PIP2 signaling,an integrator of cell polarity and vesicle trafficking in directionally migrating cells

Cell migration is a fundamental cellular process required for embryonic development to wound healing and also plays a key role in tumor metastasis and atherosclerosis. Migration is regulated at multiple strata, from cytoskeletal reorganization to vesicle trafficking. In migrating cells, signaling pathways are integrated with vesicle trafficking machineries in a highly coordinated fashion to accomplish the recruitment and trafficking of the trans-membrane proteins toward the leading edge. Different signaling molecules regulate cell migration in different physio-pathological contexts, among them, phosphatidylinositol-4,5-biphosphate (PIP2) is an integral component of the plasma membrane and pleiotropic lipid signaling molecule modulating diverse biological processes, including actin cytoskeletal dynamics and vesicle trafficking required for cell migration. In this commentary, we provide a brief overview of our current understandings on the phosphoinositide signaling and its implication in regulation of cell polarity and vesicle trafficking in migrating cells. In addition, we highlight the coordinated role of PIPKIγi2, a focal adhesion-targeted enzyme that synthesizes PIP2, and the exocyst complex, a PIP2-effector, in the trafficking of E-cadherin in epithelial cells and integrins in migrating cancer cells.     

Navigating the cell

Changes in cell shape are associated with a variety of processes including cell migration, axon outgrowth, cell division, and vesicle trafficking. C. elegans UNC-53 and its vertebrate homologs, the Navigators, are required for the migration of cells and the outgrowth of neuronal processes. The identification of novel molecular interactions and live imaging studies have revealed that UNC-53/NAVs are signal transducers associated with actin filaments, microtubules, and intermediate filaments. In addition to modulating cytoskeletal dynamics at the leading edge of migrating or outgrowing cells, both UNC-53 and the navigators are expressed in adult cells, conspicuously those with specialized roles in endocytosis or secretion. Collectively, these results suggest that UNC-53/NAVs may be a central regulator of cytoskeletal dynamics, responsible for integrating signaling cues to multiple components of the cytoskeleton to coordinate rearrangement during cell outgrowth or trafficking.     

Phosphoinositide signaling regulates the exocyst complex and polarized integrin trafficking in directionally migrating cells

Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP(2)-synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration, PIPKIγi2 via PIP(2) generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP(2) requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP(2) binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration.     

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.     

RalB mobilizes the exocyst to drive cell migration

The Ras family GTPases RalA and RalB have been defined as central components of the regulatory machinery supporting tumor initiation and progression. Although it is known that Ral proteins mediate oncogenic Ras signaling and physically and functionally interact with vesicle trafficking machinery, their mechanistic contribution to oncogenic transformation is unknown. Here, we have directly evaluated the relative contribution of Ral proteins and Ral effector pathways to cell motility and directional migration. Through loss-of-function analysis, we find that RalA is not limiting for cell migration in normal mammalian epithelial cells. In contrast, RalB and the Sec6/8 complex or exocyst, an immediate downstream Ral effector complex, are required for vectorial cell motility. RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells. We propose that RalB regulation of exocyst function is required for the coordinated delivery of secretory vesicles to the sites of dynamic plasma membrane expansion that specify directional movement.     

DENND2B activates Rab13 at the leading edge of migrating cells and promotes metastatic behavior

The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer–based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell’s leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.     

A membrane fusion protein,Ykt6, regulates epithelial cell migration via microRNA-mediated suppression of Junctional Adhesion Molecule A

Vesicle trafficking regulates epithelial cell migration by remodeling matrix adhesions and delivering signaling molecules to the migrating leading edge. Membrane fusion, which is driven by soluble N-ethylmaleimide-sensitive factor associated receptor (SNARE) proteins, is an essential step of vesicle trafficking. Mammalian SNAREs represent a large group of proteins, but few have been implicated in the regulation of cell migration. Ykt6 is a unique SNARE existing in equilibrium between active membrane-bound and inactive cytoplasmic pools, and mediating vesicle trafficking between different intracellular compartments. The biological functions of this protein remain poorly understood. In the present study, we found that Ykt6 acts as a negative regulator of migration and invasion of human prostate epithelial cells. Furthermore, Ykt6 regulates the integrity of epithelial adherens and tight junctions. The observed anti-migratory activity of Ykt6 is mediated by a unique mechanism involving the expressional upregulation of microRNA 145, which selectively decreases the cellular level of Junctional Adhesion Molecule (JAM) A. This decreased JAM-A expression limits the activity of Rap1 and Rac1 small GTPases, thereby attenuating cell spreading and motility. The described novel functions of Ykt6 could be essential for the regulation of epithelial barriers, epithelial repair, and metastatic dissemination of cancer cells.     

Planar polarity pathway and Nance-Horan syndrome-like 1b have essential cell-autonomous functions in neuronal migration

Components of the planar cell polarity (PCP) pathway are required for the caudal tangential migration of facial branchiomotor (FBM) neurons, but how PCP signaling regulates this migration is not understood. In a forward genetic screen, we identified a new gene, nhsl1b, required for FBM neuron migration. nhsl1b encodes a WAVE-homology domain-containing protein related to human Nance-Horan syndrome (NHS) protein and Drosophila GUK-holder (Gukh), which have been shown to interact with components of the WAVE regulatory complex that controls cytoskeletal dynamics and with the polarity protein Scribble, respectively. Nhsl1b localizes to FBM neuron membrane protrusions and interacts physically and genetically with Scrib to control FBM neuron migration. Using chimeric analysis, we show that FBM neurons have two modes of migration: one involving interactions between the neurons and their planar-polarized environment, and an alternative, collective mode involving interactions between the neurons themselves. We demonstrate that the first mode of migration requires the cell-autonomous functions of Nhsl1b and the PCP components Scrib and Vangl2 in addition to the non-autonomous functions of Scrib and Vangl2, which serve to polarize the epithelial cells in the environment of the migrating neurons. These results define a role for Nhsl1b as a neuronal effector of PCP signaling and indicate that proper FBM neuron migration is directly controlled by PCP signaling between the epithelium and the migrating neurons.     

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex,as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.     

Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking

Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton, and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity, and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.     

Phosphatidylinositol (4,5) bisphosphate controls T cell activation by regulating T cell rigidity and organization

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.     

磷脂酰肌醇-4,5-二磷酸调控细胞迁移的研究进展

磷脂酰肌醇-4,5-二磷酸(phosphatidylinositol-4,5-bisphosphate,PIP2)是细胞膜上一种重要的磷脂酰肌醇,通过作为第二信使前体及自身信号分子的作用,控制其效应物的靶向定位和活性从而调节细胞迁移、囊泡运输、细胞形态发生、信号传导等过程.细胞迁移异常会导致人类多种疾病包括神经发育异常、阿尔茨海默病、癌症和纤毛疾病等.作为细胞骨架的调节剂,PIP2在细胞迁移的关键作用已经被广泛证实,本文将从由PIP5KIs介导的PIP2产生与踝蛋白、Rho家族小GTP酶等效应物关联调节黏附作用和肌动蛋白聚合的角度,讨论PIP2在细胞迁移中发挥作用的具体机制.     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.Key words: BAR domain, MIM, directed cell migration, competition, membrane dynamics, actin cytoskeleton, endocytosis     

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.     

The activation loop of phosphatidylinositol phosphate kinases determines signaling specificity

Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking. However, little is known about the temporal and spatial regulation of its synthesis. Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate. These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases. While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant. Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases. Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels.     

PKCθ Regulates T Cell Motility via Ezrin-Radixin-Moesin Localization to the Uropod

Cell motility is a fundamental process crucial for function in many cell types, including T cells. T cell motility is critical for T cell-mediated immune responses, including initiation, activation, and effector function. While many extracellular receptors and cytoskeletal regulators have been shown to control T cell migration, relatively few signaling mediators have been identified that can modulate T cell motility. In this study, we find a previously unknown role for PKCθ in regulating T cell migration to lymph nodes. PKCθ localizes to the migrating T cell uropod and regulates localization of the MTOC, CD43 and ERM proteins to the uropod. Furthermore, PKCθ-deficient T cells are less responsive to chemokine induced migration and are defective in migration to lymph nodes. Our results reveal a novel role for PKCθ in regulating T cell migration and demonstrate that PKCθ signals downstream of CCR7 to regulate protein localization and uropod formation.     

Nonpolarized signaling reveals two distinct modes of 3D cell migration

We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.