Endocytosis and Exocytosis in Cellular Biology

1. BIOLOGY, Faculty of Dentistry 2017. 10. 9. 2017.10.19.. László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof.Department of Genetics, Cell- andImmunobiologySemmelweis University Endocytosis - Exocytosis

2. !

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. ! Main steps of coatedvesicleformation

11. Components of coatedvesicles – Adaptorproteins

12. Components of coated vesicles

13. Receptor-mediated endocytosis of LDL

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

15. ! 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)

16. ! 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)

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

18. Fate of LDL internalized by receptor-mediated endocytosis

19. The transferrin-cycle

20. ! 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

23. Dissociation of receptor-ligand complex in late endosomes

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

25. ! 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)

26. 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

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

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

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

30. 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.

31. ! Caveolae • 50-80 nm, bottle-likeinfoldings of thesurfacemembrane • endothels, adipocytes • caveolin • potocytosis - caveolaeclosebutnotinternalized, thematerials enter thecytoplasmby a specialcarriermoleculee.g. vitamine B4 • someothercaveolae enter thecell !!!

32. Caveolae

33. 33 AA 44 AA C 101 AA N Caveolinoligomers and caveolae assembly

35. Tumor treatmentviacaveolas • Tumor-associatedcaveolaproteins • Antibodiesdeliverdrugsselectivelytothetumors

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

37. Dynamin in the cell

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

39. 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

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

41. ! 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

42. Proteasomes

43. Ubiquitation - proteasome

44. ! „Exocytotic” processes

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

46. Exocytosis in TEM

47. Apical and basolateral targeting in epithelial cell

48. ! 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

49. Release of neurotransmitters