Understanding Endocytosis and Exocytosis in Cell Biology

1. BIOLOGY, Faculty of Pharmacy 2015. 10. 12. László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof.Department of Genetics, Cell- andImmunobiologySemmelweis University 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

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

10. Components of coated vesicles

11. Receptor-mediated endocytosis of LDL

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

13. Selective incorporation of membrane proteins Into the coated vesicles

14. 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)

15. 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)

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

17. Fate of LDL internalized by receptor-mediated endocytosis

18. The transferrin-cycle

19. 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

22. Dissociation of receptor-ligand complex in late endosomes

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

24. 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)

25. 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

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

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

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

29. 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.

30. 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 !!!

31. Caveolae

32. 33 AA 44 AA C 101 AA N Caveolin oligomers and caveolae assembly

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

35. Dynamin in the cell

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

37. 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

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

39. 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

40. Proteasomes

41. Ubiquitation - proteasome

42. „Exocytotic” processes

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

44. Exocytosis in TEM

45. Apical and basolateral targeting in epithelial cell

46. 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

47. Release of neurotransmitters