Endocytosis - Exocytosis BIOLOGY, Faculty of Pharmacy 2015. 10. 12. László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof. Department of Genetics, Cell- and Immunobiology Semmelweis University

Endocytosis Phagocytosis – solid Pinocytosis – liquid (general) Uptake of substances Transport of protein or lipid components of compartments Metabolic or division signaling Defense to microorganisms

Phagocytosis (1) Predominant cells: unicellular cells macrophages osteoslats throphoblasts Functions: uptake of food partickles immuneresponses 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 lysosomes 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. Nobel-prize - 1974 Lysosomes (TEM)

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 assembly 101 AA 33 AA 44 AA N C

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

Dynamin in the cell

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

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