Cortical Actin Network Research Articles

963-18 Reoxygenation Following Hypoxia Induces the Reorganization of the Actin Cytoskeleton of Endothelial Cells

Ischemia can induce an angiogenic response resulting in physiologically significant collateral vascularization of the myocardium. Angiogenesis requires the migration of endothelial cells, a process which depends upon the dynamic remodeling of the actin cytoskeleton. In a model of cultured bovine aorta endothelial cells exposed to hypoxic conditions (pO2 decreasing to 10 ± 5 Torr over 4 hours at 37°) and reoxygenated for up to 45 minutes, we studied the actin cytoskeleton response to changes in pO2. In these conditions, at the end of hypoxia (Hypo), the relative concentration of filamentous actin and the distribution of the actin stress fibers were unaltered. However, reoxygenation (Reox) of the cells after 4 hours of Hypo induced within 5 minutes a remarkable reorganization of the actin cytoskeleton with rapid polymerization of actin filaments leading to an increase in filamentous actin of 41% above normoxia (Normo) level. Concurrently with the actin polymerization, redistribution of the actin filaments to the cortical actin network and disappearance of central actin stress fibers were observed. Similar results were obtained using human aorta endothelial cells. Conditions Normo Hypo SEM Reox: 5’ SEM 45’ SEM Control 100% 101% ± 11% 141% ± 12% 127% ± 12% Genistein 100% 104% ± 7% 101% ± 10% 105% ± 16% Genistein (300 μ M), an established tyrosine kinase inhibitor, prevented the actin changes induced by Reox, suggesting the participation of tyrosine kinase pathway(s) in the reorganization of the actin cytoskeleton induced by Reox. Considering the pivotal role of the actin cytoskeleton in cell motility, Reox-induced actin changes may represent the first step of a phenotypic shift leading to endothelial cell migration required for the genesis of blood vessels.

37] Cell motility assay and inhibition by Rho-GDP dissociation inhibitor

This chapter describes the cell motility assays based on phagokinetic and scattering activities. Phagokinetic activity is estimated by measuring cell tracks formed by the phagocytosis of colloidal gold particles due to migrating cultured cells, and scattering activity is estimated by observing the scattering of the epithelial cells growing in colonies by phase contrast microscopy. The chapter describes the methods for microinjection of Rho– GDP-Dissociation Inhibitor (GDI) or C3, both of which inhibit cell motility by the cell motility assays. Cell motility is essential for inflammatory reactions, tissue repair, and immune system interactions in normal cells. Cell motility is essential for malignant cancer cells to infiltrate surrounding tissues and for smooth muscle cells in the media to migrate to the intima at the atherosclerotic region of vascular cells. The actomyosin system is involved in cell motility. In cultured cells, actin filaments exist principally in three types of structures: the cortical actin network, actin stress fibers, and cell surface protrusions, including membrane ruffling and microspikes. Membrane ruffling, which is caused by the polymerization of actin at the inner surface of the plasma membrane, is associated with cell motility in cultured cells.

ATP depletion: a novel method to study junctional properties in epithelial tissues: cytoskeleton: I. Rearrangement of the actin

The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction's role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.

ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K(+)-ATPase and E-cadherin.

MDCK and JTC cells were subjected to ATP depletion by treating the cells with 10 microM antimycin A and 10 mM 2-deoxyglucose. As visualized by confocal fluorescence microscopy, E-cadherin and Na+,K(+)-ATPase were rapidly internalized following depletion of the intracellular ATP stores. The time course of internalization was similar to the depolymerization of the cortical actin network and dissolution of the actin ring (see companion paper, this volume, pp. 3301-3313). Cell surface biotinylation was used to assay the amount of surface-accessible E-cadherin and Na+,K(+)-ATPase during ATP depletion. At 30 minutes of ATP depletion, 74% and 69% of E-cadherin and Na+,K(+)-ATPase were internalized, respectively, in MDCK cells. By 60 minutes of ATP depletion, internalization increased to 95% and 89%, respectively. The redistribution of both plasma membrane proteins was not microtubule dependent. Similar results were observed in JTC cells. Total biotinylated protein decreased by 67% and 82%, after 30 minutes and 60 minutes of ATP depletion, respectively. The E-cadherin internalization strongly suggests that disruption of adherens junctions occurred following ATP depletion. These results, along with the previously described loss of tight junction integrity, suggest that ATP depletion may be a useful method to study the assembly and disassembly of junctional complexes in epithelial cells.

Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium.

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.

Ponticulin is an atypical membrane protein.

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias Studies on living cells

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.

Alternative pathway activation of complement by cultured human proximal tubular epithelial cells

Human proximal tubular epithelial cells (PTEC) incubated with normal human serum (NHS) were found to fix on their surface C3, properdin, terminal complement components and C5b-9 MAC neoantigen, but not C1q and C4, by immunofluorescence. Complement fixation was abrogated if PTEC were incubated with EDTA-treated NHS or C3-deficient human serum, but not with Mg EGTA-treated NHS or C1q-deficient human serum, showing the prevalent activation of the alternative pathway of complement. This event was followed by marked cytoskeleton alterations with disruption of the actin cortical network, redistribution of actin throughout the cytoplasm and formation of blebs, and by cell cytolysis. In addition, superoxide anion and hydrogen peroxide production and chemiluminescence response were detected in consequence of MAC insertion on PTEC plasma membrane. The dependency on MAC of the observed biological effects of complement fixation on PTEC surface was shown by using sera selectively deficient of terminal components of complement (C6 or C8), and therefore unable to form the C5b-9 MAC, and by restoring the ability to form MAC after addition of purified C6 or C8. The possible pathogenetic relevance of these observations in tubulointerstitial injury occurring in patients with complementuria due to non-selective proteinuria, is discussed.

Microtubule-associated protein 2 and the organization of cellular microtubules

Microtubule-associated proteins (MAPs) are prominent components of the neuronal cytoskeleton that can promote microtubule formation and whose expression is under strong developmental regulation. They are thought to be involved in organizing the structure of microtubule fascicles in axons and dendrites, although whether they form active cross-links between microtubules or serve as strut-like spacer elements has yet to be resolved. In the experiments reported here we explored their influence on microtubules by expressing them in non-neuronal cells using DNA transfection techniques. We confirm earlier reports that microtubule-associated proteins of the MAP2/tau class can induce bundling of microtubules. In addition we find that MAP2 causes the rearrangement of microtubules in the cytoplasm in a manner that is dependent on the length of the microtubule bundles. Short bundles are straight and run across the cytoplasm whereas long bundles form a marginal band-like array at the periphery. We suggest that the latter arrangement is produced when microtubule bundles that are too long to fit inside the diameter of the cell bend under the restraining influence of the cortical cytoskeleton. In confirmation of this, we show that when the cortical actin network is depolymerized by cytochalasin B the MAP2-containing microtubule bundles push out cylindrical extensions from the cell surface. These results suggest that the induction of stiff microtubules bundles by MAP2, coupled with a breach in the cortical actin network, can confer two of the properties characteristic of neuronal processes; their cylindrical form and the presence of fasciculated microtubules.

Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans.

We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans.

Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells

Blockade of mevalonate synthesis by the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL-5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL-5 cells also lose vinculin-related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non-sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability.

Reorganization of actin and myosin II in Dictyostelium amoeba during stimulation by cAMP.

The distribution of actin and myosin II was studied by immunofluorescence microscopy in Dictyostelium discoideum cells that had been stimulated with a chemoattractant, cAMP. The amounts of actin but not of myosin II increased in the cortical region of cells after 5-10 seconds of stimulation by cAMP at 21 degrees C (the first peak). After a transient decrease in the amount of actin in the cortical region, the amounts of both actin and myosin II increased in the cortical region after 25-35 seconds of stimulation (the second peak). The amounts of myosin II decreased in the cortical region thereafter but actin became localized in the pseudopods. The stimulated cells extended many short projections that contained actin at the time of the first peak and the cell bodies contracted at the time of the second peak. Foci in the cortical actin network decreased in number at 20-30 sec and recovered thereafter. A quantitative examination using a fluorocytometer revealed that the amounts of actin filaments in the cells increased at both peaks. The calcium ionophore A23187 and Ca2+ ion induced the reorganization of actin and myosin II to a certain extent. Furthermore, Ca2+ ions inhibited the return of myosin II to the endoplasm in cAMP-stimulated cells after preincubation with A23187. Thus, Ca2+ ions may play a dual role, being involved in the regulation of both the initiation and the termination of the reorganization of cytoskeletons. The cAMP-stimulated responses of cytoskeletons were not inhibited by EGTA, La3+ ions, or ruthenium red but were inhibited by preincubation with BAPTA-AM.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation.

Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.

Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the initial rate of exocytosis in response to nicotinic receptor stimulation

Nicotinic stimulation and high K + depolarization of bovine chromaffin cells cause disassembly of cortical filamentous actin networks. Previous work from our laboratory has demonstrated that disassembly of actin filaments is Ca 2+-dependent, precedes exocytosis and occurs in cortical areas of low cytoplasmic viscosity which are the sites of exocytosis. It has also been suggested that protein kinase C is involved in catecholamine secretion from chromaffin cells. Therefore, the possibility that protein kinase C activation might be implicated in cortical filamentous actin disassembly was investigated. Here we report that phorbol myristate acetate, a protein kinase C activator, causes cortical filamentous actin disassembly. Short-term phorbol ester treatment does not alter the morphology of chromaffin cells; however, l h after phorbol ester exposure an increase in cell flattening and membrane ruffling is observed. Phorbol ester-induced cortical filamentous actin disassembly is inhibited by protein kinase C activity inhibitors, is independent of extracellular Ca 2+ and has a slower time course than that induced by either nicotinic receptor stimulation or K +-depolarization. Phorbol ester effects are likely to be mediated by activation of protein kinase C and not by any changes in intracellular Ca 2+ levels, as indicated by measurements of Ca 2+ transients. Pretreatment of chromaffin cells with phorbol myristate acetate increases the initial rate of nicotine-evoked catecholamine release. Nicotine-induced cortical actin filament disassembly and catecholamine secretion are partially (29–40%) inhibited by pretreatment of cells with either calphostin C, staurosporine or sphingosine. The results suggest that protein kinase C may be involved in the reorganization of the cortical actin filament network priming the cells for release by removing a barrier to secretory granule mobility. However, its role in exocytosis is modulatory but not essential.

Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.

Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network.

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells.

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

Astrocyte process growth induction by actin breakdown.

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.

Actin filaments regulate epithelial Na+ channel activity.

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

Calcium signalling and the triggering of secretion in adrenal chromaffin cells

The pivotal intracellular message for triggering catecholamine release from bovine adrenal chromaffin cells is an elevation in the concentration of cytosolic free Ca 2+ ([Ca 2+] i). Studies using video-imaging techniques have shown that a rise in [Ca 2+] i at the cell periphery, that is due to Ca 2+ entry, is the major activating signal for exocytosis. The cytoskeleton has been identified as a major regulatory site of exocytosis, with Ca 2+-induced disruption of the cortical actin network being required in order that previously restrained granules may have access to their exocytotic sites. The Ca 2+- and phospholipid-dependent annexin protein, calpactin, has been strongly implicated in a late stage of interaction between granules and the plasma membrane by both ultrastructural and biochemical studies.