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

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

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

The progressive ankylosis protein ANK facilitates clathrin- and adaptor-mediated membrane traffic at the trans-Golgi network-to-endosome interface

Dominant or recessive mutations in the progressive ankylosis gene ANKH have been linked to familial chondrocalcinosis (CCAL2), craniometaphyseal dysplasia (CMD), mental retardation, deafness and ankylosis syndrome (MRDA). The function of the encoded membrane protein ANK in cellular compartments other than the plasma membrane is unknown. Here, we show that ANK localizes to the trans-Golgi network (TGN), clathrin-coated vesicles and the plasma membrane. ANK functionally interacts with clathrin and clathrin associated adaptor protein (AP) complexes as loss of either protein causes ANK dispersion from the TGN to cytoplasmic endosome-like puncta. Consistent with its subcellular localization, loss of ANK results in reduced formation of tubular membrane carriers from the TGN, perinuclear accumulation of early endosomes and impaired transferrin endocytosis. Our data indicate that clathrin/AP-mediated cycling of ANK between the TGN, endosomes, and the cell surface regulates membrane traffic at the TGN/endosomal interface. These findings suggest that dysfunction of Golgi-endosomal membrane traffic may contribute to ANKH-associated pathologies

Inactivation of the Class II PI3K-C2β Potentiates Insulin Signaling and Sensitivity

In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2β kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2β inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2β inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2β as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2β as a potential drug target for insulin sensitization

PI3K Class II [alpha] Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2a is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2a-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2a rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2a regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation

Phosphatidylinositol Turnover and Receptors

Abstract Phosphoinositides are a small portion of cellular phospholipids involved in virtually all cellular processes. They play a fundamental role in cellular signaling pathways, membrane trafficking, actin cytoskeletal dynamics, and regulate ion channels and transporters. Multiple pathways including phosphoinositide kinases and phosphatases control tightly the turnover of phosphoinositides, therefore their membrane concentration and distribution. Many human diseases, including cancer, metabolic syndrome, and neurological diseases are caused by alteration in phosphoinositide dependent pathways, indicating that phosphoinositide metabolism is essential for the regulation a variety of physiological function