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Endocytosis - Exocytosis

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Endocytosis - Exocytosis Endocytosis - Exocytosis BIOLOGY, Faculty of Pharmacy 2016. 10. 10. László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof. Department of Genetics, Cell- and Immunobiology Semmelweis University Endocytosis • Phagocytosis – solid • Pinocytosis – liquid (general) Endocytosis: • Uptake of substances • Transport of protein or lipid components of compartments • Metabolic or division signaling • Defense to microorganisms Phagocytosis (1) Predominant cells: Functions: • unicellular cells • uptake of food • macrophages partickles • osteoslats • immuneresponses • throphoblasts • elimination of aged cells (RBC) Phagocytosis (2) Required: • signal • membrane receptor (Fc receptor for Ab) • formation of pseudopodium • cortical actin network The formed vesicle: phagosome (hetero-; auto-) Endocytosis • Clathrin-coated vesicles • Non-clathrin coated vesicles • Macropinocytosis • Potocytosis Clathrin coated pits/vesicles Function of clathrin coated vesicles Receptor mediated endocytosis • Selective uptake of molecules (low environmental conc.) • Membrane receptors • Concentration of ligand (1000x) Components of coated vesicles Receptor-mediated endocytosis of LDL Sorting signals of secreted and membrane proteins to transport vesicles Selective incorporation of membrane proteins Into the coated vesicles Endosomal-Lysosomal compartment Structure • tubular, vesicular • acidic pH - vacuolar H+ ATP-ase - proton pump • early-endosome (EE) and late-endosomes (LE) and lysosomes (L) • EE pH= 6; LE pH=5 • in EE no lysosomal membrane proteins or enzymes (in contrast LE) Endosomal-Lysosomal compartment Function • sorting • transport • degradation • removal of clathrin layer • formation of EE in the EE: • dissociation of receptor-ligand complex - receptor- recycling (e.g. LDL, transferrin) • receptor-ligand complex transported together - receptor down regulation (e.g. EGF) Pathway of LDL -insulin or other hormones – in receptor mediated endocytosis Fate of LDL internalized by receptor-mediated endocytosis The transferrin-cycle Late endosome • early endosomes, TGN and autophagosomes feed late endosomes • lysosomal enzymes M-6-P signal is changed, the phosphate group is cleaved - receptors can not bind enzymes • the enzyme content of vacuoles is in the lumen lysosomes Dissociation of receptor-ligand complex in late endosomes De Duve, Ch. Lysosomes (TEM) Nobel-prize - 1974 Lysosomes • enzymes - acidic hydrolases e.g. protease, nuclease, glycosidase, phosphatese • more than 40 types of enzymes • membrane proteins - highly glycosilated protects from the enzymes • transport molecules of the membranes - transports the products of proteolytic cleavage into the cytoplasm • the waste products are released or stored in the cytoplasm (inclusion - residual body) LAMP = lysosome associated membrane proteins • integrant membrane proteins of the lysosome • LAMP-2 – tarnsport of cholesterol • LAMP-2 defficiency- autophagy www.helsinki.fi/bioscience/biochemistry/eskelinen Autophagy - Autophagosome • intake of own components • regulates the number of organells • toxic effects can also induce it Formation of autophagosome www.helsinki.fi/bioscience/biochemistry/eskelinen E Non-clathrin coated vesicles • There is no receptor or clathrin in the membrane • The uptake of substances is less selective • Primairly liquide-phase endocytosis Macropinocytosis • Ruffling of the surface membrane forms inclusions • These „vacuoles” have no membrane • Size 0.2-5 mm - the mass/surface ratio is very good Significance: • Liquide-phase pinocytosis • Taking probes from the environment – antigene recognition in macrophages Film produced by F. Vilhardt and M. Grandahl. Caveolae • 50-80 nm, bottle-like infoldings of the surface membrane • endothels, adipocytes • caveolin • potocytosis - caveolae close but not internalized, the materials enter the cytoplasm by a special carrier molecule e.g. vitamine B4 • some other caveolae enter the cell !!! Caveolae Caveolin oligomers and caveolae 33 AA assembly 44 AA C 101 AA N Functions of dynamin Clathrin-mediated endocytosis Membrane retrieval Endosome- Secretory to-Golgi vesicle transport formation in TGF Caveolae Fluid phase endocytosis Dynamin in the cell Structure of dynamin Interaction with membranes Activation of Interaction with GTP-ase domain cytosceleton Dynamin requires GTP hydrolysis to pinching off coated vesicles • The not-hydrolysable GTP-gS is added • Dots represent binding of anti-dynamin antibodies • The long neck shows that however the coated pit was formed, in the absence of GTP hydrolysis its pinching off is absence Carrier mediated proteolysis • some molecules can enter lysosome directly from the cytoplasm • the signal of entry: KFERQ (Lys-Phe-Glu-Arg- Gln) Proteasome • non-lysosomal cleavage of proteins • cylindric, multienzyme complex • parts: ATP binding-, substrate binding-, regulator-domain • location: close to the external part of ER-translocon • ubiquitin - degradation-signal - is required • the non-properly folded or damaged proteins • regulator - eliminator - role e.g. cyclins • cystic fibrosis - Cl- fac. transp. is affected as the responsible membrane protein is broken down in proteosome Proteasomes Ubiquitation - proteasome „Exocytotic” processes The mannose-6-P pathway and lysosomal enzymes Exocytosis in TEM Apical and basolateral targeting in epithelial cell Transcytosis • the ligands walk around the endosomal compartment • ligands transported from one surface to the other • e.g. immunoglobulins of the colostrum cross the intestinal epithelium by transcytosis Release of neurotransmitters .
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