Endocytosis - ExocytosisEndocytosis - Exocytosis

BIOLOGY, Faculty of Pharmacy

2014. 10. 13.

László KŐHIDAI, Med. Habil. MD, PhD., Assoc. Prof.Department of Genetics, Cell- and

ImmunobiologySemmelweis University

EndocytosisEndocytosis

• Phagocytosis – solid Phagocytosis – solid

• Pinocytosis – liquid (general)Pinocytosis – liquid (general)

EndocytosisEndocytosis::

• Uptake of substancesUptake of substances

• Transport of protein or lipid components of Transport of protein or lipid components of compartmentscompartments

• Metabolic or division signalingMetabolic or division signaling

• Defense to microorganismsDefense to microorganisms

Phagocytosis (1)Phagocytosis (1)

Predominant cells:Predominant cells:

• unicellular cells unicellular cells

• macrophages macrophages

• osteoslats osteoslats

• throphoblaststhrophoblasts

Functions:Functions:

• uptake of food uptake of food particklespartickles

• immuneresponsesimmuneresponses

• elimination of aged elimination of aged cells (RBC)cells (RBC)

Phagocytosis (2)Phagocytosis (2)

Required:Required:

• signal signal

• membrane receptor (Fc receptor for Ab)membrane receptor (Fc receptor for Ab)

• formation of pseudopodiumformation of pseudopodium

• cortical actin networkcortical actin network

The formed vesicle: phagosome (hetero-; auto-)The formed vesicle: phagosome (hetero-; auto-)

EndocytosisEndocytosis

• Clathrin-coated vesiclesClathrin-coated vesicles

• Non-clathrin coated vesiclesNon-clathrin coated vesicles

• MacropinocytosisMacropinocytosis

• Potocytosis Potocytosis

Clathrin coated Clathrin coated pits/vesiclespits/vesicles

Function of clathrin coated vesiclesFunction of clathrin coated vesicles

Receptor mediated endocytosisReceptor mediated endocytosis

• Selective uptake of moleculesSelective uptake of molecules (low environmental conc.)(low environmental conc.)

• Membrane receptorsMembrane receptors• Concentration of ligand (1000x)Concentration of ligand (1000x)

Components of coated vesicles Components of coated vesicles

Receptor-mediated endocytosis of LDL Receptor-mediated endocytosis of LDL

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

Selective incorporationSelective incorporationof membrane proteinsof membrane proteinsInto the coated vesiclesInto the coated vesicles

Endosomal-Lysosomal compartmentEndosomal-Lysosomal compartmentStructureStructure

• tubular, vesiculartubular, vesicular

• acidic pH - vacuolar Hacidic pH - vacuolar H++ ATP-ase - proton pump ATP-ase - proton pump

• early-endosome (EE) and late-endosomes (LE) early-endosome (EE) and late-endosomes (LE) and lysosomes (L)and lysosomes (L)

• EE pH= 6; LE pH=5EE pH= 6; LE pH=5

• in EE in EE nono lysosomal membrane proteins or lysosomal membrane proteins or enzymes (in contrast LE) enzymes (in contrast LE)

Endosomal-Lysosomal compartmentEndosomal-Lysosomal compartmentFunctionFunction

• sortingsorting

• transporttransport

• degradation degradation

• removal of clathrin layerremoval of clathrin layer

• formation of EEformation of EE

in the EE:in the EE:

• dissociation of receptor-ligand complex - dissociation of receptor-ligand complex - receptor-recyclingreceptor-recycling (e.g. (e.g. LDL, transferrinLDL, transferrin))

• receptor-ligand complex transported together - receptor-ligand complex transported together - receptor down receptor down regulation regulation (e.g. (e.g. EGFEGF))

Pathway of LDLPathway of LDL-insulin or otherinsulin or otherhormones – hormones – in receptorin receptormediatedmediatedendocytosis endocytosis

Fate of LDL Fate of LDL internalized by internalized by receptor-mediated receptor-mediated endocytosis endocytosis

The transferrin-cycle The transferrin-cycle

Late endosomeLate endosome

• early endosomes, TGN and autophagosomes feed early endosomes, TGN and autophagosomes feed late endosomeslate endosomes

• lysosomal enzymes M-6-P signal is changed, the lysosomal enzymes M-6-P signal is changed, the phosphate group is cleaved - receptors can not phosphate group is cleaved - receptors can not bind enzymesbind enzymes

• the enzyme content of vacuoles is in the lumen the enzyme content of vacuoles is in the lumen

lysosomeslysosomes

Dissociation of receptor-ligand complex in Dissociation of receptor-ligand complex in late endosomes late endosomes

Lysosomes (TEM)Lysosomes (TEM)De Duve, Ch.

Nobel-prize - 1974

LysosomesLysosomes• enzymesenzymes - acidic hydrolases - acidic hydrolases

e.g. protease, nuclease, glycosidase, phosphatese e.g. protease, nuclease, glycosidase, phosphatese

• more than 40 types of enzymesmore than 40 types of enzymes

• membrane proteins membrane proteins - highly glycosilated protects - highly glycosilated protects from the enzymesfrom the enzymes

• transport molecules transport molecules of the membranes - transports of the membranes - transports the products of proteolytic cleavage into the the products of proteolytic cleavage into the cytoplasmcytoplasm

• the waste products are released or stored in the the waste products are released or stored in the cytoplasm (inclusion - residual body)cytoplasm (inclusion - residual body)

LAMP = LAMP = lysosome associated membrane proteinslysosome 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 - AutophagosomeAutophagy - Autophagosome

• intake of own componentsintake of own components

• regulates the number of organellsregulates the number of organells

• toxic effects can also induce ittoxic effects can also induce it

E

Formation of autophagosomeFormation of autophagosome

www.helsinki.fi/bioscience/biochemistry/eskelinen

Non-clathrin coated vesiclesNon-clathrin coated vesicles

• There is no receptor or clathrin in the membraneThere is no receptor or clathrin in the membrane

• The uptake of substances is less selectiveThe uptake of substances is less selective

• Primairly liquide-phase endocytosisPrimairly liquide-phase endocytosis

MacropinocytosisMacropinocytosis

• Ruffling of the surface membrane forms inclusionsRuffling of the surface membrane forms inclusions• These „vacuoles” have no membraneThese „vacuoles” have no membrane• Size 0.2-5 m - the mass/surface ratio is very good

SignificanceSignificance::

• Liquide-phase pinocytosisLiquide-phase pinocytosis• Taking probes from the Taking probes from the

environment environment – – antigene recognitionantigene recognition in macrophagesin macrophages

Film produced by F. Vilhardt and M. Grandahl.

CaveolaeCaveolae

• 50-80 nm, bottle-like infoldings of the surface 50-80 nm, bottle-like infoldings of the surface membranemembrane

• endothels, adipocytesendothels, adipocytes

• caveolincaveolin

• potocytosis - caveolae close but not internalized, potocytosis - caveolae close but not internalized, the materials enter the cytoplasm the materials enter the cytoplasm

by by a special carrier molecule a special carrier molecule e.g. e.g. vitamine B4vitamine B4

• some other caveolae enter the cell !!!some other caveolae enter the cell !!!

CaveolaeCaveolae

Caveolin oligomers and caveolae Caveolin oligomers and caveolae assemblyassembly

101 AA101 AA

33 AA

44 AA

N

C

Functions of dynaminFunctions of dynamin

Fluid phaseFluid phaseendocytosisendocytosis

CaveolaeCaveolae

Membrane Membrane retrievalretrieval

Clathrin-mediated endocytosisClathrin-mediated endocytosis

Endosome-Endosome-to-Golgito-Golgitransporttransport

Secretory Secretory vesiclevesicle formationformation in TGFin TGF

Dynamin in the cellDynamin in the cell

Structure of dynaminStructure of dynamin

Interaction with Interaction with membranesmembranes

Activation of Activation of GTP-ase domainGTP-ase domain

Interaction with Interaction with

cytosceletoncytosceleton

Dynamin requires GTP hydrolysis to pinching off Dynamin requires GTP hydrolysis to pinching off coated vesiclescoated vesicles

• The not-hydrolysable GTP-gS is addedThe not-hydrolysable GTP-gS is added• Dots represent binding of anti-dynamin antibodiesDots represent binding of anti-dynamin antibodies• The long neck shows that however the coated pit was formed, The long neck shows that however the coated pit was formed, in the absence of GTP hydrolysis its pinching off is absence in the absence of GTP hydrolysis its pinching off is absence

Carrier mediated proteolysisCarrier mediated proteolysis

• some molecules can enter lysosomesome molecules can enter lysosome directly directly from from the cytoplasmthe cytoplasm

• the signal of entry: KFERQ (Lys-Phe-Glu-Arg-the signal of entry: KFERQ (Lys-Phe-Glu-Arg-Gln)Gln)

ProteasomeProteasome• non-lysosomal cleavage of proteinsnon-lysosomal cleavage of proteins

• cylindric, multienzyme complexcylindric, multienzyme complex

• parts: ATP binding-, substrate binding-, regulator-domainparts: ATP binding-, substrate binding-, regulator-domain

• location: close to the external part of ER-transloconlocation: close to the external part of ER-translocon

• ubiquitinubiquitin - degradation-signal - is required - degradation-signal - is required

• the non-properly folded or damaged proteinsthe non-properly folded or damaged proteins

• regulator - eliminator - role e.g. regulator - eliminator - role e.g. cyclinscyclins

• cystic fibrosis cystic fibrosis - Cl- Cl-- fac. transp. is affected as the responsible fac. transp. is affected as the responsible membrane protein is broken down in proteosomemembrane protein is broken down in proteosome

ProteasomesProteasomes

Ubiquitation - proteasomeUbiquitation - proteasome

„„Exocytotic” processesExocytotic” processes

The mannose-6-P pathway and lysosomal enzymesThe mannose-6-P pathway and lysosomal enzymes

Exocytosis in TEMExocytosis in TEM

Apical and basolateral targeting in epithelial cellApical and basolateral targeting in epithelial cell

TranscytosisTranscytosis

• the ligands walk around the endosomal the ligands walk around the endosomal compartmentcompartment

• ligands transported from one surface to the otherligands transported from one surface to the other

• e.g. e.g. immunoglobulinsimmunoglobulins of the colostrum cross the of the colostrum cross the intestinal epithelium by transcytosisintestinal epithelium by transcytosis

Release of neurotransmittersRelease of neurotransmitters