Endocytosis - Exocytosis

1. László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof.Department of Genetics, Cell- andImmunobiologySemmelweis University 2008 Endocytosis - Exocytosis

4. 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

5. Predominant cells: unicellular cells macrophages osteoslats throphoblasts Functions: uptake of food partickles immuneresponses elimination of aged cells (RBC) Phagocytosis (1)

6. Phagocytosis (2) Required: • signal • membrane receptor (Fc receptor for Ab) • formation of pseudopodium • cortical actin network The formed vesicle: phagosome (hetero-; auto-)

7. Endocytosis • Clathrin-coated vesicles • Non-clathrin coated vesicles • Macropinocytosis • Potocytosis

8. Clathrin coated pits/vesicles

10. Function of clathrin coated vesicles • Receptor mediated endocytosis • Selective uptake of molecules • (low environmental conc.) • Membrane receptors • Concentration of ligand (1000x)

11. Components of coated vesicles

12. Receptor-mediated endocytosis of LDL

13. Sorting signals of secreted and membrane proteins to transport vesicles

14. Selective incorporation of membrane proteins Into the coated vesicles

15. LRO dER TGN LE EE L RE

16. -AP-2 -AP180 -clathrin -PIP2 -actin -synaptotagmin -dynamin

17. Endosomal-Lysosomal compartmentStructure • 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)

18. Endosomal-Lysosomal compartmentFunction • 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)

19. Pathway of LDL • insulin or other • hormones – • in receptor • mediated • endocytosis

20. Fate of LDL internalized by receptor-mediated endocytosis

21. The transferrin-cycle

22. Partcipating components in budding of coated vesicles

23. 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

26. Dissociation of receptor-ligand complex in late endosomes

27. De Duve, Ch. Nobel-prize - 1974 Lysosomes (TEM)

28. 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)

29. 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

30. Autophagy - Autophagosome • intake of own components • regulates the number of organells • toxic effects can also induce it

31. Formation of autophagosome www.helsinki.fi/bioscience/biochemistry/eskelinen E

32. Non-clathrin coated vesicles • There is no receptor or clathrin in the membrane • The uptake of substances is less selective • Primairly liquide-phase endocytosis

33. 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.

34. 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 !!!

35. Caveolae

36. 33 AA 44 AA C 101 AA N Caveolin oligomers and caveolae assembly

37. Dynamincs of caveolae-formation

39. Functions of dynamin Clathrin-mediated endocytosis Membrane retrieval Endosome- to-Golgi transport Secretory vesicle formation in TGF Caveolae Fluid phase endocytosis

40. Dynamin in the cell

41. Structure of dynamin Interaction with membranes Interaction with cytosceleton Activation of GTP-ase domain

42. 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

43. Carrier mediated proteolysis • some molecules can enter lysosome directly from the cytoplasm • the signal of entry: KFERQ (Lys-Phe-Glu-Arg-Gln)

44. 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

45. Proteasomes

46. Ubiquitation - proteasome

47. „Exocytotic” processes

48. The mannose-6-P pathway and lysosomal enzymes

49. Exocytosis in TEM

50. Apical and basolateral targeting in epithelial cell