PIP3-dependent macropinocytosis is incompatible with chemotaxis

In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3) has been particularly controversial. PIP3 has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP3 is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP3. PIP3 patches in these cells show no directional bias, and overall only PIP3-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP3 patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP3 patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP3 promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells

Crystal structure of the dynamin tetramer

The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover

Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch

Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature

The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP–WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX–BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease–associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.J.L. Gallop is supported by a Wellcome Trust Research Career Development Fellowship (grant WT095829AIA). F.  Daste, A.  Walrant, J.R. Gadsby, and J. Mason are supported by an H2020 European Research Council Starting Grant (281971) awarded to J.L. Gallop. Gurdon Institute funding is provided by the Wellcome Trust (grant 092096) and Cancer Research UK (grant C6946/A14492). The Swedish Medical Research Council and the Swedish Foundation for Strategic Research supported the work of M.R. Holst and R. Lundmark. S.F. Lee is funded by a Royal Society University Research Fellowship (grant UF120277). M. Mettlen is funded by grant MH73125 to Sandra L. Schmid (University of Texas Southwestern Medical Center)

Crystal structure of nucleotide-free dynamin

Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function

Inactivation of class II PI3K-C2 alpha induces leptin resistance, age-dependent insulin resistance and obesity in male mice

AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request

Local actin polymerization during endocytic carrier formation

Extracellular macromolecules, pathogens and cell surface proteins rely on endocytosis to enter cells. Key steps of endocytic carrier formation are cargo molecule selection, plasma membrane folding and detachment from the cell surface. While dedicated proteins mediate each step, the actin cytoskeleton contributes to all. However, its role can be indirect to the actual molecular events driving endocytosis. Here, we review our understanding of the molecular steps mediating local actin polymerization during the formation of endocytic carriers. Clathrin-mediated endocytosis (CME) is the least reliant on local actin polymerization, as it is only engaged to counter forces induced by membrane tension or cytoplasmic pressure. Two opposite situations are coated pit formation in yeast and at the basolateral surface of polarized mammalian cells which are respectively dependent and independent on actin polymerization. Conversely, Clathrin-independent endocytosis (CIE) forming both nanometer (CLIC/GEEC, caveolae, FEME, IL2β uptake) and micrometer carriers (macropinocytosis) are dependent on actin polymerization to power local membrane deformation and carrier budding. A variety of endocytic adaptors can recruit and activate the Cdc42/N-WASP or Rac1/WAVE complexes, which in turn engage the Arp2/3 complex, thereby mediating local actin polymerization at the membrane. However, the molecular steps for RhoA and formin-mediated actin bundling during endocytic pit formation remains unclear

Räumlich-zeitliche Kontrolle der Clathrin-vermittelten Endozytose durch PI3K C2α und Phosphatidylinositol-3,4-bisphosphat

Phosphoinositides (PIs) are rare lipids of the cytoplasmic leaflet of eukaryotic membranes. They serve as spatiotemporal signposts directing proteins to distinct subcellular compartments or membrane domains and thereby contribute to defining membrane identity. Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is most abundant at the plasma membrane where it regulates signaling, ion channels, actin dynamics, and clathrin-mediated endocytosis (CME). The nucleation and assembly of clathrin-coated pits (CCPs) depends on PI(4,5)P2. After membrane fission, PI(4,5)P2 hydrolysis triggers vesicle uncoating and newly formed vesicles fuse with the early endosomal compartment. Endosomes are highly enriched in phosphatidylinositol-3-phosphate [PI(3)P] and maintenance and functionality of these organelles both require PI(3)P. However, the mechanism of PI conversion on the endocytic route from a PI(4,5)P2 to a PI(3)P enriched membrane is still enigmatic. Here, we show that phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] is a novel lipid regulator of CME that is required for the maturation of CCPs. Cells depleted of PI(3,4)P2 by means of the PI(3,4)P2-specific 4-phosphatase INPP4B display endocytic defects characterized by long-lived CCPs. This effect is different from PI(4,5)P2-controlled initiation of CCP formation and demonstrates a dual lipid requirement for the sequential generation of PI(4,5)P2 and PI(3,4)P2 during CME. We further identify the clathrin-associated class II phosphatidylinositol-3-kinase, PI3K C2α, as the enzyme that synthesizes PI(3,4)P2 at CCPs. RNA interference-mediated depletion of PI3K C2α phenocopies effects seen in cells enzymatically depleted of PI(3,4)P2 and results in a partial loss of PI(3,4)P2 from the plasma membrane. Detailed characterization suggested a late maturation defect of CCPs preceding dynamin-mediated fission. A comprehensive analysis of endocytic proteins revealed the PX-BAR domain protein sorting nexin 9 (SNX9) as an effector of PI(3,4)P2. The recruitment of SNX9 to CCPs arrested at a late stage due to dynamin2-depletion strictly depends on both PI3K C2α and PI(3,4)P2. Furthermore, depletion of SNX9 and its close homolog SNX18 causes endocytic defects akin to loss of PI3K C2α that cannot be rescued by PI-binding deficient mutants of SNX9. Taken together, these observations identify a novel lipid requirement in CME and suggest a continuous mechanism for PI conversion along the endocytic route. Instead of de novo formation of PI(3)P on early endosomes, endocytic vesicle formation is coupled to PI(3,4)P2 synthesis. The phosphatase INPP4A, an effector of the early endosomal GTPase Rab5, would directly generate PI(3)P on membranes en route to early endosomes. These findings establish a novel role of PI(3,4)P2 as a product of a class II PI3K in a central cell physiological process and thus significantly advance our understanding of membrane traffic, the roles of class II PI3Ks, and the physiological importance of PI(3,4)P2.Phosphoinositide (PIs) sind seltene Lipide der zytoplasmatischen Seite eukaryo-tischer Membranen, die als räumliche und zeitliche Orientierungspunkte für Proteine dienen. Dadurch definieren PIs wesentlich die Identität distinkter zellulärer Membranen. Phosphatidylinositol-4,5-bisphosphat [PI(4,5)P2] ist in der Plasmamembran (PM) konzentriert und dort von essentieller Bedeutung für verschiedene Prozesse, darunter die Clathrin- vermittelte Endozytose (CME). Für die Nukleation und Assemblierung Clathrin- ummantelter Membranvertiefungen (CCPs) ist PI(4,5)P2 unverzichtbar. Nach der Abschnürung wird PI(4,5)P2 hydrolysiert und das Vesikel fusioniert mit dem frühen endosomalen Kompartiment. In Endosomen ist Phosphatidy- linositol-3-phosphat [PI(3)P] die dominierende PI Spezies und für die Funktion dieser Organellen unabdingbar. Jedoch ist der Mechanismus der PI Konversion auf der endozytischen Route, die Umwandlung einer PI(4,5)P2- zu einer PI(3)P-reichen Membran, bisher nur unzureichend verstanden. In der vorliegenden Arbeit etablieren wir Phosphatidyl¬inositol-3,4-bisphosphat [PI(3,4)P2] als neuen Lipidregulator der CME, der für die Reifung der CCPs von Bedeutung ist. Mittels der PI(3,4)P2-spezifischen 4-Phosphatase INPP4B von PI(3,4)P2 depletierte Zellen zeigen einen endozytischen Defekt, der durch langlebige CCPs charakterisiert ist. Dieser Effekt unterscheidet sich von der PI(4,5)P2-kontrollierten Initiation der CCP Bildung und zeigt ein Erfordernis der sequentiellen Generierung von PI(4,5)P2 und PI(3,4)P2 auf. Weiterhin identifizieren wir die Clathrin-assoziierte Klasse II Phosphatidylinositol-3-Kinase (PI3K), PI3K C2α, als das Enzym, das PI(3,4)P2 an CCPs synthetisiert. Die RNA Interferenz-vermittelte Depletion von PI3K C2α führte zu einer Phänokopie der Effekte, die in PI(3,4)P2-defizienten Zellen auftraten und zu einem partiellen Verlust PI(3,4)P2s von der PM. Detaillierte Untersuchungen deuteten auf einen späten, jedoch vor der Membranfission liegenden, Maturierungsdefekt von CCPs hin. Eine Analyse endozytischer Proteine zeigte das PX-BAR-Domänen Protein sorting nexin 9 (SNX9) als Effektor von PI(3,4)P2 auf. Die Rekrutierung von SNX9 zu arretierten Spätstadium-CCPs in Dynamin2-depletierten Zellen ist strikt von PI3K C2α und PI(3,4)P2 abhängig. Weiterhin führen die Depletion von SNX9 und dessen eng verwandtem Protein SNX18 zu einem dem in PI3K C2α-depletierten Zellen ähnlichen endozytischen Defekt, der nicht von PI-Bindungs-defizienten Mutanten von SNX9 kompensiert werden kann. Diese Daten identifizieren ein neues Lipid- Erfordernis in der CME und legen einen kontinuierlichen Mechanismus der PI Konversion nahe. Anstatt der de novo Bildung von PI(3)P in frühen Endosomen findet PI(3,4)P2-Synthese während der Vesikelbildung statt. Die Phosphatase INPP4A, ein Effektor der früh-endosomalen GTPase Rab5, könnte PI(3)P direkt auf endozytischen Vesikeln generieren, bevor diese mit dem frühen endosomalen Kompartiment fusionieren. Diese Ergebnisse etablieren eine neue Rolle von PI(3,4)P2 als Produkt einer Klasse II PI3K in einem zentralen zellphysiologischen Prozess und tragen daher signifikant zu unserem Verständnis des Membranverkehrs, der Rollen der Klasse II PI3Ks und der physiologischen Bedeutung von PI(3,4)P2 bei