EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP₂; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP₂ in living cells. We show that EGF induces a rapid loss of PIP₂ through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.     

A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP2) Inhibition

Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP₂). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP₂- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP₂. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP₂ inhibition, resulting in enhanced actin dynamics.     

Nonmuscle myosin IIA is required for lamellipodia formation through binding to WAVE2 and phosphatidylinositol 3,4,5-triphosphate

Investigation of the mechanism underlying cell membrane-targeted WAVE2 capture by phosphatidylinositol 3,4,5-triphosphate (PIP₃) through IRSp53 revealed an unidentified 250-kDa protein (p250) bound to PIP₃. We identified p250 as nonmuscle myosin IIA heavy chain (MYH9) by mass spectrometry and immunoblot analysis using anti-MYH9 antibody. After stimulation with insulin-like growth factor I (IGF-I), MYH9 colocalized with PIP₃ in lamellipodia at the leading edge of cells. Depletion of MYH9 expression by small interfering RNA (siRNA) and inhibition of myosin II activity by blebbistatin abrogated the formation of actin filament (F-actin) arcs and lamellipodia induced by IGF-I. MYH9 was constitutively associated with WAVE2, which was dependent on myosin II activity, and the MYH9-WAVE2 complex colocalized to PIP₃ at the leading edge after IGF-I stimulation. These results indicate that MYH9 is required for lamellipodia formation since it provides contractile forces and tension for the F-actin network to form convex arcs at the leading edge through constitutive binding to WAVE2 and colocalization with PIP₃ in response to IGF-I.     

Interaction of maize actin-depolymerising factor with actin and phosphoinositides and its inhibition of plant phospholipase C.

We have previously reported the isolation of three Zea mays genes that encode actin-depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of Acanthamoeba actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP₂), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP₂ is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.     

Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.     

Regulation of sperm motility by PIP2(4,5) and actin polymerization

Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. In previous studies, we demonstrated that the increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP₂). Here, we showed for the first time the involvement of PIP₂/gelsolin in human sperm motility before and during capacitation. Activation of gelsolin by causing its release from PIP₂ inhibited sperm motility, which could be restored by adding PIP₂ to the cells. Reduction of PIP₂ synthesis inhibited actin polymerization and motility, and increasing PIP₂ synthesis enhanced these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP₂ and F-actin. During capacitation there was an increase in PIP₂ and F-actin levels in the sperm head and a decrease in the tail. In sperm with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends on its binding to PIP₂. Activation of phospholipase C by Ca²⁺-ionophore or by activating the epidermal-growth-factor-receptor inhibits tyrosine phosphorylation of gelsolin. In conclusion, the data indicate that the increase of PIP₂ and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result the decrease of gelsolin in the tail allows keeping high level of F-actin in the tail, which is essential for the development of HA motility.     

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1

Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H⁺ efflux by a Na⁺-H⁺ exchanger (NHE; Ddnhe1⁻), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1⁻ cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1⁻ cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H⁺ efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1⁻ cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1⁻ cells is determined primarily by loss of cofilin-dependent actin dynamics.     

Activation of F-actin binding capacity of ezrin: synergism of PIP₂ interaction and phosphorylation

Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP₂). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP₂ binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP₂, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP₂ was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP₂, which is enhanced by the phosphorylation.     

Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.     

The Switch-associated Protein 70 (SWAP-70) Bundles Actin Filaments and Contributes to the Regulation of F-actin Dynamics

Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.     

Identification of a cofilin-like actin-binding site on translationally controlled tumor protein (TCTP)

Translationally controlled tumor protein (TCTP) expression is suppressed during cancer cell reversion to a non-malignant phenotype. We identified a primary sequence of TCTP with homology to ADF/cofilin. We confirm that a synthetic peptide corresponding to this sequence binds specifically to actin and is displaced from actin by cofilin. TCTP peptide has higher affinity for G-actin than F-actin and does not block actin-filament depolymerization by cofilin. These results suggest that TCTP may channel active cofilin to F-actin, enhancing the cofilin-activity cycle in invasive tumor cells. Loss of TCTP may result in sequestration of active cofilin by a monomeric pool of actin.     

Molecular cloning and functional expression of chromaffin cell scinderin indicates that it belongs to the family of Ca2+-dependent F-actin severing proteins

Scienderin is a Ca⁺-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca²⁺ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP₂) and actin in a Ca⁺-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP₂ binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca²⁺-dependent F-actin severing proteins which includes gelsolin and villin.Abbreviations PIP₂ phosphatidylinositol 4,5 bisphosphate - PKC protein kinase C - Sc scinderin - PS phosphatidyl serine - F-Sc scinderin fusion protein - PCR polymerase chain reaction     

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂‐generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4‐phosphate 5‐kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.     

Phosphatidylinositol 4,5-Bisphosphate Alters the Number of Attachment Sites between Ezrin and Actin Filaments: A COLLOIDAL PROBE STUDY*

Direct linkage between the plasma membrane and the actin cytoskeleton is controlled by the protein ezrin, a member of the ezrin-radixin-moesin protein family. To function as a membrane-cytoskeleton linker, ezrin needs to be activated in a process that involves binding of ezrin to phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphorylation of a conserved threonine residue. Here, we used colloidal probe microscopy to quantitatively analyze the interaction between ezrin and F-actin as a function of these activating factors. We show that the measured individual unbinding forces between ezrin and F-actin are independent of the activating parameters, in the range of approximately 50 piconewtons. However, the cumulative adhesion energy greatly increases in the presence of PIP₂ demonstrating that a larger number of bonds between ezrin and F-actin has formed. In contrast, the phosphorylation state, represented by phosphor-mimetic mutants of ezrin, only plays a minor role in the activation process. These results are in line with in vivo experiments demonstrating that an increase in PIP₂ concentration recruits more ezrin to the apical plasma membrane of polarized cells and significantly increases the membrane tension serving as a measure of the adhesion sites between the plasma membrane and the F-actin network.     

Modeling the Synergy of Cofilin and Arp2/3 in Lamellipodial Protrusive Activity

Rapid polymerization of actin filament barbed ends generates protrusive forces at the cell edge, leading to cell migration. Two important regulators of free barbed ends, cofilin and Arp2/3, have been shown to work in synergy (net effect greater than additive). To explore this synergy, we model the dynamics of F-actin at the leading edge, motivated by data from EGF-stimulated mammary carcinoma cells. We study how synergy depends on the localized rates and relative timing of cofilin and Arp2/3 activation at the cell edge. The model incorporates diffusion of cofilin, membrane protrusion, F-actin capping, aging, and severing by cofilin and branch nucleation by Arp2/3 (but not G-actin recycling). In a well-mixed system, cofilin and Arp2/3 can each generate a large pulse of barbed ends on their own, but have little synergy; high synergy occurs only at low activation rates, when few barbed ends are produced. In the full spatially distributed model, both synergy and barbed-end production are significant over a range of activation rates. Furthermore, barbed-end production is greatest when Arp2/3 activation is delayed relative to cofilin. Our model supports a direct role for cofilin-mediated actin polymerization in stimulated cell migration, including chemotaxis and cancer invasion.     

Modeling the Synergy of Cofilin and Arp2/3 in Lamellipodial Protrusive Activity

Rapid polymerization of actin filament barbed ends generates protrusive forces at the cell edge, leading to cell migration. Two important regulators of free barbed ends, cofilin and Arp2/3, have been shown to work in synergy (net effect greater than additive). To explore this synergy, we model the dynamics of F-actin at the leading edge, motivated by data from EGF-stimulated mammary carcinoma cells. We study how synergy depends on the localized rates and relative timing of cofilin and Arp2/3 activation at the cell edge. The model incorporates diffusion of cofilin, membrane protrusion, F-actin capping, aging, and severing by cofilin and branch nucleation by Arp2/3 (but not G-actin recycling). In a well-mixed system, cofilin and Arp2/3 can each generate a large pulse of barbed ends on their own, but have little synergy; high synergy occurs only at low activation rates, when few barbed ends are produced. In the full spatially distributed model, both synergy and barbed-end production are significant over a range of activation rates. Furthermore, barbed-end production is greatest when Arp2/3 activation is delayed relative to cofilin. Our model supports a direct role for cofilin-mediated actin polymerization in stimulated cell migration, including chemotaxis and cancer invasion.     

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.     

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP₃) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP₃ or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP₃ that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP₃-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP₃. IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP₃ at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP₃ that is produced near locally activated IGF-IR in response to IGF-I.     

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.