Endocytosis - Exocytosis

László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof.

Department of Genetics, Cell- and Immunobiology Semmelweis University 2008

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: 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

dER TGN LRO LE L RE EE

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

• • • • •

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 -

recycling

(e.g.

LDL, transferrin

)

receptor-

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

Partcipating components in budding of coated vesicles

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.

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 m m - 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

33 AA 44 AA C 101 AA N

Dynamincs of caveolae-formation

Functions of dynamin

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

Dynamin in the cell

Structure of dynamin

Interaction with membranes Activation of GTP-ase domain Interaction with 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