vesicles in a sensitive quasi-elastic light-scattering assay. Further, we found no specific AQP4 binding of a fluorescently labeled oligopeptide containing the putative adhesion sequence in the second extracellular loop of AQP4. These data provide evidence against involvement of AQP4 in cell-cell adhesion.     

Nucleation and growth of cadherin adhesions

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.     

Cooperative role of nectin-nectin and nectin-afadin interactions in formation of nectin-based cell-cell adhesion

The nectin cell adhesion molecules interact in trans with each other through their extracellular regions and with afadin through their cytoplasmic tails, forming adherens junctions in cooperation with cadherins. In a single cell, Necl-5 (nectin-like molecule-5) localizes at the leading edge and regulates directional cell movement in response to a chemoattractant. In such a single cell, afadin also localizes at the leading edge without interacting with nectins or Necl-5. It remains unknown how the nectin-nectin and nectin-afadin interactions are initiated when moving cells contact each other to initiate the formation of adherens junctions. We show here that the Necl-5-nectin interaction induced by cell-cell contact enhances the nectin-afadin interaction. This interaction then enhances the nectin-nectin interaction, which further enhances the nectin-afadin interaction in a positive feedback manner. Thus, the Necl-5-nectin, nectin-nectin, and nectin-afadin interactions cooperatively increase the clustering of the nectin-afadin complex at the cell-cell contact sites, promoting the formation of the nectin-based cell-cell adhesion.     

Sequential steps of carbohydrate signaling mediate sensory afferent differentiation

Differences in carbohydrate signaling control sequential steps in synaptic growth of sensory afferents in the leech. The relevant glycans are constitutive and developmentally regulated modifications of leechCAM and Tractin (family members of NCAM and L1) that are specific to the surface of sensory afferents. A mannosidic glycosylation mediates the dynamic growth of early afferents as they explore their target region through sprouting sensory arbors rich with synaptic vesicles. Later emerging galactosidic glycosylations serve as markers for subsets of the same sensory afferents that correlate with different sensory modalities. These developmentally regulated galactose markers now oppose the function of the constitutive mannose marker. Sensory afferents gain cell-cell contact with central neurons and self-similar afferents, but lose filopodia and synaptic vesicles. Extant vesicles are confined to sites of en passant synapse formation. The transformation of sensory afferent growth, progressing from mannose- to galactose-specific recognition, is consistent with a change from cell-matrix to cell-cell contact. While the constitutive mannosidic glycosylation promotes dynamic growth, developmentally regulated galactosidic glycosylations of the same cell adhesion molecules promote tissue stability. The persistence of both types of neutral glycans beyond embryonic age allows their function in synaptic plasticity during habituation and learning.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes

A human autoantiserum (5051) directed against pericentriolar material (PCM) was used to study the distribution of microtubule-organizing centers (MTOCs) in the oocyte and during the first cell cycle of mouse development. In oocytes, the PCM was found not only at the poles of the barrel-shaped metaphase II spindle but also at many discrete loci around the cytoplasm near the cell cortex. The spindle poles were also composed of several PCM foci. In metaphase-arrested eggs only the PCM foci located near the chromosomes acted as MTOCs. However, after reduction of the critical concentration for tubulin polymerization by taxol, the cytoplasmic PCM foci were also found to be associated with nucleation of microtubules. After fertilization the cortical PCM foci remained in a peripheral position until the end of the S phase, when they appeared to migrate centrally towards the pronuclei. At prometaphase of the first mitotic division, numerous MTOCs were found around the two sets of chromosomes; these MTOCs then aligned to form two bands on either side of the metaphase plate of the first mitosis.     

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.     

Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta- catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549- 557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E- cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.     

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.     

Cell-cell communication via extracellular membrane vesicles and its role in the immune response

The host immune response involves a variety of cell types, including specialized immune and non-immune cells. The delicate coordination among these cells via close communication is central for the proper operation of immune system. Cell-cell communication is mediated by a complex network that includes soluble factors such as cytokines, chemokines, and metabolites exported from cells, as well as membrane-bound receptors and their ligands. Cell-cell communication is also mediated by membrane vesicles (e.g., exosomes, ectosomes), which are either shed by distant cells or exchanged by cells that are making direct contact. Intercellular communication via extracellular membrane vesicles has drawn much attention recently, as they have been shown to carry various biomolecules that modulate the activities of recipient cells. In this review, I will discuss current views on cell-cell communication via extra-cellular membrane vesicles, especially shedded membrane vesicles, and their effects on the control of the immune system.