RanBP9 at the intersection between cofilin and Aß pathologies: rescue of neurodegenerative changes by RanBP9 reduction.

Molecular pathways underlying the neurotoxicity and production of amyloid ß protein (Aß) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aß production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin-actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aß-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aß-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin-actin pathology, synaptic damage, gliosis, and Aß accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aß accumulation but also in mediating the neurotoxic actions of Aß at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

ADF/Cofilin-actin rods in neurodegenerative diseases.

Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.

Sperm incorporation in Xenopus laevis: characterisation of morphological events and the role of microfilaments.

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells.

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

Calcium-dependent regulation of interactions of caldesmon with calcium-binding proteins found in growth cones of chick forebrain neurons.

1. This study was undertaken to determine if caldesmon, calmodulin, S100beta, and neurocalcin delta were present in chick forebrain neurons, and if so, to investigate the interactions of these proteins in the presence of different concentrations of calcium. 2. Immunocytochemistry was used to determine the presence and localization of these proteins in cultured forebrain neurons. Western blotting, gel electrophoresis in the presence of different concentrations of calcium, chemical cross-linking, and affinity chromatography were used to investigate the interactions of these proteins with each other. 3. Our data show that caldesmon and three calcium-binding proteins (S100beta, calmodulin, and neurocalcin 3) are localized in growth cones and neurites of chick forebrain neurons in culture. In the presence of different concentration of calcium, these calcium-binding proteins have different affinities to caldesmon and to each other. S100beta binds with greater affinity than calmodulin to caldesmon, and its ability to bind to caldesmon is regulated by neurocalcin delta. 4. These findings suggest a specific calcium-dependent regulatory pathway for modulating actomyosin during growth cone motility.

Intracellular pH modulation of ADF/cofilin proteins.

The ADF/cofilin (AC) proteins are necessary for the high rates of actin filament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 and pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation to alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intracellular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocalization analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increases. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), when overexpressed as a GFP chimera, uniquely produces aberrant cellular phenotypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate different phenotypes in response to pH changes. Phospho-AC levels increase with cell density, and in motile cells, phospho-AC increases with alkalization, suggesting a homeostatic mechanism that compensates for increased AC activity and filament turnover. These results show that the behavior of AC proteins with pH-sensitivity in vitro is affected by pH in vivo.

Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.

Inclusions containing actin-depolymerizing factor (ADF) and cofilin, abundant proteins in adult human brain, are prominent in hippocampal and cortical neurites of the post-mortem brains of Alzheimer's patients, especially in neurites contacting amyloid deposits. The origin and role of these inclusions in neurodegeneration are, however, unknown. Here we show that mediators of neurodegeneration induce the rapid formation of transient or persistent rod-like inclusions containing ADF/cofilin and actin in axons and dendrites of cultured hippocampal neurons. Rods form spontaneously within neurons overexpressing active ADF/cofilin, suggesting that the activation (by dephosphorylation) of ADF/cofilin that occurs in response to neurodegenerative stimuli is sufficient to induce rod formation. Persistent rods that span the diameter of the neurite disrupt microtubules and cause degeneration of the distal neurite without killing the neuron. These findings suggest a common pathway that can lead to loss of synapses.

Regulating actin dynamics in neuronal growth cones by ADF/cofilin and rho family GTPases.

Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.

Cdc42 stimulates neurite outgrowth and formation of growth cone filopodia and lamellipodia.

To assess the role of cdc42 during neurite development, cmyc-tagged constitutively active (CA) and dominant negative (DN) cdc42 were expressed in dissociated primary chick spinal cord neurons using adenoviral-mediated gene transfer. Three days after infection, >85% of the neurons in infected cultures expressed cdc42 proteins, as detected by indirect immunofluorescence against cmyc. Growth cones of infected neurons displayed 1.83- (CAcdc42) and 1.93-fold (DNcdc42) higher cmyc immunofluorescence per square micrometer than uninfected controls. CAcdc42 expression stimulated growth cones, almost doubling growth cone size and number of filopodia, and increased neurite growth rates by 65-89%. In neurons plated onto fibronectin, the percent of growth cones with both filopodia and lamellipodia increased from 71 to 92%. Total Texas Red-phalloidin staining in these growth cones doubled, and the percent of growth cones with F-actin localized to peripheral regions increased from 52% in controls to 78% after CAcdc42 expression. Expression of DNcdc42 did not significantly alter growth cone morphology or neurite growth rates. Addition of soluble laminin to spinal cord neurons resulted in the identical phenotype as CAcdc42 expression, including changes in growth cone morphology, F-actin localization, and neurite growth rates. Significantly, expression of DNcdc42 blocked the effects of laminin on growth cones. These results show that cdc42 promotes neurite outgrowth and filopodial and lamellipodial formation in growth cones and suggests that cdc42 and laminin share a common signaling pathway during neurite development. Addition of laminin to CAcdc42-expressing neurons is inhibitory to growth cones, indicating that laminin also may activate some other pathways.

Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor.

Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpressed ADF in primary neurons and analyzed neurite lengths. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cultured rat cortical neurons. Peak expression was approximately 75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected with a green fluorescent protein-expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth.

Regulating actin-filament dynamics in vivo.

The assembly and disassembly (i.e. turnover) of actin filaments in response to extracellular signals underlie a wide variety of basic cellular processes such as cell division, endocytosis and motility. The bulk turnover of subunits is 100-200 times faster in cells than with pure actin, suggesting a complex regulation in vivo. Significant progress has been made recently in identifying and clarifying the roles of several cellular proteins that coordinately regulate actin-filament turnover.

Proteins of the ADF/cofilin family: essential regulators of actin dynamics.

Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.

Putting a new twist on actin: ADF/cofilins modulate actin dynamics.

The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.

Neuronal survival activity of s100betabeta is enhanced by calcineurin inhibitors and requires activation of NF-kappaB.

S100betabeta is a calcium binding, neurotrophic protein produced by nonneuronal cells in the nervous system. The pathway by which it enhances neuronal survival is unknown. Here we show that S100betabeta enhances survival of embryonic chick forebrain neurons in a dose-dependent manner. In the presence of suboptimal amounts of S100betabeta, neuronal survival is enhanced by the immunosuppressants FK506 and cyclosporin A at concentrations that inhibit calcineurin, which is present in these cells. Rapamycin, an immunosuppressant that does not inhibit calcineurin, did not enhance cell survival. Cypermethrin, a direct and highly specific calcineurin inhibitor, mimicked the immunophilin ligands in its neurotrophic effect. None of the drugs stimulated neuronal survival in the absence of S100betabeta. In the presence of suboptimal amounts of S100betabeta, FK506, cyclosporin A, and cypermethrin (but not rapamycin) also increased NF-kappaB activity, as measured by immunofluorescence of cells stained with antibody to the active subunit (p65) and by immunoblotting of nuclear extracts. Antioxidant and glucocorticoid inhibitors of NF-kappaB decreased both the amount of active NF-kappaB and the survival of neurons caused by S100betabeta alone or in the presence of augmenting drugs. We conclude that S100betabeta enhances the survival of chick embryo forebrain neurons through the activation of NF-kappaB.

Cotransport of glyceraldehyde-3-phosphate dehydrogenase and actin in axons of chicken motoneurons.

1. To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [35S]methionine and, 1-20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I-Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography. 2. In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 3. The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods. 4. Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3-5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity. 5. Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.

A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain.

The large G-actin pool in individual actively motile cells has been shown to be maintained primarily by the actin sequestering protein thymosin beta four (Tbeta4). It is not clear whether Tbeta4 or an isoform also plays a primary role in neural tissue containing highly motile axonal growth cones. To address this question we have made a definitive analysis of the relative contributions of all the known G-actin sequestering proteins: Tbeta4, Tbeta10, profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-actin pool in developing chick brain at embryonic days 13 and 17. From our measurements we estimate the intracellular concentration of G-actin as 30-37 microM and of Tbeta4 as 50-60 microM in an 'average' brain cell in embryonic chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to Tbeta4 is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cofilin concentrations are an order of magnitude lower than Tbeta4. Combining the contributions of Tbeta4, unphosphorylated ADF and unphosphorylated cofilin, we estimate a mean G-actin critical concentration of approximately 0.45 microM and approximately 0.2 microM, respectively, in day 13 and day 17 embryonic brain extracts, suggesting a significant developmental decrease. We conclude that (a) Tbeta4 is the major actin sequestering protein in embryonic chick brain and the only beta thymosin isoform present; (b) ADF may play a significant developmental role, as its concentration changes significantly with age; (c) the known G-actin binding proteins can adequately account for the G-actin pool in embryonic chick brain.

Myelin and collapsin-1 induce motor neuron growth cone collapse through different pathways: inhibition of collapse by opposing mutants of rac1.

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.

Rac1-dependent actin filament organization in growth cones is necessary for beta1-integrin-mediated advance but not for growth on poly-D-lysine.

The activity of filopodia and lamellipodia determines the advance, motility, adhesion, and sensory capacity of neuronal growth cones. The shape and dynamics of these highly motile structures originate from the continuous reorganization of the actin cytoskeleton in response to extracellular signals. The small GTPases, Rac1, Rho, and CDC42, regulate the organization of actin filament structures in nonneuronal cells; yet, their role in growth cone motility and neurite outgrowth is poorly understood. We investigated in vitro the function of Rac1 in neurite outgrowth and differentiation by introducing purified recombinant mutants of Rac1 into primary chick embryo motor neurons via trituration. Endogenous Rac1 was expressed in growth cone bodies as well as in the tips and shafts of filopodia, where it often colocalized with actin filament structures. The introduction of constitutively active Rac1 resulted in an increase in rhodamine-phalloidin staining, presumably from an accumulation of actin filaments in growth cones, while dominant negative Rac1 caused a decrease in rhodamine-phalloidin staining. Nevertheless, both Rac1 mutants retarded growth cone advance, and hence attenuated neurite outgrowth and inhibited differentiation of neurites into axons and dendrites on laminin and fibronectin. In contrast, on poly-D-lysine, neither Rac1 mutant affected growth cone advance, neurite outgrowth, or neurite differentiation despite inducing similar changes in the amount of rhodamine-phalloidin staining in growth cones. Our data demonstrate that Rac1 regulates actin filament organization in neuronal growth cones and is pivotal for beta1 integrin-mediated growth cone advance, but not for growth on poly-D-lysine.

Actin disassembles reversibly during electrically induced recycling of synaptic vesicles in cultured neurons.

We have studied depolarization-induced regulation of actin assembly in exocytotically active areas of dissociated chick sympathetic neurons. Active areas were identified with the fluorescent dye FM1-43 which labels synaptic vesicles that recycle in these regions. Exocytosis (electrically stimulated) was monitored in real time through depletion of FM1-43 fluorescence. To study depolarization-induced disassembly of actin in the FM1-43-stained regions, the cells were fixed after different periods of depolarization and stained with rhodamine phalloidin, which binds preferentially to the filamentous form of actin. In active regions, actin disassembles and reassembles during continuous 2 min depolarization. Actin disassembly that occurs after the first 25 s of depolarization was detected by a reduction in rhodamine phalloidin staining and confirmed by correlative electron microscopy. Immunogold staining revealed that actin is abundant throughout resting terminals. In some experiments, actin filaments were stabilized by loading cells with unlabelled phalloidin before stimulating secretion. Stabilizing the filaments does not alter the initial release but strongly reduces the release rate at later stages. These data are consistent with a model in which partial disassembly of actin filaments is necessary for facilitating the transport of vesicles within the terminal and reassembly is necessary for limiting that movement.