Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+.

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca(2+)-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 microM, elicited rounding (or prevented spreading) of cells cultured for 16-38 h in 0.5-2.0 mM Ca2+. Addition of 0.5-2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 microM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.     

Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.     

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.     

Determination of cell form in cultured fibroblasts : Role of surface components and cytokinetic elements

Functions involved in the determination of the morphology of mouse embryo fibroblasts have been studied using as a system the cell rounding and reflattening related to the presence or absence of a protein component of the cell surface [18]. Cells that had undergone rounding following the removal of the surface protein gradually reflattened if their protein synthesis was not impaired but could rapidly revert to the flattened state if a fraction containing the surface protein was returned to the cells. Studies with sodium azide and potassium cyanide showed that both the rounding and reflattening were energy-dependent. When the rounded state was induced the organised appearance of the microfilaments present in the cortical layer adjacent to the plasma membrane was lost. However, microfilament structures reappeared when rounded cells respread over the glass substrate. On the other hand no reflattening occurred if microfilament function was impaired by treatment with cytochalasin B (CB), showing a requirement of microfilament integrity for the flattened form to be manifest. Microtubules also were shown to be involved in shape determination as their integrity was necessary for cells to progress from the flattened form to the rounded state, but they did not appear to be primarily involved in the process of reflattening. The distribution of macromolecular components of the plasma membrane was also shown to affect changes of cell shape as cross-linking of surface receptors by concanavalin A (ConA) or specific anti-cell immunoglobulins prevented cells from acquiring the rounded form and treatment of cells with media of low pH, which causes aggregation of intra-membrane particles, also prevented rounding. A comparison between quiescent cells and cells in S phase showed the changes of morphology to be similar in nature, but more prompt and marked in the S population. The indication of the studies presented is that determination of cell form requires the cooperative interaction between the protein component of the cell surface, integral constituents of the plasma membrane and cytokinetic organelles.     

Contact relationships between chick embryo cells growing in monolayer culture after infection with Rous sarcoma virus

An electron microscopic examination was made of cell contacts and associated microfilament arrays in subconfluent cultures of chick embryo fibroblasts (CEF) and chick embryo retinal pigmented epithelium cells (RPE) transformed by strains of Rous sarcoma virus (RSV) imparting a rounded (Morph r) or fusiform (Morph f) transformed morphology. A few cell substrate contact specializations were found in Morph r-transformed CEF and RPE cells. These resembled cell/substrate plaques of uninfected fibroblasts, but lacked associated microfilament tracts. In contrast Morph f-transformed CEF and RPE resembled untransformed fibroblasts having well developed cell/substrate and cell/cell contact specializations with extensive associated microfilament arrays. Morph r- and Morph f-transformed RPE cells had lost the junctional complex typical of untransformed RPE cultures and additionally no melanosomes were found. SEM and TEM demonstrated differences in adhesive properties of CEF and RPE cell surfaces, few virions adhering to the free cell surface of RPE cells but being found in clumps and singly on CEF cells.     

Overexpression of SPARC in stably transfected F9 cells mediates attachment and spreading in Ca(2+)-deficient medium.

The Ca(2+)-binding protein SPARC is one of a group of proteins that function in vitro to promote the rounding of cells. To assess whether the modulation of cell shape by SPARC is affected by extracellular Ca2+, we used F9 cell lines that had been stably transfected with sense or antisense SPARC DNA. Sense-transfected (S) lines that overexpress SPARC are aggregated and rounded, whereas antisense (AS) lines that express low levels of the protein are flat and spread. We tested whether the cell lines would exhibit these altered morphologies in Ca(2+)-deficient media. When cultured under these conditions, S lines attached and spread, whereas AS lines attached but remained round, with no subsequent spreading. Addition of CaCl2 or purified SPARC to the Ca(2+)-deficient medium resulted in spreading of the AS and control lines and a reappearance of the altered morphologies. Expression of the Ca(2+)-binding cadherin uvomorulin by the cell lines correlated with neither their morphology nor their level of SPARC expression. We conclude that the altered phenotypes of the transected lines reflect, in part, the concentration of extracellular Ca2+ and that the spreading exhibited by the S lines under Ca(2+)-deficient conditions is directly related to their enhanced expression of SPARC. SPARC might, therefore, mediate interactions between cells and matrix that are permissive for adhesion when levels of extracellular Ca2+ are diminished.     

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.     

High and selective resistance to mecillinam in adenylate cyclase-deficient or cyclic adenosine 3'',5''-monophosphate receptor protein-deficient mutants of Escherichia coli.

Adenylate cyclase-deficient (cya) mutants of Escherichia coli K-12 were selectively and highly resistant to mecillinam (FL1060) among several beta-lactam antibiotics in the absence of cyclic adenosine 3',5'-monophosphate (cAMP). They became sensitive to the drug in the presence of cAMP. Also, cAMP receptor protein-negative (crp) mutants, with the exception of strain 5333, were highly resistant to mecillinam in the presence and in the absence of cAMP. Mecillinam exerted two distinct and sequential effects in both cya+ strains and cya strains supplemented with cAMP: (i) rounding of cells and (ii) cessation of cell division. The first effect was accompanied by a decrease in growth rate, whereas the second effect was accompanied by enlargement and lysis of the rounded cells. The second effect of mecillinam was dependent on inoculum size and cAMP. When the cell density was above about 10(6) cells per ml, the rounded cells stopped dividing but did not lyse. In the absence of cAMP, cya strains neither stopped dividing nor lysed; they were resistant to the second, lethal effect of mecillinam.     

Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells.

The effects of ommission of Ca2+ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaCl2 or MgCl2. Incubation of the cells in the (Ca2+ + Mg2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca2+ and Mg2+ but was not abolished, even in the presence of EDTA. Ca2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg2+, Sr2+ and Ba2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.     

Influence of proteolytic enzymes and calcium-binding agents on nuclear and cell surface topography

Summary Transmission electron microscopy was used to study the effects of proteolytic enzymes (collagenase, trypsin, clostripain), the calcium chelator ethyleneglycol-bis-(-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), and the calcium ionophore A 23187 on substrate adhesion and fine structure of chondrocytes and fibroblasts. Monolayer cultured cells responded to treatment with the proteolytic enzymes followed by EGTA or A 23187 by rounding and detaching from the substrate. This was accompanied by the formation of a microvillous surface, deep nuclear folds, and numerous cytoplasmic vacuoles. Labeling experiments with colloidal thorium dioxide indicated that the vacuoles were formed by endocytosis and fusion of endocytic vesicles with preexisting lysosomes. To a variable extent, similar changes were produced by trypsin or EGTA alone. The cells regained their normal fine structure after withdrawal of the reagents and when seeded onto a substrate. In suspension culture, recovery was incomplete; the cells retained a rounded shape and an increased number of cytoplasmic vacuoles.The results suggest that changes in plasma membrane composition and its permeability to calcium represent the primary signal for cell rounding and detachment. The cellular mechanisms responsible for the associated folding of the nuclear envelope and the cell surface remain unidentified. Nevertheless, this is believed to represent a means of handling of excess membrane during sudden transition from a flattened to a rounded shape. Membrane stored in folds and vacuoles is reutilized when the cells reattach and spread out on a substrate.Expert technical assistance was provided by Karin Blomgren and Anne-Marie Motakefi. Financial support was obtained from the Swedish Medical Research Council (06537), the King Gustaf V 80th Birthday Fund and from the Funds of Leiden University     

The relationship between extracellular K+ and Ca2+ on aminopyrine accumulation in rat parietal cells.

The effects of extracellular K+ in relation to extracellular Ca2+ on acid production were studied. Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. In the absence of K+ in the incubation medium histamine-stimulated AP accumulation ratios were significantly decreased independently in the presence or absence of extracellular Ca2+. Under basal conditions, in the absence of extracellular Ca2+, increasing concentrations of extracellular K+ enhanced AP accumulation ratios to significantly higher than those found in the presence of Ca2+. In histamine-, cAMP-, and carbachol-stimulated parietal cells, high K+ concentrations increased AP accumulation significantly less in Ca(2+)-free than in Ca(2+)-containing media. High K+ also induced significantly both an increase in cytosolic free Ca2+ concentration and 45Ca2+ uptake. The present results confirmed the importance of K+ in gastric acid production and suggested a role for Ca2+ as a modulator of mechanisms of parietal cell stimulation.     

The modulation of cellular contractility and adhesion by trypsin and EGTA

The relationship of cell to substrate and the organisation of the cellular contractile system has been studied using whole-cell mount transmission electron microscopy (TEM). The cellular response to trypsin is compared with that induced by treatment with the Ca²⁺-specific chelating agent EGTA. Trypsinisation results in a breakdown of the ordered contractile system and both cell-cell and cell-substrate adhesion. Studies with the respiratory inhibitor 2,4 dinitrophenol (DNP) have shown that cellular rounding mediated by trypsinisation is energy independent. The response to EGTA is confluence modulated. Confluent monolayers are induced to contract and detach as sheets, a response related here to the overlapping of the peripheral cytoplasm. Subconfluent cells contract centripetally withdrawing organelles to the perinuclear region. Withdrawal of organelles is accompanied by a redistribution of the contractile system. Contraction results in a rounded cell anchored to the substrate by numerous retraction fibrils. The response to EGTA is energy dependent. The observations suggest that a chelation of Ca²⁺ results in an active contraction of the actomyosin system. We propose a mechanism for this response based on ultrastructural and biochemical characterisation of the actomyosin system.     

Enzymatic properties of human aminopeptidase A. Regulation of its enzymatic activity by calcium and angiotensin IV

Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up- or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.     

Mechanically induced electrical and intracellular calcium responses in normal and cancerous mammary cells.

Mechanically induced channel activities and increase of intracellular calcium ([Ca2+]i) in normal and cancerous murine mammary cells (MMT 060562) were investigated using the patch clamp technique and Fura-2 fluorescence. Both cell types showed similar properties. Upon mechanical stimulation, activation of the Ca(2+)-dependent K+ channel or outward membrane current was recorded in cells which were several cells distant from the stimulated cell. Mechanical stimulation also induced an increase of [Ca2+]i in the touched cell, and this increase of [Ca2+]i spread to the surrounding cells. The [Ca2+]i signal travelled a distance of 100-200 microns within 20-40 s and then diminished. The presence of cell-to-cell communication between adjacent mammary cells through gap junction was indicated by injection of lucifer yellow and measurements of electrical coupling (coupling constant = 0.2-0.3). The mechanically induced increase of the [Ca2+]i signal spread to adjacent cells even when the stimulated cell had no physical contact with them. In the absence of fluid movement, the pattern of the spread of the [Ca2+]i signal was a concentric circle. However, in the presence of fluid movement, the pattern changed to elongate to the direction of the flow. These findings suggested that a certain factor was released from the mechanically stimulated cell to the extracellular space, and this factor induces the increase of [Ca2+]i in surrounding cells.     

Local calcium transients contribute to disappearance of pFAK, focal complex removal and deadhesion of neuronal growth cones and fibroblasts

Cell adhesion is crucial for migration of cells during development, and cell-substrate adhesion of motile cells is accomplished through the formation and removal of focal complexes that are sites of cell-substrate contact. Because Ca2+ signaling regulates the rate of axon outgrowth and growth cone turning, we investigated the potential role of Ca2+ in focal complex dynamics. We describe a novel class of localized, spontaneous transient elevations of cytosolic Ca2+ observed both in Xenopus neuronal growth cones and fibroblasts that are 2-6 mum in spatial extent and 2-4 s in duration. They are distributed throughout growth cone lamellipodia and at the periphery of fibroblast pseudopodia, which are regions of high motility. In both cell types, these Ca2+ transients lead to disappearance of phosphorylated focal adhesion kinase (pFAK) and deadhesion from the substrate as assessed by confocal and internal reflection microscopy, respectively. The loss of pFAK is inhibited by cyclosporin A, suggesting that these Ca2+ transients exert their effects via calcineurin. These results identify an intrinsic mechanism for local cell detachment that may be modulated by agents that regulate motility.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

Can retinal adhesion mechanisms determine cell-sorting patterns: a test of the differential adhesion hypothesis

Embryonic chick neural retina cells possess two classes of adhesion mechanism, one Ca2+-independent, one Ca2+-dependent, responsible for short-term cell aggregation. This study investigates the role of these mechanisms in the long-term cell sorting potentially relevant to in vivo histogenesis. Retina cells are prepared either with both (E cells) or with only one mechanism (TC cells, CD; LTE cells, CI), respectively. The two types of cell preparations are differentially labelled using fluorescein or rhodamine isothiocyanate, mixed and allowed to aggregate in the presence or absence of cycloheximide at 0.5 microgram ml-1 to retard metabolic recovery of the removed adhesive mechanism. When observed by fluorescence and phase-contrast microscopy, the aggregates formed in cycloheximide show cell sorting, the cells with both mechanisms assuming a more interior position relative to those with a single adhesion mechanism. In parallel hanging-drop experiments, preformed aggregates of cells with a single adhesion mechanism are seen to spread upon aggregates of cells with both mechanisms. No sorting occurs amongst cells from a given stage prepared using any single dissociation protocol. The observed cell sorting would thus seem to derive exclusively from differential cell adhesiveness dependent upon the different dissociation conditions and maintained in the presence of cycloheximide. The experiments support the hypothesis that the dual CI and CD adhesion mechanisms in question can play a central role in governing cell-sorting behaviour during normal histogenesis.