RESUMEN
Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Quinasa 5 Dependiente de la Ciclina/genética , Endocitosis/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Clatrina/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Dinamina I/genética , Dinamina I/metabolismo , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Células HeLa , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuronas/metabolismo , Unión Proteica , Interferencia de ARNRESUMEN
In the version of this Letter originally published, the name of co-author Safa Lucken-Ardjomande Häsler was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.
RESUMEN
Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells1, but several clathrin-independent endocytic routes exist in parallel2,3. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the ß1 adrenergic receptor4. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin4,5. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5'-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5-10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Endocitosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Unión a Ácidos Grasos , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Antígenos de Histocompatibilidad Menor/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Transducción de Señal , Factores de Tiempo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Clathrin-independent endocytosis (CIE) mediates the cellular uptake of many extracellular ligands, receptors, and pathogens, including several life-threatening bacterial toxins and viruses. So far, our understanding of CIE carrier formation has lagged behind that of clathrin-coated vesicles. Impediments have been the imprecise definition of some CIE pathways, the lack of specific cargoes being transported and of exclusive cytosolic markers and regulators. Notwithstanding these limitations, three distinct molecular mechanisms by which CIE carriers form can be defined. Cargo capture by cytosolic proteins is the main mechanism used by fast endophilin-mediated endocytosis (FEME) and interleukin 2 receptor (IL-2R) endocytosis. Acute signaling-induced membrane remodeling drives macropinocytosis. Finally, extracellular lipid or cargo clustering by the glycolipid-lectin (GL-Lect) hypothesis mediates the uptake of Shiga and cholera toxins and receptors by the CLIC/GEEC pathway. Here, we review these mechanisms and highlight current gaps in knowledge that will need to be addressed to complete our understanding of CIE.
Asunto(s)
Vesículas Cubiertas por Clatrina/genética , Clatrina/genética , Endocitosis/genética , Transporte Biológico/genética , Clatrina/química , Vesículas Cubiertas por Clatrina/química , Humanos , Transducción de Señal/genéticaRESUMEN
Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate-produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2-recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α2a- and ß1-adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME).
