Involvement of slingshot in the Rho-mediated dephosphorylation of ADF/cofilin during Xenopus cleavage

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.     

Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes

We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer–sequestering drug. On the other hand, jasplakinolide, an F-actin–stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor–arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis.     

Induction of increase in intracellular calcium concentration of embryonic cells and acceleration of morphogenetic cell movements during amphibian gastrulation by a 50-Hz magnetic field

The influence of an alternating electromagnetic field (EMF) on early development of amphibian embryos was examined. When the embryos developed under the influence of a low-frequency EMF (50 Hz, 5-30 mT), the rate of early development was accelerated. The effect of EMF was exerted preferentially at the gastrula stage, and the period of gastrulation was shortened. Histological observations showed that EMF promoted morphogenetic cell movements during the gastrulation. The concentration of intracellular free Ca2+ ([Ca2+]i) in the embryonic cells under the influence of EMF was analyzed using Fura-2, an indicator of the intracellular concentration of calcium ions. The influence of EMF on [Ca2+]i was analyzed in embryonic cells isolated from blastula, gastrula, and neurula, EMF increased a [Ca2+]i particularly in the cells isolated from gastrula. Our results suggest that EMF specifically increased the [Ca2+]i of gastrula cells, thereby, accelerating the rate of morphogenetic cell movements during gastrulation.     

Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation.     

Epibolic extension of the presumptive ectodermal layer of embryos of the newt Cynops pyrrhogaster before and during gastrulation.

Epibolic extension of the presumptive ectodermal layer (PEL) was investigated in embryos of the newt Cynops pyrrhogaster before and during gastrulation. The PEL was composed of only one layer of columnar cells at all stages examined. The cells of the PEL became elongated from the blastula to the early gastrula stage. They were most elongated at the early gastrula stage and then shortened during gastrulation. Present observations suggest that changes in cell shape of the PEL play an important role in the control of the epibolic extension of the newt embryos. The morphology and movement of the isolated cells from the PEL were examined in an attempt to elucidate the role of cell movement in epibolic extension of the PEL. Blebbing and vermiform cells which showed active cell movement appeared at the early blastula stage. The blebbing cells, which formed large hyaline blebs that moved around the circumference of each cell, appeared in large numbers at the early blastula stage. The frequency of the blebbing cells decreased from the early blastula to the early gastrula stage and increased again during gastrulation. The vermiform cells, which had an elongated cell body and moved in a worm-like manner, increased in frequency from the early blastula to the early gastrula stage. The relative number of such vermiform cells was maximal at the early gastrula stage and decreased abruptly during gastrulation. These results suggest that the elongation of the cells of the PEL is controlled by the active cell movement which resembles that of a worm.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.     

Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation- regulated protein essential for development

Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST- fusion proteins have pH-dependent actin-depolymerizing and F-actin- binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration- dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.     

ADF/cofilin controls cell polarity during fibroblast migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.     

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.     

A radioautographic study of protein synthesis in loach blastoderm]

The dynamics of protein synthesis in the loach embryos has been studied by means of autoradiography at the stages of cleavage, blastula and gastrula. During the synchronous cleavage divisions, nuclear proteins are mainly synthesized. From the early blastula stage until the early gastrula stage, the intensity of nuclear protein synthesis increases 2.5 times whereas the intensity of cytoplasmic and total protein synthesis is low and relatively constant. After the onset of gastrulation the intensity of nuclear and cytoplasmic protein synthesis increases 3-4 times and at the late gastrula stage it decreases twice as compared with that at the midgastrula stage. During blastulation, no regional differences in the intensity of nuclear and cytoplasmic protein synthesis were found. With the onset of gastrulation, a vegeto-animal gradient of labeled aminoacid incorporation into nuclear and cytoplasmic proteins appears. During gastrulation, reliable differences were found between the intensity of labeled aminoacid incorporation into proteins of the cells of intact and dissociated blastoderms. During this period, the intensity of protein synthesis in embryonic shield is higher than that in the extraembryonic part of blastoderm.     

Xoom is maternally stored and functions as a transmembrane protein for gastrulation movement in Xenopus embryos

Xoom has been identified as a novel gene that plays an important role in gastrulation of Xenopus laevis embryo. Although Xoom is actively transcribed during oogenesis, distribution and function of its translation product have not yet been clarified. In the present study, the polyclonal antibody raised against Xoom was generated to investigate a behavior of Xoom protein. Anti-Xoom antibodies revealed that there are two forms of Xoom protein in Xenopus embryos: (i) a 45 kDa soluble cytoplasmic form; and (ii) a 44 kDa membrane-associated form. Two forms of Xoom protein were ubiquitously detected from unfertilized egg to tadpole stage, with a qualitative peak during blastula and gastrula stages. Immunohistochemical examination showed that Xoom protein is maternally stored in the animal subcortical layer and divided into presumptive ectodermal cells during cleavage stages. Enzymatic digestion of membrane protein and immunologic detection of Xoom showed that Xoom exists as a membrane-associated protein. To examine a function of Xoom protein, anti-Xoom antibodies were injected into blastocoele of stage 7 blastula embryo. Anti-Xoom antibodies caused gastrulation defect in a dose- dependent manner. These results suggest that maternally prepared Xoom protein is involved in gastrulation movement on ectodermal cells.     

Ultrastructural Study of Changes in Cell Morphology and Extracellular Matrix in Prospective Endodermal Cells of Newt Embryos (Cynops pyrrhogaster) before and during Gastrulation

The cell morphology, cell-to-cell contact behavior and extracellular matrix (ECM) of inner cells (prospective endodermal cells) of newt ( Cynops pyrrhogaster ) embryos were examined from the morula to gastrula stage by light and electron microscopy. The inner cells showed increased cell-to-cell contact from the early blastula to early gastrula stage. The cells formed blebs (5–15 μm in diameter) during the blastula stage, and started to form filopodia and lamellipodia before gastrulation. Alcian blue and lanthanum nitrate treatment revealed ECM components on the cell surface in the early blastula stage and these components increased in amount from the late blastula to early gastrula stage. It is suggested that the increase in ECM components on the cell surface may have some relation with changes in cell-to-cell contact and formation of processes on the cell surface. Besides the cell surface ECM components, glycogen-like granules were observed in intercellular spaces. From the distribution of granules in gastrulae, it is suggested that these may be important in maintaining intercellular spaces for migration of invaginating cells.     

Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze-Dried Newt Embryos Before and During Gastrulation

A scanning electron-microscopic study was carried out on the extracellular matrices (ECMs) in freeze-dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin

The ADF (actin-depolymerizing factor)/cofilin family is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1), much less is known about its reactivation through dephosphorylation. Here we report Slingshot (SSH), a family of phosphatases that have the property of F actin binding. In Drosophila, loss of ssh function dramatically increased levels of both F actin and phospho-cofilin (P cofilin) and disorganized epidermal cell morphogenesis. In mammalian cells, human SSH homologs (hSSHs) suppressed LIMK1-induced actin reorganization. Furthermore, SSH and the hSSHs dephosphorylated P cofilin in cultured cells and in cell-free assays. Our results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo.