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.

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.

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.

FBP17-mediated finger-like membrane protrusions in cell competition between normal and RasV12-transformed cells.

At the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic. We first show by electron microscopic analysis that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface. In addition, FBP17, a member of the F-BAR proteins, accumulates in RasV12 cells, as well as surrounding normal cells, which plays a positive role in the formation of finger-like protrusions and apical elimination of RasV12 cells. Furthermore, cdc42 acts upstream of these processes. These results suggest that the cdc42/FBP17 pathway is a crucial trigger of cell competition, inducing "protrusion to protrusion response" between normal and RasV12-transformed cells.

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.

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.

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.

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.

A curvature-dependent membrane binding by tyrosine kinase Fer involves an intrinsically disordered region.

Tyrosine kinases are important enzymes that mediate signal transduction at the plasma membrane. While the significance of membrane localization of tyrosine kinases has been well evaluated, the role of membrane curvature in their regulation is unknown. Here, we demonstrate that an intrinsically disordered region in the tyrosine kinase Fer acts as a membrane curvature sensor that preferentially binds to highly curved membranes in vitro. This region forms an amphipathic α-helix upon interaction with curved membranes, aligning hydrophobic residues on one side of the helical structure. Further, the tyrosine kinase activity of Fer is significantly enhanced by the membrane in a manner dependent on curvature. We propose a model for the regulation of Fer based on an intramolecular interaction and the curvature-dependent membrane binding mediated by its intrinsically disordered region.

IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.

Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2.

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.

Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIß, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus.

srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity.

The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

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.

The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration.

Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front-rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2-containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and 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.

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes.

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.

Regulation of IGF-1/PI3K/Akt signalling by the phosphoinositide phosphatase pharbin.

Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.

Mutation of ARHGAP9 in patients with coronary spastic angina.

Coronary artery spasm has an important function in the etiology of variant angina and other acute coronary syndromes. Abnormal activation of Rho-family GTPases has been observed in cardiovascular disorders, but the function of genetic variability in Rho-family GTPases remains to be evaluated in cardiovascular disorders. We examined the genetic variability of Rho-family GTPases and their regulators in coronary artery spasm. We performed a comprehensive candidate gene analysis of 67 single nucleotide polymorphisms with amino-acid substitution in Rho-family GTPases and their regulators in 103 unrelated Japanese patients with acetylcholine-induced coronary artery spasm and 102 control Japanese subjects without acetylcholine-induced coronary artery spasm. We noted an association of the single nucleotide polymorphism of ARHGAP9 (rs11544238, Ala370Ser) with coronary artery spasm (odds ratio =2.67). We found that ARHGAP9 inactivated Rac as RacGAP and that the mRNA level of ARHGAP9 was strongly detected in hematopoietic cells. ARHGAP9 negatively regulated cell migration. The Ala370Ser polymorphism counteracted ARHGAP9-reduced cell migration, spreading and adhesion. The Ala370Ser polymorphism in the ARHGAP9 gene is associated with coronary artery spasm. These data suggest that the polymorphism of ARHGAP9 has a critical function in the infiltration of hematopoietic cells into the endothelium and inflammation leading to endothelial dysfunction.

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.