Endocytosistom [email protected] 20061Vesicular-based transport of proteins and lipid • Hallmark of eukaryotic cells • Extraordinarily large & fast membrane fluxes along endocytic and secretory pathways• Traffic is selective• Preserves distinct identities of membranous organelles throughout the life of the cell21a. basic considerations in vesicular transport31a. basic considerations in vesicular transport• selection: cargo sorting before budding31a. basic considerations in vesicular transport• selection: cargo sorting before budding• budding: assembly of a cytosolic coat31a. basic considerations in vesicular transport• selection: cargo sorting before budding• budding: assembly of a cytosolic coat• traffic: find destination31a. basic considerations in vesicular transport• selection: cargo sorting before budding• budding: assembly of a cytosolic coat• fusion: dock and release cargo • traffic: find destination34anterograde vesicular traffic: VSVGTS GFP from the ER to the GolgI56VSVGTS - EGFP : GOLGI > PLASMA MEMBRANE7Cholera Toxin - Mechanism of Action GolgiApical Membrane EndosomecAMPERAdenylylCyclaseGsαBasolateral MembraneForskolin8GolgiCholera Toxin entry of Cholera Toxin followed by live-cell imaging 9Clathrin / AP-1,3,4 / GGA’s Clathrin / AP-2 1011coated pitendosomeLDLRTfRENDOCYTOSIS12coated pitendosomeLDLRTfRENDOCYTOSISJ. Heuser12coated pitendosomeLDLRTfRENDOCYTOSISJ. Heuser12131414CLATHRIN COAT @ 7.9 Å resolutionAlex Fotin15CLATHRIN COAT @ 7.9 Å resolutionlight chainAlex Fotin151617σ2 - EGFP (AP-2) Ehrlich et al 200418Ehrlich et al 200419...FDNPVY......FDNPVC...J.D. PATIENTLDLr20DiI - LDL YFP - LCa Clathrin Ehrlich et al 200421DiI - LDL YFP - LCa Clathrin capture of LDL by a clathrin coated pitat the plasma membraneEhrlich et al 2004210 40 80TIME (s)CLATHRIN INTENSITY0 10 20 300.00.51.01.52.0TIME (s)DISPLACEMENT( nm x 10+3 )n = 53TIME 1/2 (s)DISPLACEMENT( nm x 10+3 )-4 -3 -2 -1 00.00.51.01.52.0n = 41T1T3LDLT2T4Ehrlich et al 200422coatcomponentsformation of clathrin coated pits and vesiclesbudding nucleation&growth 23coatcomponentsformation of clathrin coated pits and vesiclesuncoating budding nucleation&growth 2324recruitmentof auxilin24recruitmentof auxilinrecruitmentof Hsc70 - ATP24recruitmentof auxilinrecruitmentof Hsc70 - ATP24recruitmentof auxilinrecruitmentof Hsc70 - ATPATP hydrolysisHsc70 - ADPauxilin releaseuncoating24recruitmentof auxilinrecruitmentof Hsc70 - ATPATP hydrolysisHsc70 - ADPauxilin releaseuncoating24recruitmentof auxilinrecruitmentof Hsc70 - ATPATP hydrolysisHsc70 - ADPauxilin releaseuncoatingADP - ATPexchangeHsc70 - ATP24GAKPTENJ-domainAuxilin 1(brain)Auxilin -- domain organizationAuxilin 2Massol et al 200625GAKPTENJ-domainHSC70Auxilin 1(brain)Auxilin -- domain organizationAuxilin 2Massol et al 200625GAKPTENJ-domainHSC70clathrinAuxilin 1(brain)Auxilin -- domain organizationAuxilin 2Massol et al 200625GAKPTENJ-domainHSC70clathrinAuxilin 1(brain)Auxilin -- domain organizationdynaminAuxilin 2Massol et al 200625GAKPTENJ-domainHSC70clathrinS / TAuxilin 1(brain)Auxilin -- domain organizationdynaminAuxilin 2Massol et al 200625CLATHRIN COAT @ 12 Å resolutionAlex Fotin26AuxilinCLATHRIN COAT @ 12 Å resolutionAlex Fotin26Alex Fotin27Alex Fotin2728282828Hsc7028GAKJ-domainHSC70clathrinS / TAuxilin 1(brain)Auxilin -- domain organizationdynaminAuxilin 2Massol et al 200629GAKPTENJ-domainHSC70clathrinS / TAuxilin 1(brain)Auxilin -- domain organizationdynaminAuxilin 2Massol et al 200629GAKPTENJ-domainHSC70clathrinPIPsS / TAuxilin 1(brain)Auxilin -- domain organizationdynaminesentialfor final recruitmentAuxilin 2Massol et al 200629AuxilinClathrin 30Phosphoinositides31Phosphoinositides31Phosphoinositides31323334Jurkat T - cells34Jurkat T - cellsREGULATION OF CELL SIZE DURING CELL DIVISIONEmmanuel Boucrot34cell division35Area = 27 Area = 1Vol = 2 Vol = 2rd = 3.0 rsh = 0.1 rd36metaphaseinterphasecytokinesispro-metaphase02004006008001 0000 0,1 0,2 0,3 0,4InterphaseMetaphaseCytokinesisFM 1-43 Fluorescence (a.u) BSC1 cellsn = 45area37metaphaseinterphasecytokinesispro-metaphase02004006008001 0000 0,1 0,2 0,3 0,4InterphaseMetaphaseCytokinesisFM 1-43 Fluorescence (a.u) BSC1 cellsn = 45area37metaphaseinterphasecytokinesispro-metaphase38metaphaseinterphasecytokinesispro-metaphase38interphase39during mitosis:• clathrin endocytosis does not changeinterphasemetaphaseanaphase > cytokinesis39interphase prophase metaphase anaphase telophase cytokinesisendocytosis of transferrin continues during cell divisiontransferrin: at the surfaceinternalized40- dynasore+ dynasoredynasore prevents mitotic roundup41endocytosis is required to reduce cell surface42endocytosis is required to reduce cell
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