Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers

Identification of multiple actin-binding sites in cofilin-phosphatase Slingshot-1L

AbstractSlingshot-1L (SSH1L) is a phosphatase that specifically dephosphorylates and activates cofilin, an actin-severing and -depolymerizing protein. SSH1L binds to and is activated by F-actin in vitro, and co-localizes with F-actin in cultured cells. We examined the F-actin-binding activity, F-actin-mediated phosphatase activation, and subcellular distribution of various mutants of SSH1L. We identified three sites involved in F-actin binding of SSH1L: Trp-458 close to the C-terminus of the phosphatase domain, an LHK motif in the N-terminal region, and an LKR motif in the C-terminal region. These sites play unique roles in the control of subcellular localization and F-actin-mediated activation of SSH1L

Cloning of CRP2, a novel member of the cysteine-rich protein family with two repeats of an unusual LIM/double zinc-finger motif

AbstractThe cDNA coding for a novel member of the cysteine-rich protein family was isolated from a rat brain cDNA library. It encodes a protein, denoted cysteine-rich protein 2 (CRP2), of 208 amino acid residues containing two tandem repeats of an unusual LIM/double zinc-finger-like motif. The ubiquitous tissue distribution and high level of expression of CRP2 mRNA suggest an important role for CRP2 in cell functions

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)–1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement

Cofilin Phosphorylation by Protein Kinase Testicular Protein Kinase 1 and Its Role in Integrin-mediated Actin Reorganization and Focal Adhesion Formation

科研費報告書収録論文(課題番号:11480213・基盤研究(B)(2)・H11～H12/研究代表者:水野, 健作/LIMキナーゼによるアクチン細胞骨格の制御機構の解明

A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases ∼10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1β induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin–rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1β and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm

Intercellular Adhesion Molecule-5 Induces Dendritic Outgrowth by Homophilic Adhesion

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte b2-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4–5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5–expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes