SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor.

Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanism underlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting into MVBs in a manner dependent on its C-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB sorting and EGFR degradation mediated by ESCRT complexes.

Non-cell-autonomous migration of RasV12-transformed cells towards the basal side of surrounding normal cells.

Oncogenic transformation enables cells to behave differently from their neighboring normal cells. Both cancer and normal cells recognize each other, often promoting the extrusion of the former from the epithelial cell layer. Here, we show that RasV12-transformed normal rat kidney 52E (NRK-52E) cells are extruded towards the basal side of the surrounding normal cells, which is concomitant with enhanced motility. The active migration of the basally extruded RasV12 cells is observed when surrounded by normal cells, indicating a non-cell-autonomous mechanism. Furthermore, specific inhibitor treatment and knockdown experiments elucidate the roles of PI3K and myosin IIA in the basal extrusion of Ras cells. Our findings reveal a new aspect of cancer cell invasion mediated by functional interactions with surrounding non-transformed cells.

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.

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

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.

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.

Exploring membrane mechanics: The role of membrane-cortex attachment in cell dynamics.

The role of plasma membrane (PM) tension in cell dynamics has gained increasing interest in recent years to understand the mechanism by which individual cells regulate their dynamic behavior. Membrane-to-cortex attachment (MCA) is a component of apparent PM tension, and its assembly and disassembly determine the direction of cell motility, controlling the driving forces of migration. There is also evidence that membrane tension plays a role in malignant cancer cell metastasis and stem cell differentiation. Here, we review recent important discoveries that explore the role of membrane tension in the regulation of diverse cellular processes, and discuss the mechanisms of cell dynamics regulated by this physical parameter.

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.

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins.

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

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 1alpha1), 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)P(2)). Whereas Coronin 1A co-localizes with PI(4,5)P(2) at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P(2) signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P(2)-containing liposomes and that PI(4,5)P(2) 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.

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis.

The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.

PTEN is required for the migration and invasion of Ras-transformed MDCK cells.

The balance between phosphoinositides distributed at specific sites in the plasma membrane causes polarized actin polymerization. Oncogenic transformations affect this balance by regulating phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), causing metastatic behavior in cancer cells. Here, we show that the PTEN tumor suppressor gene is required for epithelial cancer cell invasion. Loss of PTEN in Ras-transformed MDCK cells suppressed their migratory phenotype in collagen gel and invasion through Matrigel. Rescue experiments showed a requirement for the C2 domain-mediated membrane recruitment of PTEN, which is typically observed at the rear side of invading cancer cells. These findings support the role of PTEN in suppression of unwanted leading edges necessary for efficient migration of epithelial cancer cells.

Homeostatic membrane tension constrains cancer cell dissemination by counteracting BAR protein assembly.

Malignancy is associated with changes in cell mechanics that contribute to extensive cell deformation required for metastatic dissemination. We hypothesized that the cell-intrinsic physical factors that maintain epithelial cell mechanics could function as tumor suppressors. Here we show, using optical tweezers, genetic interference, mechanical perturbations, and in vivo studies, that epithelial cells maintain higher plasma membrane (PM) tension than their metastatic counterparts and that high PM tension potently inhibits cancer cell migration and invasion by counteracting membrane curvature sensing/generating BAR family proteins. This tensional homeostasis is achieved by membrane-to-cortex attachment (MCA) regulated by ERM proteins, whose disruption spontaneously transforms epithelial cells into a mesenchymal migratory phenotype powered by BAR proteins. Consistently, the forced expression of epithelial-mesenchymal transition (EMT)-inducing transcription factors results in decreased PM tension. In metastatic cells, increasing PM tension by manipulating MCA is sufficient to suppress both mesenchymal and amoeboid 3D migration, tumor invasion, and metastasis by compromising membrane-mediated mechanosignaling by BAR proteins, thereby uncovering a previously undescribed mechanical tumor suppressor mechanism.

An influenza-derived membrane tension-modulating peptide regulates cell movement and morphology via actin remodeling.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.

Thin layer chromatography-blotting, a novel method for the detection of phosphoinositides.

Phosphoinositides are believed to be involved in fundamental cellular events such as signal transduction and vesicular trafficking. Aberrant metabolisms of this lipid, caused by mutations in phosphoinositide kinases, phosphatases and lipases are known to be related to variety of human disorders such as diabetes and cancer. While the majority of such information is obtained by analyzing genetic and biochemical properties of phosphoinositide-metabolic enzymes, direct measurement of cellular content of the lipid is hindered by the lack of a simple method that is sensitive enough to measure phosphoinositides present in trace amounts in vivo. Here, we describe a novel, thin layer chromatography (TLC)-based method by which cellular phosphoinositides are separated, transferred and detected by specific phosphoinositide-binding domains. This method was applied to follow the generation of minor phosphoinositides, such as PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to insulin and to compare PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels in several cancer cell lines. The method has potential application not only in investigating the physiological roles of phosphoinositides, but also in diagnosing metabolic disease and cancer by directly assessing phosphoinositide levels in samples obtained from patients.

Feedback regulation between plasma membrane tension and membrane-bending proteins organizes cell polarity during leading edge formation.

Tension applied to the plasma membrane (PM) is a global mechanical parameter involved in cell migration. However, how membrane tension regulates actin assembly is unknown. Here, we demonstrate that FBP17, a membrane-bending protein and an activator of WASP/N-WASP-dependent actin nucleation, is a PM tension sensor involved in leading edge formation. In migrating cells, FBP17 localizes to short membrane invaginations at the leading edge, while diminishing from the cell rear in response to PM tension increase. Conversely, following reduced PM tension, FBP17 dots randomly distribute throughout the cell, correlating with loss of polarized actin assembly on PM tension reduction. Actin protrusive force is required for the polarized accumulation, indicating a role for FBP17-mediated activation of WASP/N-WASP in PM tension generation. In vitro experiments show that FBP17 membrane-bending activity depends on liposomal membrane tension. Thus, FBP17 is the local activator of actin polymerization that is inhibited by PM tension in the feedback loop that regulates cell migration.

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

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity-dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1-Arf1/5 pathway.

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

Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

Nebulin and N-WASP cooperate to cause IGF-1-induced sarcomeric actin filament formation.

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-3ß 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.

The tyrosine kinase Fer is a downstream target of the PLD-PA pathway that regulates 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.