Roles of cofilin in development and its mechanisms of regulation.

The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2015) 57, 275–290

doi: 10.1111/dgd.12213

Review Article

Roles of cofilin in development and its mechanisms of regulation Kazumasa Ohashi* Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan

Reorganization of the actin cytoskeleton is essential for cellular processes during animal development. Cofilin and actin depolymerizing factor (ADF) are potent actin-binding proteins that sever and depolymerize actin filaments, acting to generate the dynamics of the actin cytoskeleton. The activity of cofilin is spatially and temporally regulated by a variety of intracellular molecular mechanisms. Cofilin is regulated by cofilin binding molecules, is phosphorylated at Ser-3 (inactivation) by LIM-kinases (LIMKs) and testicular protein kinases (TESKs), and is dephosphorylated (reactivation) by slingshot protein phosphatases (SSHs). Although studies of the molecular mechanisms of cofilin-induced reorganization of the actin cytoskeleton have been ongoing for decades, the multicellular functions of cofilin and its regulation in development are just becoming apparent. This review describes the molecular mechanisms of generating actin dynamics by cofilin and the intracellular signaling pathways for regulating cofilin activity. Furthermore, recent findings of the roles of cofilin in the development of several tissues and organs, especially neural tissues and cells, in model animals are described. Recent developmental studies have indicated that cofilin and its regulatory mechanisms are involved in cellular proliferation and migration, the establishment of cellular polarity, and the dynamic regulation of organ morphology. Key words: actin cytoskeleton, cofilin, development, LIM kinase, slingshot.

Introduction The organization of cell populations within animal tissues is essential for the morphogenesis of organs during development. Cells recognize three-dimensional positions with respect to the whole organism, and regulate cell shape, motility, migration, polarization, growth, differentiation, gene expression, and cell death according to extracellular signals. These signals include various soluble factors and molecules that direct cell– cell and cell–substrate adhesion. Remodeling of the actin cytoskeleton is essential for these cellular responses and morphological changes, and must be regulated spatially and temporally by a number of complex signaling pathways (Etienne-Manneville & Hall 2002). The actin cytoskeleton consists of bundles and combinations of actin filaments. Its reorganization occurs through the assembly and the disassembly of actin filaments, involving numerous actin-binding proteins (Moon & Drubin 1995; Pantaloni et al. 2001;

*Author to whom all correspondence should be addressed. Email: [email protected] Received 21 February 2015; revised 18 March 2015; accepted 19 March 2015. ª 2015 Japanese Society of Developmental Biologists

Etienne-Manneville & Hall 2002). The molecular mechanisms and the signaling pathways required for actin cytoskeleton remodeling have been elucidated by many studies over the last decades (Ayscough 1998; Etienne-Manneville & Hall 2002; Ono 2007). One of major breakthroughs was the identification of the Rho family of small GTPases as key molecules (Jaffe & Hall 2005). Thereafter, a number of the downstream effectors of Rho family molecules were identified and a broad understanding of the roles of these molecules has been established (Bustelo et al. 2007). The roles of regulators of the actin cytoskeleton and its signaling pathways during tissue development and organ morphogenesis have been actively studied in recent years. The cofilin/actin-depolymerizing factor (ADF) family proteins (hereafter referred to as cofilin) are conserved from yeast to human. Cofilin, one of the essential actin regulating proteins, binds to both monomeric globular (G)-actins and filamentous (F)-actins, and severs the F-actin, which causes the depolymerization of the F-actin (Moon & Drubin 1995; Bamburg et al. 1999; Andrianantoandro & Pollard 2006; Bernstein & Bamburg 2010). Since cofilin has a higher affinity for ADP-bound actin filaments than ATP-bound filaments, the “aged” actin filaments are selectively disassembled (Pollard & Borisy 2003). These activities are essential

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for generating actin cytoskeleton dynamics in cells. It has been found that the activity of cofilin is spatiotemporally regulated by phosphorylation, lipid binding, and the binding proteins downstream of various signaling pathways (Ono 2007). LIM-kinases and Slingshot phosphatases phosphorylate and dephosphorylate cofilin, respectively (Mizuno 2013). The level of phosphorylation of cofilin in cells is altered dynamically during various cellular responses, and the change in the phosphorylation level is critical for the regulation of actin cytoskeleton dynamics (Kiuchi et al. 2011). The studies of the role of cofilin and its phospho-regulation in the development and morphogenesis of tissues and organs have also been ongoing over the last decade (Mizuno 2013). In this review, we focus on the role of cofilin in development. Since there are many brilliant reviews that describe the molecular mechanisms of cofilin activity and the regulation of cofilin in detail (Ono 2007; Bernstein & Bamburg 2010; Mizuno 2013), the roles of cofilin and its regulators in actin cytoskeleton remodeling are briefly described. The functions of cofilin in developing neural tissues and cells will be described in detail. Studies of animal models with loss- or gain-offunction mutations in cofilin or its regulators will be also described.

Cofilin preferentially binds to ADP-bound F-actin and destabilizes and severs the actin filaments, resulting in depolymerization of the short actin filaments (Fig. 1) (Pollard & Borisy 2003). One of the roles of the actin severing activity of cofilin is to generate free actin monomers for polymerization (Pollard & Borisy 2003; Kiuchi et al. 2007, 2011). Cofilin promotes both actin filament disassembly, by severing, and actin polymerization, by supplying actin monomers, resulting in enhanced dynamics of the actin cytoskeleton. An alternative model was proposed in which the severing activity of cofilin generates short filaments and new barbed ends for actin assembly (Fig. 1) (Ghosh et al. 2004; DesMarais et al. 2005; Oser & Condeelis 2009). Furthermore, it was proposed that the high concentration of cofilin promotes the nucleation of actin, leading to actin assembly (Andrianantoandro & Pollard 2006). When the cellular morphology changes, cofilin promotes the generation of actin monomers, nuclei, and newly barbed ends for the assembly of actin structures

Structure and regulation of cofilin Cofilin was purified and identified as a 20-kDa protein, which induces depolymerization of actin filaments in an extract from avian or porcine brain (Bamburg et al. 1980; Nishida et al. 1984). The cofilin family is composed of non-muscle type cofilin-1 (also named n-cofilin), muscle type cofilin-2 (also named m-cofilin) and actin depolymerizing factor (ADF, also named destrin) in mammals (Ono 2007; Poukkula et al. 2011). At least one cofilin protein exists in all eukaryotic cells (Ono 2007). Cofilins are globular proteins with a core consisting of four or five b-sheets, which is surrounded by four or five helices (Hatanaka et al. 1996; Ono 2007). Cofilin amino acid sequence and structure are highly conserved from human to yeast (Ono 2007). The ADF-homology (ADF-H) domain, which contains a homologous sequence to cofilin, is conserved in five actin-related protein families, including twinfilin, coactosin actin-binding protein 1 (ABP1), drebrin, and glia maturation factor-like protein (GMF). Although these proteins exhibit actin or actin-related protein 2/3 (Arp2/3) complex binding activities, they do not have actin depolymerizing and severing activities, except for yeast twinfilin (Poukkula et al. 2011). The functions of the ADF-H domain diversified during evolution. ª 2015 Japanese Society of Developmental Biologists

Fig. 1. Model of the actin assembly and disassembly by cofilin. Cofilin preferentially binds to ADP-bound actin in an actin filament and severs the actin filament into short filaments. In one pathway, the short filaments are depolymerized and regenerated to ATP-bound actin monomers. The regenerated actin monomers contribute to assembly of the actin cytoskeleton by increasing the concentration of actin monomers. In an alternate pathway, the short filaments contribute to increasing the barbed ends and nuclei for actin polymerization. In other pathways, the high concentration of cofilin promotes the nucleation of actin, leading to actin assembly. Cofilin promotes the dynamics of the actin cytoskeleton by disassembling actin filaments and by supplying actin monomers, nuclei, and barbed ends.

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2000; Gohla et al. 2005; Oleinik et al. 2010) (Fig. 2). However, it is not well understood how regulation of cofilin’s phosphorylation status by NRK, CIN, PP1, and PP2 affects normal cellular processes. Although, cofilin is found in all eukaryotes, from yeast to human, including plants, orthologous LIMK and SSH genes are not found in yeast, Caenorhabditis elegans (C. elegans), Dictyostelium, or in plants (Mizuno 2013). The phospho-regulation of cofilin may have evolved as a mechanism for actin cytoskeleton reorganization during complex multicellular processes in some higher-level organisms. Fig. 2. The regulatory molecules of cofilin activity. Cofilin is phosphorylated and inactivated by LIM-kinases (LIMKs), testicular protein kinases (TESKs), and Nck-interacting kinase (NIK)-related kinase (NRK), and by binding of PIP2 and cortactin. In contrast, cofilin is dephosphorylated and reactivated by slingshot protein phosphatases (SSHs), PP1, PP2A, and chronophin (CIN). Cofilin is also activated by the binding of Aip1 and CAP1.

such as lamellipodia, filopodia, and stress fibers, and also promotes the turnover of these structures. In contrast, the inactivation of cofilin in response to extracellular signals decelerates the actin turnover and contributes to the assembly of actin structures (Ohashi et al. 2011, 2014). Cofilin activity is regulated by several molecular mechanisms (Fig. 2). Cofilin is inactivated by phosphorylation at an N-terminal Serine-3 (Ser-3) residue (Moriyama et al. 1996), and by the binding of phosphatidylinositol 4,5-bisphosphate (PI (4,5)P2) (Yonezawa et al. 1990; van Rheenen et al. 2007) and cortactin (Oser et al. 2009). Likewise, cofilin is activated by actin-interacting protein-1 (Aip1) and cyclase-associated protein-1 (CAP1) (Fig. 2) (Moriyama & Yahara 2002; Ono 2003). The phosphorylation level of cofilin is regulated by a number of extracellular signals. Cofilin is phosphorylated at Ser-3 by several kinases. The major kinases of cofilin in mammals are LIM-kinase family proteins, which consist of LIM domain containing protein kinase 1 (LIMK1), LIMK2, Testicular protein Kinase 1 (TESK1), and TESK2 (Arber et al. 1998; Yang et al. 1998; Sumi et al. 1999; Toshima et al. 2001a,b). Nck-interacting kinase (NIK)-related kinase (NRK) also phosphorylates cofilin (Nakano et al. 2003) (Fig. 2). On the other hand, cofilin is dephosphorylated by members of the Slingshot (SSH) protein phosphatase family, which includes SSH1, SSH2, and SSH3 in mammals (Niwa et al. 2002; Ohta et al. 2003; Mizuno 2013). It was also reported that the haloacid dehalogenase, chronophin (CIN), and the general protein phosphatases, protein phosphatase1 (PP1) and protein phosphatase 2A (PP2A), also dephosphorylate cofilin (Ambach et al.

Signal transduction-mediated phosphoregulation of cofilin A number of studies have investigated the functions of LIMKs and SSHs, and their respective signaling pathways, during the response to extracellular signals. LIMK1 was discovered while cloning the c-Sea receptor tyrosine kinase from a human cDNA library (Mizuno et al. 1994). The other members, LIMK2 (Nunoue et al. 1995; Okano et al. 1995), TESK1, and TESK2 (Toshima et al. 1995, 2001b) were cloned by low-stringency DNA hybridization, using the LIMK1 cDNA as a probe. The LIMK proteins contain tandem, N-terminal LIM domains, which contain Zn-finger motifs, an internal PDZ-like domain and a C-terminal protein kinase domain (Fig. 3). (Okano et al. 1995). The TESK proteins are related to the LIMKs and contain the highly homologues kinase domain of LIMKs at the N-terminus and a unique C-terminal proline-rich domain (Fig. 3) (Toshima et al. 2001b). LIMKs and TESKs phosphorylate cofilin at Ser-3 and inactivate its actin binding activity. LIMK1 is activated and contributes to lamellipodia or stress fiber formation downstream of Rho signaling. LIMKs are phosphorylated at a conserved threonine residue (Thr-508 in human LIMK1 or Thr-505 in human LIMK2) in the activation loop of the kinase domain and activated by Rho-associated kinase (ROCK) (Maekawa et al. 1999; Ohashi et al. 2000; Sumi et al. 2001a), p21-activated kinase-1, -2, -4 (PAK1, PAK2, and PAK4), and myotonic dystrophy kinase-related Cdc42-binding kinase-a (MRCKa) (Edwards et al. 1999; Dan et al. 2001; Sumi et al. 2001b; Ahmed et al. 2008), which are downstream effectors of RhoA, Rac1 and Cdc42, respectively (Fig. 4). LIMK1 is also activated downstream of Ca2+ signaling. LIMK1 is phosphorylated at the same Thr508 residue by Ca2+/calmodulin-dependent protein kinase (CaMK) II and IV (Fig. 4) (Takemura et al. 2009; Saito et al. 2013). Another phosphorylation site for the activation of LIMK1 has been identified. LIMK1 is phosphorylated at the Ser-323 residue by p38 mitoª 2015 Japanese Society of Developmental Biologists

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Fig. 3. Structures of human LIM-kinases (LIMKs), testicular protein kinases (TESKs), and slingshot protein phosphatases (SSHs). The numbers on both sides of the schemata indicate the length of the amino acid sequences of the LIMKs and SSHs. The percentages indicate the identities of amino acid sequences in the kinase domains. LIM, LIM domain; PDZ, PDZ domain; PK, protein kinase domain; Pro-rich, proline-rich region; A and B, N-terminal conserved domains in SSHs; DSP, dual specificity protein phosphatase domain; Serrich, Serine-rich region.

Fig. 4. The regulatory mechanisms of LIM-kinases (LIMKs), testicular protein kinases (TESKs), and slingshot protein phosphatases (SSHs). LIMKs are phosphorylated and activated by the indicated upstream kinases (upper left) and are inactivated by molecules with their indicated actions (upper right). TESK1 is activated downstream of an integrin signal. SSHs is activated and inactivated by the indicated molecules (bottom). Activation of LIMKs and inactivation of SSHs stabilize the actin cytoskeleton, and, conversely, inactivation of LIMKs and activation of SSHs accelerate the dynamics of the actin cytoskeleton via a mechanism that corresponds to the dephosphorylation of cofilin.

gen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2/MK2) downstream of p38-MAP kinase in response to vascular endothelial growth factor (VEGF) in vascular endothelial cells (Fig. 4) (Kobayashi et al. 2006). BMP signal also regulates LIMK1 activity. Bone morphogenic protein (BMP)-4 and -7 stimulation activates LIMK1 (Foletta et al. 2003; LeeHoeflich et al. 2004). However, two groups reported opposite mechanisms. One group reported that binding of LIMK1 to the cytoplasmic C-terminal domain of BMP receptor II (BMPRII) inhibits LIMK1 and that LIMK1 is activated when released from BMPRII by the ª 2015 Japanese Society of Developmental Biologists

binding of BMP4 to BMPRII (Foletta et al. 2003). The second group reported that LIMK1 binds to the cytoplasmic domain of BMPRII and is activated downstream of Cdc42 when BMP7 activates Cdc42 through BMP receptor I (Lee-Hoeflich et al. 2004) (Fig. 4). In both models, LIMK1 is activated by the BMP stimulation. In contrast, LIMK1 is inactivated by several mechanisms (Fig. 4). LIMK1 is dephosphorylated and inactivated by Slingshot-1 phosphatase (Soosairajah et al. 2005). LIMKs are also inactivated by the binding of polarity protein Par-3 (Chen & Macara 2006), Nischarin (Ding et al. 2008), b-arrestin

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Davis 2005; Sese et al. 2006; Chu et al. 2012). Tissue defects in several developing stages were analyzed using a thermolabile mutant and an inducible mosaic analysis in the loss- and gain-of-function mutants. The phenotypes of the mutants tend to be more severe than the phenotypes in mammalian mutants because the functional redundancy of related genes is not so strong in Drosophila. The functions of cofilin and its regulators in Drosophila development are listed in Table 1. Neural tissue development and neurite growth The roles of cofilin and its regulators in nervous system were analyzed using the Drosophila mutants named above and transgene experiments. The cofilin mutant impairs proliferation of neuroblast cells and axons growth in the mushroom body of the Drosophila brain (Ng & Luo 2004). Slingshot is required for axon extension, and mutation of limk weakly inhibits the axon growth. Neither Slingshot nor LIMK is required for the proliferation of the neuroblast cells in the mushroom body. Expression of the constitutively active S3A-cofilin mutant partially rescues the proliferation of neuroblast cells and axon growth (Ng & Luo 2004). These results indicate that the cofilin activity is essential for the proliferation of neural cells and axon growth, and that proper axon extension requires phospho-regulation of cofilin. Studies have described the molecular mechanism by which cofilin phosphoregulation regulates axon growth. Loss of sickie, a Drosophila ortholog of the mammalian microtubuleassociated protein neuron navigator 2 (NAV2 MAP), suppresses axon growth of neural cells in the mushroom body (Abe et al. 2014). Sickie, which is a Rac effector protein, is required for decreasing of the phosphorylation level of cofilin in developing neurons via a mechanism that depends on Rac but not on Pak. Cofilin phosphorylation increases in the neural cells of sickie mutants and induces actin over-polymerization, resulting in the inhibition of the axonal outgrowth. Furthermore, the defect caused by the sickie mutation is rescued by overexpression of the active forms of cofilin or slingshot, but not their inactive forms (Abe et al. 2014). These results indicate that axonal outgrowth requires cofilin dephosphorylation by the Rac-Sickie-slingshot pathway, which functions in parallel to the Rac-Pak-LIMK pathway. [Correction added on 8 May 2015, after first online publication: ‘stickie’ has been corrected to ‘sickie’ in the above paragraph.] Ovary development and oogenesis The function of cofilin during the development of the ovary and during oogenesis was investigated using a ª 2015 Japanese Society of Developmental Biologists

thermolabile mutant of cofilin (Chen et al. 2001). Convergent extension of the ovarian terminal-filament cells in the third larval instar is impaired by downregulation of cofilin. At later stages, migration of border cells in the egg chamber during oogenesis is also suppressed (Chen et al. 2001), and the expression of wild type and constitutively active S3A-cofilin, but not dominant negative S3E-cofilin, in which the serine-3 is replaced with glutamic acid, rescues to the migration defect of mutant border cells (Zhang et al. 2011). CAP1 is involved in egg chamber development. Loss of CAP1 causes F-actin accumulation in the apical side of epithelial follicle cells in the egg chamber and inhibits apical construction, resulting in the perturbation of oocyte polarity (Baum et al. 2000; Baum & Perrimon 2001). Spermatogenesis Since cofilin is required for the formation of the cleavage furrow during cytokinesis (Abe et al. 1996), meiotic failure during spermatogenesis is one of the major defects in cofilin mutants (Gunsalus et al. 1995). Although TESK is predominantly expressed in mammalian testes in mammal, its function during spermatogenesis is not well established. Rac1 is required for sperm maturation during Drosophila spermatogenesis. Hemizygotic deletion of tesk enhances the sterility of male rac1 flies. The mutant sperm are motile but cannot enter the seminal vesicle. These results indicate that the Rac-TESK pathway is required for the late stage of Drosophila spermatogenesis (Raymond et al. 2004). Development of epithelial morphogenesis The proper spatiotemporal regulation of actin cytoskeleton reorganization is required for the development of the wing, eye, and epithelial tissues and also for apico-basal and planar cell polarity (PCP). The lethality in cofilin null mutants is rescued by the transformation of thermolabile cofilin mutants (tsr139, tsrV27Q) (Blair et al. 2006). The rescued mutant is viable at 18°C and in these animals the PCP of wing, eye and several epithelia is perturbed. Frizzled and flamingo, which are PCP-related proteins, mislocalize in the mutant epithelial cells. Expression of a constitutively active form of LIMK also perturbs the wing PCP (Blair et al. 2006). Furthermore, cofilin activity is required for retinal cell elongation and morphogenesis (Pham et al. 2008). Slingshot was originally isolated as a gene involved in bristle morphogenesis. Phenotypes of the adult animals that were rescued for the ssh null mutant include bifurcated and twisted bristles and wing hairs, disordered ommatidia, and a lack of interommatidial bristles (Niwa et al. 2002). Other reports show that the

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(Agrawal et al. 2012), adf (Bellenchi et al. 2007; Kuure et al. 2010; Flynn et al. 2012), cap1-related cap2 (Peche et al. 2013), limk1 (Meng et al. 2002), limk2 (Takahashi et al. 2002; Rice et al. 2012), and slingshot-3 (Kousaka et al. 2008) have been generated. cofilin-1; adf (Bellenchi et al. 2007; Kuure et al. 2010; Flynn et al. 2012) and limk1; limk2 (Meng et al. 2004) double knockout mice also have been generated. Although ssh3 knockout mice were generated, these animals have no distinct phenotype and appear normal (Kousaka et al. 2008). Knockout mice for ssh1, ssh2, tesk1, and tesk2 have not been reported. Although the cofilin family genes are highly and ubiquitously expressed in all tissues, the expression of limk1 is most abundant in the developing and adult nervous system. Furthermore, the hemizygotic deletion of limk1 in humans is associated with the spatial recognition defect in William’s syndrome patients (Frangiskakis et al. 1996). After this finding, the functions of LIMK1 in neural cells and in neural tissue development have been the main focus of recent studies. In the following section, the roles of cofilin and its regulators, especially the LIMKs and SSHs, in mammalian neural cell morphogenesis and neural tissue development are discussed, and then their roles in the development of other tissues are also described. Thereafter, the analysis of Drosophila mutants will be described. Table 1 summarizes the developmental processes involving cofilin and its regulators in model animals. The functions of cofilin, LIMKs and SSHs in the development of neural tissues and cells Neurites, including axons and dendrites, extend and develop in neural tissues during development. The neurite is led by the growth cone, a tip of the neurite, which has dynamic actin-rich structures. The growth cone is similar to the leading edge of a migrating cell containing lamellipodia, filopodia, and the contractile actomyosin fibers (Luo 2000). The spatiotemporal regulation of cofilin activity is involved in the neurite extension, retraction, and turning (Fass et al. 2004). Another important function of neurites is the formation of the neural circuit through a specialized cell–cell junction, called a synapse (Harris 1999). A dendritic spine, which is the mushroom-like, actin-rich protrusion of the postsynaptic membrane, matures and develops in response to the nerve stimuli-induced reorganization of the actin cytoskeleton (Bosch & Hayashi 2012). The regulation of cofilin activity is also involved in spine formation and spine function. Cofilin-1, non-muscle type cofilin, and ADF are expressed in whole brain, but the level of ADF is lower than the level of cofilin (Gurniak et al. 2005). cofilin-1 knockout mice are embryonic lethal at stage E9. The ª 2015 Japanese Society of Developmental Biologists

major defects in cofilin-1 knockout mice include the failure of neural crest cell migration and the failure of neural tube closure, resulting in the lack of development of neural crest-derived tissues (Gurniak et al. 2005). Since cofilin activity is essential for cell viability, the cofilindeficient embryo could be partially rescued by the redundancy of ADF. To investigate the neural functions of cofilin-1 and ADF, brain-specific, conditional knockouts of cofilin-1 and adf were generated in mice using nestin-Cre mediated gene deletions (Bellenchi et al. 2007). The deletion of cofilin-1 in the E10.5 brain causes the inhibition of the radial migration of neural cells into the cortex and the suppression of neurite extension by the cortical neurons, resulting in absence of intermediate cortical layers in the brain (Bellenchi et al. 2007). Although the effect of adf knockdown in the brain does not cause visible defects, cofilin/adf double knockout mice have similar and more severe defects than cofilin-1 knockout mice (Bellenchi et al. 2007). This indicates that ADF may partially contribute to brain development. There are several studies of the roles of cofilin in dendritic spine formation and in the electrophysiological spine functions. When a spine forms and develops in response to glutamate via the NMDA receptor, cofilin accumulates into spines via a mechanism that depends on b-arrestin 2 (Pontrello et al. 2012). Conditional knockout of cofilin-1 in mice, using CaMKII-Cre mediated gene deletion, impairs both long-term potentiation (LTP) and long-term depression (LTD), and enhances the stability of AMPA receptor in dendrites (Rust et al. 2010). Despite the severe defects in the brain of cofilin-1 knockout mice, knockout of limk1 causes mild brain defects. The limk1 knockout hippocampal neurons have thinner spines, and LTP, but not LTD, is enhanced (Meng et al. 2002). Although limk2 knockout mice observe no distinguishable defects in brain, both the limk1/limk2 double knockout mice have similar and more severe defects than limk1 knockout mice (Meng et al. 2004). These results suggest that proper synaptic plasticity requires not only cofilin activity but also its phospho-regulation. The functions of the signaling pathways involved in cofilin phospho-regulation during neural development have been reported. Knockout of moma-gap, which is a Cdc42 specific GTPase-activating protein (GAP), results in a thin cortex and impairs dendrite branching of cortex neurons (Rosario et al. 2012). Cdc42 is hyper-activated and the phosphorylation level of cofilin increases in moma-gap knockout cortical neurons. Expression of a nonphosphorylatable S3A-cofilin mutant, in which serine-3 is replaced with alanine, rescues the dendrite branching defect in moma-gap knockout cortical neurons (Rosario et al. 2012). The pak1/pak3 double knockout mouse has a normal brain in neonatal animals,


Table 1. Summary of the functions of cofilin and its regulators in development

Protein Cofilin-1 (Mammalian)

Effect on actin cytoskeleton

Functions in development

Severance and Migration of neural crest depolymerization cells (Gurniak et al. 2005) of actin filaments Migration and neurite extension of a cortical neurons (Bellenchi et al. 2007) Spine morphogenesis and the electrophysiological spine functions (Pontrello et al. 2012) Myelination of Schwann cells (Sparrow et al. 2012) Kidney development (Kuure et al. 2010) Podocyte development (Kidney) (Garg et al. 2010)

ADF (Mammalian) Cofilin-2 (Mammalian)

Kidney development (Kuure et al. 2010) Skeletal and cardiac muscle development (Agrawal et al. 2012)

Cofilin (Twinstar “Tsr” in Drosophila)

Proliferation of neuroblast cells and axons growth in Mushroom body (Ng & Luo 2004) The convergence extension of the terminalfilament cells of ovary and migration of border cells in egg chamber (Chen et al. 2001) Spermatogenesis (Gunsalus et al. 1995) Planar cell polarity formation (Blair et al. 2006) Eye development (Blair et al. 2006)

Muscle type Cofilin (UNC-60B in C. elegans) Aip1 (Fluer “Flr” in Drosophila)

CAP1/2 (Capulet “cap”/Act up “Acu” in Drosophila)

Muscle development (Ono et al. 1999)

Activation of cofilin by binding

Eye development (Chu et al. 2012) Planar cell polarity formation (Ren et al. 2007) Eye development (Benlali et al. 2000) Apical construction of egg chamber and oocyte polarity (Baum et al. 2000; Baum & Perrimon 2001)

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Table 1. (continued) Effect on actin cytoskeleton

Protein CAP2 (Mammalian) CAP1/2 (CAS-1 in C. elegans) LIMK1 (Mammalian)

Functions in development Cardiac development (Peche et al. 2013) Muscle development (Nomura et al. 2012)

Phophorylation Spine morphogenesis of cofilin at Ser-3 and the electrophysiological spine functions (Meng et al. 2002) Extension of axonal growth cone (Endo et al. 2003) Growth cone collapse (Aizawa et al. 2001; Hsieh et al. 2006) Dendritogenesis (LeeHoeflich et al. 2004; Saito et al. 2013)

LIMK2 (Mammalian)

Spermatogenesis (Takahashi et al. 2002) Collective migration of the epidermal keratinocytes (Rice et al. 2012)

LIMK (Drosophila)

Planar cell polarity formation (Blair et al. 2006)

TESK (Center divider “cdi” in Drosophila)

Spermatogenesis (Raymond et al. 2004) Apico-basal polarity formation (Sese et al. 2006)

SSH1 (Mammalian)

SSH1 (Drosophila)

Dephophorylation Extension of of cofilin at Ser-3 axonal growth cone (Endo et al. 2007) Eye development, epithelial morphogenesis, and planar cell polarity formation (Niwa et al. 2002)

but dendrite arborization is impaired during postnatal development of the mutant brain, resulting in reduced brain size (Huang et al. 2011). The knockout of both pak1 and pak3 reduces cofilin phosphorylation and the inhibition of cofilin dephosphorylation rescues the abnormal morphology of dendritic spines in mutant hippocampal neurons (Huang et al. 2011). Extension of ª 2015 Japanese Society of Developmental Biologists

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commissural spinal axons is suppressed by BMPs in the midline of floor plate of spinal cord (Augsburger et al. 1999). This BMP-mediated repulsive response of commissural axons is inhibited by the expression of mutant BMPRII, in which the LIMK1 binding site is deleted in the cytoplasmic region, or by expression of a constitutively active form of S3A-cofilin (Phan et al. 2010). Since BMP signals activate LIMK1, the repulsive response is required for the activation of LIMK1 and the inactivation of cofilin (Yamauchi et al. 2013). The functions of cofilin and its phospho-regulation were analyzed in more detail using isolated neural cells. Overexpression of LIMK1 or knockdown of SSH1 in chick dorsal root ganglion (DRG) neurons suppresses the extension of axonal growth cones (Endo et al. 2003, 2007). In contrast, knockdown or inhibition of LIMK1, using short hairpin RNAs or a synthetic S3-peptide that contains 16 N-terminal residues of cofilin plus a penetrable peptide sequence, in chick DRG neurons also suppresses the extension of axonal growth cones (Endo et al. 2003). The former result suggests that inactivation of cofilin causes excess polymerization of actin and the loss of actin cytoskeleton dynamics, resulting in inhibition of growth cone motility. The later result indicates that the suppression of cofilin phosphorylation fails to sufficiently polymerize actin for the formation of lamellipodia and filopodia and fails to allow actomyosin structures in the growth cones to generate contractile force. The proper regulation of LIMK1 and SSH1 activity is required for normal growth cone extension, while repulsive responses of neurites require LIMK1 activity but not SSH1. Semaphorin-induced axonal growth cone collapse is suppressed by inhibition of LIMK1 activity using the S3-peptide (Aizawa et al. 2001). The dominant-negative LIMK1 mutant, but not the phosphatase-inactive SSH1 mutant, suppresses Nogo-66-induced axonal growth cone collapse in chick DRG neurons (Hsieh et al. 2006). The attractive or repulsive response of growth cones to a guidance cue is switched by altering the intracellular conditions, such as the intracellular concentration of cyclic GMP (Song et al. 1998). A growth cone of Xenopus spinal neuron is attracted to the gradient of BMP7 during 4–8 h of culture, but after 20 h, the growth cone becomes repulsed by the BMP7 gradient (Wen et al. 2007). Inhibition of LIMK1 activity or overexpression of dominant-negative LIMK1 suppresses the attractive turning in neurons that are cultured between 4 and 8 h, and overexpression of dominant-negative SSH1 prevents the switch from attractive turning to repulsive turning in neurons cultured for 20 h. The switch in the turning response requires the transient receptor potential (TRP) channel-Ca2+-calcineurinSSH1 pathway (Wen et al. 2007). These data suggest that a balance of LIMK1 and SSH1 activity determines ª 2015 Japanese Society of Developmental Biologists

the direction of growth cone turning, and that the spatiotemporal regulation of LIMK1 and SSH1 is required for formation of an accurate neural network. [Correction added on 26 May 2015, after first online publication: ‘NGO-66’ and ‘calcinurin’ have been corrected to ‘Nogo-66’ and ‘calcineurin’ in the above paragraph.] Developing neural cells protrude neurites and grow dendrites or axons in response to neurotrophic factors. Brain-derived neurotrophic factor (BDNF) induces the increase in the number of primary dendrites in cortical neurons (McAllister et al. 1997). Depletion of LIMK1 suppresses this BDNF-dependent increase through the phospholipase Cc (PLCc)-Ca2+/calmodulin-dependent protein kinase IIb (CaMKIIb) pathway (Saito et al. 2013). BMP also induces an increase in the number of primary dendrites in cortical neurons (Lee-Hoeflich et al. 2004). It is required for the binding of LIMK1 to BMPRII and for the activation of LIMK1 downstream of Cdc42, which is activated by BMPRI upon BMP stimulation, as described above. These results indicate that dephosphorylation of cofilin facilitates the extension and branching of neurites and destabilizes spines by increasing the dynamics of the actin cytoskeleton, while, in contrast, phosphorylation of cofilin induces the retraction and stabilization of neurites and spines by decreasing the dynamics of the actin cytoskeleton. The spatiotemporal regulation of both signals is required for the normal development of neural tissues and the formation of proper neural circuits. The functions of cofilin, LIMKs and SSHs in the development of other tissues The roles of cofilin and its phospho-regulators in the development of several organs and the differentiation of cells have been reported. A Schwann cell aligns to an axon and expands the leading edge, and the resulting, enlarged membrane wraps the axon repeatedly, resulting in the formation of myelin sheath (Fernandez-Valle et al. 1997). cofilin knockdown in Schwann cells fail to myelinate the axons of co-cultured sensory neurons, and inhibition of the kinase activity of LIMKs suppresses the alignment of Schwann cells to axons (Sparrow et al. 2012). These observations suggest that LIMKs activity is required for the phosphorylation of cofilin, which stabilizes the actin cytoskeleton and facilitates the alignment of Schwann cells to the axon. These observations also suggest that the activation of cofilin by SSHs is required to generate the dynamics of the actin cytoskeleton, which causes the membrane of Schwann cells to elongate to wrap around the axon. Muscle consists of specialized structures of actin and myosin II. The importance of cofilin-2 in skeletal muscle


development was shown in humans with congenital myopathy. Point mutations in cofilin-2 in a family of congenital myopathy patients decrease the amount of cofilin-2 protein in muscle and prevent the development of skeletal muscle (Agrawal et al. 2007). To investigate the roles of cofilin-2 in muscle development, a musclespecific cofilin-2 conditional knockout mouse was generated (Agrawal et al. 2012). In cofilin-2 knockout mice, the body and muscle are similar to wild type at birth, but these mice die by postnatal day 8. In embryos, cofilin-1 is expressed in muscle and can compensate for the deletion of cofilin-2. The cofilin-2 knockout muscle shows ballooning degeneration of myofibers, sarcomeric disruptions, and actin accumulation, similar to the muscle of myopathy patients (Agrawal et al. 2012). A study using a C. elegans mutant also showed that the protein uncoordinated (UNC)-60B, the muscle-specific cofilin orthologue, is required for the formation of myofibril in body wall muscle (Ono et al. 1999). CAP1 and related CAP2 are also involved in muscle development. cap2 knockout mice show a cardiac defect characterized by dilated cardiomyopathy (Peche et al. 2013). Loss of CAS-1, which is the C. elegans orthologue of CAP, causes a defect in myofibril assembly of the body wall muscle (Nomura et al. 2012). The roles of cofilin-1 and ADF in kidney development also have been reported. The elongated- and branched-epithelial tubules, including the Wolffian ducts and the ureteric buds, develop in kidney, and the renal epithelial cells form specialized-morphologies in order to function (Dressler 2006). Both cofilin-1 and ADF are expressed in kidney. Knockout of adf causes a duplicated ureter in some mutant mice (Kuure et al. 2010). Although, tissue-specific knockout of cofilin-1 in the Wolffian ducts and the ureteric buds of mice did not affect the development of renal morphology, deletion of both cofilin-1 and adf completely prevented kidney development. Both the hemizygotic deletion of adf gene and the homozygous deletion of cofilin-1 gene caused hypoplasia, resulting in small kidneys and failure to branch the ureteric bud (Kuure et al. 2010). These results indicate that cofilin-1 and ADF play redundant roles in kidney development. The activity of cofilin is essential for kidney development, and a minimal level of cofilin is required for the ureteric buds branching. The podocyte cells, which regulate glomerular filtration, cover the visceral side of the glomerulus with interdigitating, foot-like, actin-rich processes (foot process), resulting in the formation of the slit diaphragm as a filtration barrier at the intercellular junction (Pavenstadt et al. 2003). The intercellular junction receptor nephrin is required for development of the foot processes. Nephrin binds to cofilin and PI3K, and the engagement of nephrin induces dephosphorylation of cofilin via

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SSH1 downstream of PI3K (Garg et al. 2010). The podocyte-specific cofilin-1 knockout mouse gradually develops proteinuria at the age of 3 months and the foot processes of the glomerulus are impaired after the age of 6 months. The defect is not observed until after mice reach 3 months of age because ADF expression, which can functionally substitute for cofilin-1 in early development, gradually decreases after birth (Garg et al. 2010). In zebrafish, the knockdown of cofilin also prevents development of the podocyte foot processes and induces proteinuria (Ashworth et al. 2010). Thus, the activity of cofilin is essential for kidney development, including epithelial tubule formation and branching, and for the barrier function of the glomerulus. The function of ADF in corneal epithelial cells during eye development was found through the presence of a natural variant in mouse (Ikeda et al. 2003). The abnormal thickening of the corneal epithelium is observed in corneal disease-1 (corn1) mutant mouse. Positional cloning identified Adf as the disease gene of corn1. The mutation of adf is a missense mutation in which proline106 is exchanged for serine. The activity of ADF is reduced by the mutation, and F-actin accumulates in the mutant corneal epithelial cells, resulting in blindness (Ikeda et al. 2003). Although, knockout of limk2 did not cause defects in neural tissues or severe defects in any other tissues, mild defects in the testes and in the collective migration of eyelid dermal keratinocyte were reported. The testes of limk2 knockout mice are smaller than those of wild type, and the spermatogenic cells are partially degenerated and more susceptible to heat stress when cultured in vitro (Takahashi et al. 2002). The eyes of mice at birth are closed by eyelid dermal tissue, and LIMK2 is expressed in the eyelid during eye development. The eyelids of newborn limk2 knockout mouse are not closed because the collective migration of epidermal keratinocytes is suppressed (Rice et al. 2012). Cell movements are regulated by the coordinated expression of SSHs and LIMKs. When the activity of SSHs is higher, the dynamics of the actin cytoskeleton increase and cell motility is induced. In contrast, when the activity of LIMKs is higher, the actin cytoskeleton is stabilized and cell motility is inhibited.

The function of cofilin and its regulators in Drosophila development Single genes code for cofilin (Twinstar, Tsr named in Drosophila), Aip1 (Fluer, Flr), CAP (Capulet, cap/Act up, Acu), LIMK, TESK (center divider, Cdi), and SSH in the Drosophila genome. The loss of function mutants of cofilin, aip1, cap1, tesk and slingshot, but not limk, are lethal from embryonic to pupal stages (Benlali et al. 2000; Chen et al. 2001; Niwa et al. 2002; Eaton & ª 2015 Japanese Society of Developmental Biologists

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Davis 2005; Sese et al. 2006; Chu et al. 2012). Tissue defects in several developing stages were analyzed using a thermolabile mutant and an inducible mosaic analysis in the loss- and gain-of-function mutants. The phenotypes of the mutants tend to be more severe than the phenotypes in mammalian mutants because the functional redundancy of related genes is not so strong in Drosophila. The functions of cofilin and its regulators in Drosophila development are listed in Table 1. Neural tissue development and neurite growth The roles of cofilin and its regulators in nervous system were analyzed using the Drosophila mutants named above and transgene experiments. The cofilin mutant impairs proliferation of neuroblast cells and axons growth in the mushroom body of the Drosophila brain (Ng & Luo 2004). Slingshot is required for axon extension, and mutation of limk weakly inhibits the axon growth. Neither Slingshot nor LIMK is required for the proliferation of the neuroblast cells in the mushroom body. Expression of the constitutively active S3A-cofilin mutant partially rescues the proliferation of neuroblast cells and axon growth (Ng & Luo 2004). These results indicate that the cofilin activity is essential for the proliferation of neural cells and axon growth, and that proper axon extension requires phospho-regulation of cofilin. Studies have described the molecular mechanism by which cofilin phosphoregulation regulates axon growth. Loss of sickie, a Drosophila ortholog of the mammalian microtubuleassociated protein neuron navigator 2 (NAV2 MAP), suppresses axon growth of neural cells in the mushroom body (Abe et al. 2014). Sickie, which is a Rac effector protein, is required for decreasing of the phosphorylation level of cofilin in developing neurons via a mechanism that depends on Rac but not on Pak. Cofilin phosphorylation increases in the neural cells of sickie mutants and induces actin over-polymerization, resulting in the inhibition of the axonal outgrowth. Furthermore, the defect caused by the sickie mutation is rescued by overexpression of the active forms of cofilin or slingshot, but not their inactive forms (Abe et al. 2014). These results indicate that axonal outgrowth requires cofilin dephosphorylation by the Rac-Sickie-slingshot pathway, which functions in parallel to the Rac-Pak-LIMK pathway. [Correction added on 8 May 2015, after first online publication: ‘stickie’ has been corrected to ‘sickie’ in the above paragraph.] Ovary development and oogenesis The function of cofilin during the development of the ovary and during oogenesis was investigated using a ª 2015 Japanese Society of Developmental Biologists

thermolabile mutant of cofilin (Chen et al. 2001). Convergent extension of the ovarian terminal-filament cells in the third larval instar is impaired by downregulation of cofilin. At later stages, migration of border cells in the egg chamber during oogenesis is also suppressed (Chen et al. 2001), and the expression of wild type and constitutively active S3A-cofilin, but not dominant negative S3E-cofilin, in which the serine-3 is replaced with glutamic acid, rescues to the migration defect of mutant border cells (Zhang et al. 2011). CAP1 is involved in egg chamber development. Loss of CAP1 causes F-actin accumulation in the apical side of epithelial follicle cells in the egg chamber and inhibits apical construction, resulting in the perturbation of oocyte polarity (Baum et al. 2000; Baum & Perrimon 2001). Spermatogenesis Since cofilin is required for the formation of the cleavage furrow during cytokinesis (Abe et al. 1996), meiotic failure during spermatogenesis is one of the major defects in cofilin mutants (Gunsalus et al. 1995). Although TESK is predominantly expressed in mammalian testes in mammal, its function during spermatogenesis is not well established. Rac1 is required for sperm maturation during Drosophila spermatogenesis. Hemizygotic deletion of tesk enhances the sterility of male rac1 flies. The mutant sperm are motile but cannot enter the seminal vesicle. These results indicate that the Rac-TESK pathway is required for the late stage of Drosophila spermatogenesis (Raymond et al. 2004). Development of epithelial morphogenesis The proper spatiotemporal regulation of actin cytoskeleton reorganization is required for the development of the wing, eye, and epithelial tissues and also for apico-basal and planar cell polarity (PCP). The lethality in cofilin null mutants is rescued by the transformation of thermolabile cofilin mutants (tsr139, tsrV27Q) (Blair et al. 2006). The rescued mutant is viable at 18°C and in these animals the PCP of wing, eye and several epithelia is perturbed. Frizzled and flamingo, which are PCP-related proteins, mislocalize in the mutant epithelial cells. Expression of a constitutively active form of LIMK also perturbs the wing PCP (Blair et al. 2006). Furthermore, cofilin activity is required for retinal cell elongation and morphogenesis (Pham et al. 2008). Slingshot was originally isolated as a gene involved in bristle morphogenesis. Phenotypes of the adult animals that were rescued for the ssh null mutant include bifurcated and twisted bristles and wing hairs, disordered ommatidia, and a lack of interommatidial bristles (Niwa et al. 2002). Other reports show that the


depletion of ssh or expression of tesk suppresses the generation of extra R7 photoreceptors that is caused by transforming the active form sevenless. TESK also is required for apico-basal polarity in photoreceptor cells (Sese et al. 2006). The phenotypes of aip1 mutants were weaker, but similar to those of the cofilin mutants. The wing hairs of aip1 mutant are aplasia, small or bent, and have abnormal polarity (Ren et al. 2007). Ommatidial precluster formation is also perturbed by mutation of aip1, and the defects are rescued by overexpression of cofilin (Chu et al. 2012). Mutation of aip1 and cofilin induces the polymerization and stabilization of the actin cytoskeleton, and decreases the dynamics of the actin cytoskeleton in epithelial cells, resulting in the inhibition of the reorganization of the actin cytoskeleton and the asymmetric localization of PCP proteins. The distinct phenotype of cofilin mutants was also found in cap1 mutants. cap1 loss of function induces over-polymerization of actin in cells in an eye disc and prevents the apical construction of its morphogenetic furrow. This result suggests that CAP1 suppresses the differentiation of premature photoreceptors (Benlali et al. 2000). The relations of upstream signals to the phosphoregulation of cofilin in epithelial morphogenesis have been reported. The overexpression of paxillin (named DpaxA in Drosophila), which is involved in focal adhesion formation, during Drosophila development, disrupts wing and leg morphology through the activation of Rac and the inactivation of Rho. The effects of paxillin expression are suppressed by the expression of LIMK and TESK and by the defective mutation of Slingshot and cofilin (Chen et al. 2005). Loss of function Rho mutants have enlarged lumens of the salivary gland, and the defect is rescued by decreasing the expression of cofilin. Therefore, cofilin and its regulators are required for proper epithelial tissue morphogenesis and for apico-basal and planar cell polarity through actin cytoskeleton reorganization.

Conclusion and perspective Cofilin is primarily essential for the generation of actin cytoskeleton dynamics in cells. Properly controlled cofilin activity spatially and temporally regulates the dynamics of cell morphology and contributes to the morphogenesis of developing tissues and organs. Excess cofilin activity destabilizes various structures in cells such as pseudopodia, polarized cell structures, and cell–substrate and cell–cell adhesions. In contrast, cofilin defect results in the immobilization of the actin cytoskeleton and inhibits the proper morphological changes in cells. The various cofilin-related molecules and the signaling pathways involved in the

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spatiotemporal regulation of cofilin activity in development are described above. The differences in phylogenetic distribution of cofilin, Aip1, CAP, LIMKs, TESKs and SSHs suggest that the original functions of cofilin, such as the regulation of cell division, are critical for the cell viability, and that the phospho-regulation of cofilin was acquired for the more complex function of regulation of actin cytoskeleton reorganization during the evolution of more advanced multicellular organisms. Therefore, although the molecular mechanisms and the functions of cofilin and its regulators in actin filament dynamics in cellular processes of single cultured cells have been unraveled over decades, the roles of the regulation of cofilin activity in multicellular processes and in the morphogenesis in development remain unknown. One result that is still not well understood is why limk1 and limk2 knockouts in mice and flies did not cause severe defects even though depletion of LIMK1 inhibits cell migration, neurite extension, and neurite retraction in cultured cells. It remains unclear whether other molecules compensate for the lack of LIMKs. The functions of TESKs and SSHs in spermatogenesis and epithelial morphogenesis are also unclear. To further characterize these functions, further studies of the tissue- and the developmental stage-specific depletion of LIMKs, TESKs and SSHs are required. Other unknown functions of cofilin also need to be revealed. Recently, cofilin was shown to be involved in the Hippo pathway, which regulates organ size during development. The depletion of cofilin in mammary epithelial cells induces the nuclear import of YAP/TAZ, a key molecule of the Hippo pathway, and promotes cell proliferation through actin polymerization (Aragona et al. 2013). The regulation of cofilin activity in the cells of an organ may determine the volume of the organ by regulating cell number. Furthermore, recent advances in techniques that allow live imaging of cell migration, morphological changes, polarization, proliferation, and death in developing tissues will significantly advance the understanding of the functions of cofilin in development.

Acknowledgments I thank Professor K. Mizuno for helpful suggestions. This work was supported by a Grant-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (23112005 and 25440076 to K.O.).

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Cytochrome P450IIE1: roles in nitrosamine metabolism and mechanisms of regulation.

The roles and regulation of Polycomb complexes in neural development.

The DEK oncoprotein and its emerging roles in gene regulation.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation.

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis.

Structural pathways of cytokines may illuminate their roles in regulation of cancer development and immunotherapy.

Mechanisms of complex transcriptional regulation: implications for brain development.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases.

Regulatory mechanisms for the development of the migratory phenotype: roles for photoperiod and the gonad.

Roles of EphA2 in Development and Disease.

Mechanisms of beta-adrenergic receptor regulation in lungs and its implications for physiological responses.

Roles and regulation of phospholipid scramblases.

Analysis, regulation, and roles of endosomal phosphoinositides.

Activity-dependent dendritic spine shrinkage and growth involve downregulation of cofilin via distinct mechanisms.

Roles for Ca2+ mobilization and its regulation in mast cell functions: recent progress.

Emerging roles and underlying molecular mechanisms of DNAJB6 in cancer.

mTOR signaling and its roles in normal and abnormal brain development.

Molecular mechanisms for generation of neural diversity and specificity: roles of polypeptide factors in development of postmitotic neurons.

Mechanisms of Hsp90 regulation.

[Neuropeptide-mediated transmission of nociceptive information and its regulation. Novel mechanisms of analgesics].

Effects of honey and its mechanisms of action on the development and progression of cancer.

Cofilin-2 phosphorylation and sequestration in myocardial aggregates: novel pathogenetic mechanisms for idiopathic dilated cardiomyopathy.