Kazuya Tsujita

The Tyrosine Kinase Fer Is a Downstream Target of the PLD-PA Pathway that Regulates Cell Migration

Changes in membrane composition stimulate Fer activity to regulate actin remodeling and drive cell migration. Migration FX A key regulator of cell motility is the negatively charged lipid phosphatidic acid (PA), which can be generated from the membrane lipid phosphatidylcholine by phospholipase D (PLD). The mechanisms by which PA and PLD regulate cell migration are not well understood. Focusing on the cytosolic tyrosine kinase Fer, which promotes actin polymerization, Itoh et al. identified a PA-binding domain in Fer that they termed the FX domain. PA binding to Fer enhanced its kinase activity and required positively charged residues in the FX domain. Forced expression of Fer increased lamellipodia formation and migration compared to cells expressing a Fer mutant lacking the positively charged residues in the FX domain; these effects were dependent on PLD activity. Thus, Fer may link changes in membrane composition to actin remodeling events that drive cell migration. Phosphatidic acid (PA), which can be produced by phospholipase D (PLD), is involved in various signaling events, such as cell proliferation, survival, and migration. However, the molecular mechanisms that link PA to cell migration are largely unknown. Here, we show that PA binds to the tyrosine kinase Fer and enhances its ability to phosphorylate cortactin, a protein that promotes actin polymerization. We found that a previously unknown lipid-binding module in Fer adjacent to the F-BAR [Fes-Cdc42–interacting protein 4 (CIP4) homology (FCH) and bin-amphiphysin-Rvs] domain mediated PA binding. We refer to this lipid-binding domain as the FX (F-BAR extension) domain. Overexpression of Fer enhanced lamellipodia formation and cell migration in a manner dependent on PLD activity and the PA-FX interaction. Thus, the PLD-PA pathway promotes cell migration through Fer-induced enhancement of actin polymerization.

Nebulin and N-WASP Cooperate to Cause IGF-1–Induced Sarcomeric Actin Filament Formation

Muscle Building The signaling mechanisms involved in actin filament formation for myofibril formation, which is required for growth factor-induced muscle maturation and hypertrophy, remain unclear. Takano et al. (p. 1536; see the Perspective by Gautel and Ehler) now show that the mechanism involves the interaction of nebulin and N-WASP. N-WASP is an activator of the Arp2/3 complex, which induces branched actin filaments in nonmuscle cells. The nebulin–N-WASP complex formed in muscle, however, causes nucleation of unbranched actin filaments within myofibrils without the Arp2/3 complex. Nebulin–N-WASP–mediated myofibrillar actin filament formation is required for muscle hypertrophy and might explain a congenital hereditary neuromuscular disorder caused by nebulin gene mutation: nemaline myopathy. An alternative signaling mechanism for nucleating unbranched actin filaments is required for skeletal muscle maturation. Insulin-like growth factor 1 (IGF-1) induces skeletal muscle maturation and enlargement (hypertrophy). These responses require protein synthesis and myofibril formation (myofibrillogenesis). However, the signaling mechanisms of myofibrillogenesis remain obscure. We found that IGF-1–induced phosphatidylinositol 3-kinase–Akt signaling formed a complex of nebulin and N-WASP at the Z bands of myofibrils by interfering with glycogen synthase kinase-3β in mice. Although N-WASP is known to be an activator of the Arp2/3 complex to form branched actin filaments, the nebulin–N-WASP complex caused actin nucleation for unbranched actin filament formation from the Z bands without the Arp2/3 complex. Furthermore, N-WASP was required for IGF-1–induced muscle hypertrophy. These findings present the mechanisms of IGF-1–induced actin filament formation in myofibrillogenesis required for muscle maturation and hypertrophy and a mechanism of actin nucleation.

SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain

The newly identified SYLF lipid-binding domain of SH3YL1 mediates phosphoinositide binding during dorsal membrane morphogenesis.

SGIP1α Is an Endocytic Protein That Directly Interacts with Phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1α. SGIP1α bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1α furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1α was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1α was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1α at clathrin-coated pits/vesicles. SGIP1α overexpression reduced transferrin and EGF endocytosis. SGIP1α knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1α plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.

Phosphoinositides in the regulation of actin cortex and cell migration.

In order for the cell to function well within a multicellular system, the mechanical properties of the plasma membrane need to meet two different requirements: cell shape maintenance and rearrangement. To achieve these goals, phosphoinositides play key roles in the regulation of the cortical actin cytoskeleton. PI(4,5)P₂is the most abundant phosphoinositide species in the plasma membrane. It maintains cell shape by linking the actin cortex to the membrane via interactions with Ezrin/Radixin/Moesin (ERM) proteins and class I myosins. Although the role of D3-phosphoinositides, such as PI(3,4,5)P₃, in actin-driven cell migration has been a subject of controversy, it becomes evident that the dynamic turnover of the phosphoinositide by the action of metabolizing enzymes, such as 5-phosphatases, is necessary. Recent studies have revealed an important role of PI(3,4)P₂in podosome/invadopodia formation, shedding new light on the actin-based organization of membrane structures regulated by phosphoinositide signaling. This article is part of a Special Issue entitled Phosphoinositides.

Physicochemical analysis from real-time imaging of liposome tubulation reveals the characteristics of individual F-BAR domain proteins.

The Fer-CIP4 homology-BAR (F-BAR) domain, which was identified as a biological membrane-deforming module, has been reported to transform lipid bilayer membranes into tubules. However, details of the tubulation process, the mechanism, and the properties of the generated tubules remain unknown. Here, we successfully monitored the entire process of tubulation and the behavior of elongated tubules caused by four different F-BAR domain family proteins (FBP17, CIP4, PSTPIP1, and Pacsin2) using direct real-time imaging of giant unilamellar liposomes with dark-field optical microscopy. FBP17 and CIP4 develop many protrusions simultaneously over the entire surface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only a few protrusions from a narrow restricted part of the surface of individual liposomes. Tubules formed by FBP17 or CIP4 have higher bending rigidities than those formed by PSTPIP1 or Pacsin2. The results provide striking evidence that these four F-BAR domain family proteins should be classified into two groups: one group of FBP17 and CIP4 and another group of PSTPIP1 and Pacsin2. This classification is consistent with the phylogenetic proximity among these proteins and suggests that the nature of the respective tubulation is associated with biological function. These findings aid in the quantitative assessment with respect to manipulating the morphology of lipid bilayers using membrane-deforming proteins.

Proteome of Acidic Phospholipid-binding Proteins SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Whereas Coronin 1A co-localizes with PI(4,5)P2 at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P2 signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P2-containing liposomes and that PI(4,5)P2 antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.

ARAP1 regulates the ring size of circular dorsal ruffles through Arf1 and Arf5

Circular dorsal ruffles (CDRs) are highly dynamic F-actin–based membrane structures involved in bulk endocytosis of membrane receptors and macropinocytosis. A key role of ARAP1, an Arf GAP protein, is found to be in the control of the ring size of CDRs.

Characterization of the EFC/F-BAR domain protein, FCHO2.

We have previously shown that SGIP1α is an endocytic protein specifically expressed in neural tissues. SGIP1α has a lipid‐binding domain called the MP domain, which shows no significant homology to any other domains. In this study, we characterized FCHO2, a protein with a high level of homology to SGIP1α. FCHO2 lacks the MP domain but has another lipid‐binding domain, the EFC/F‐BAR domain. FCHO2 was ubiquitously expressed. The FCHO2 EFC domain bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubes. FCHO2 localized to clathrin‐coated pits at the plasma membrane and bound to Eps15, an important adaptor protein in clathrin‐mediated endocytosis. FCHO2 knockdown reduced transferrin endocytosis. These results suggest that FCHO2 regulates clathrin‐mediated endocytosis through its interactions with membranes and Eps15. These properties of FCHO2 are similar to those of SGIP1α. FCHO2 is likely to be a ubiquitous homologue of SGIP1α. We furthermore found that FCHO2 was subjected to monoubiquitination, and gel filtration analysis showed that FCHO2 formed an oligomer. These new properties might also contribute to the role of FCHO2 in clathrin‐mediated endocytosis.

Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

Summary FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.