Endocytosis, vesicular transport Anna L. Kiss Deparment of Human Morphology and Develpomentall Biology , Semmelweis University 2015 Endocytosis: phagocytosis, pinocytosis; primary lysosomes, secondary lysosomes, phagosomes, autophagy, lyosomal desaeses, receptor-mediated endocytosis; clathrin coated vesivle, caveolae, endosomes (early. Late, recycling) transcytosis

Endocytosis Endocytosis: - active transport - uptake of substances into the cell by invagination of the plasma membrane - the endocytosed substance or particle is separated from the cytosol by a membrane (derivative of the plasma membrane). Phagocytosis Pinocytosis

II. Pinocytosis Uptake of particles smaller than 0,1 μm, or macromolecules or dissolved substances by endocytosis. The resulting vesicles are 50-100 μm large. Fluid phase pinocytosis: the particles are in a dissolved state (solution) and are taken up into the cell by invagination of the plasma membrane (endocytic vesicles). Adsorptive pinocytosis. The small particles are bound to the plasma membrane (enrichment of the particles on the surface of the plasma membrane), therefore uptake is more effective even at low concentrations of the particles. Non-specific binding sites (e.g. glycocalyx, non-specific receptors). Some bacterial toxins (Diphteria), viruses (e.g. Influenza, VSV, Semliki Forest virus) are taken up into the cell this way. Specific binding sites (receptors): receptor-mediated pinocytosis (or endocytosis). Ligands: immunoglobulins, α2-makroglobulin, transferrin, LDL , asialoglikoproteins, … Receptors: integral membrane proteins

Receptors

Endocytotic pathways Phagocytosis („eating”): >0,25µm Pinocytosis („drinking”) Macropinocytosis: >1µm Endocytosis via clathrin-coated vesicles: ~120nm Endocytosis via caveolae: ~60nm Endocytosis clathrin- és caveolin-independent and/or diynamin independent: ~90nm Nature 422, 37 - 44 (06 March 2003); doi:10.1038/nature01451 <> Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Figure 1 Multiple portals of entry into the mammalian cell. The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation. Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Nature 422, 37 - 44 (06 March 2003)

Macropinocytosis Depends on ruffling

Endocytotic pathways Phagocytosis („eating”): >0,25µm Pinocytosis („drinking”) Macropinocytosis: >1µm Endocytosis via clathrin-coated vesices: ~120nm Endocytosis via caveolae: ~60nm Endocytosis clathrin- és caveolin-independent and/or diynamin independent: ~90nm Nature 422, 37 - 44 (06 March 2003); doi:10.1038/nature01451 <> Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Figure 1 Multiple portals of entry into the mammalian cell. The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation. Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Nature 422, 37 - 44 (06 March 2003)

Receptor-mediated endocytosis via clathrin coated vesicles Highly regulated, specific uptake precess! Clatrhin-coated vesicles: a basket-like coat is forming around the vesicle Clathrin: heavy and light chain triskelion pentagons and hexagons

Clatrhin coated vesicles mediated endocytosis coated pit Receptor proteins with the bound substance, adaptin proteins and clathrin assemble in the membrane. The GTP-binding protein dynamin separates the vesicle from the plasma membrane. The clathrin coat is then shed from the vesicle in the cytosol.

Clathrin-mediated endocytosis Pinching off the vesicle by dynamin (small GTP-ase)

Clathrin-coated pits and vesicles under the cell membrane, seen from inside the cell, electron micrograph, freeze fractured and deep etched Heuser technique. clathrin-coated vesicle clathrin-coated pit)

Uncoating and fusion with early endosomes

Receptor-mediated endocytosis Endosomes: acidification early endosome late endosome recycling endosome

The early endosome is a sorting organelle plasma membrane vesicles with unloaded receptors on the way back to the plasma membrane (recycling) transport vesicles with ligand-loaded receptors early endosome multivesicularis test late endosome (multi- vesicular body) Lysosome 

Endocytotic pathways Phagocytosis („eating”): >0,25µm Pinocytosis („drinking”) Macropinocytosis: >1µm Endocytosis via clathrin-coated vesices: ~120nm Endocytosis via caveolae: ~60nm Endocytosis clathrin- és caveolin-independent and/or diynamin independent: ~90nm Nature 422, 37 - 44 (06 March 2003); doi:10.1038/nature01451 <> Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Figure 1 Multiple portals of entry into the mammalian cell. The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation. Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Nature 422, 37 - 44 (06 March 2003)

Caveolae-mediated endocytosis Caveolae: d: 50-100nm flask-or omega-shaped invaginations Dr L. Kiss Anna felvétele Dr L. Kiss Anna felvétele

caveolae

Caveolae: caveolin-containing lipid rafts Highly hidrophobic membrane domenes: cholesterol, sphyngolipids, glycosyl-phosphatydil-inositol; lipid rafts Caveolin: caveolin-1 caveolin-2 caveolin-3 Cavin (PTRF): accessory protein

Lipid raft is invaginating Caveolae Lipid rafts: highly hydriphobic membrane domains rich in sphyndolipids, cholesterol, glycolipids proteins: caveolin (1, 2, 3) Lipid raft is invaginating

Caveola: „signalling organelle” scaffolding domene: at the N terminus; binds signaling molecules

Caveolae-mediated endocytosis Uptake of SV40 virus : occurs with caveolae Virus binds to MHC I present in the host cell plasma membrane MHCI (the receptor for virus) is present in caveolae Caveolin is highly phosphorylated by Src kinase, (tyrosine phosphorylation) Reorganization of the cortical cytoskeleton (actin filaments) actin-tail is formed Dynamin (small GTPase) associates to the neck region of caveolae Caveola is pinching off

Caveola-mediated endocytosis Parton , R:G: and Simons, K. Nature Reviews 2007.

Caveolae-mediated endocytosis:caveosome-like structures

caveolin-115, albumin10 Caveosomes??? caveolin-115, CD6310

Caveola-mediated endocytosis CD63: late endosomal marker Colocalization of caveolin and CD63 Caveola-mediated endocytosis can terminate in lysosomes

Caveola-mediated endocytosis Parton , R:G: and Simons, K. Nature Reviews 2007.

Endocytotic pathways Phagocytosis („eating”): >0,25µm Pinocytosis („drinking”) Macropinocytosis: >1µm Endocytosis via clathrin-coated vesices: ~120nm Endocytosis via caveolae: ~60nm Endocytosis clathrin- és caveolin-independent and/or diynamin independent: ~90nm Nature 422, 37 - 44 (06 March 2003); doi:10.1038/nature01451 <> Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Figure 1 Multiple portals of entry into the mammalian cell. The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation. Regulated portals of entry into the cell SEAN D. CONNER AND SANDRA L. SCHMID Nature 422, 37 - 44 (06 March 2003)

The fate of pinocytotic vesicles and of the interiorized substance. The pinocytotic vesicle sheds its coat and fusions with the early endosome. Endosome: A membrane-bound cell organelle of variable size and shape. Its interior is gradually acidified by the action of proton pumps in the endosomal membrane. Early endosomes: mostly present in the marginal cytoplasm. pH-values 6.5-5, at this pH most ligands are released from their receptors (ligand-receptor uncoupling). Important sorting organelle towards plasma membrane, (recycling endosome) Golgi-apparatus and late endosome – lysosome. Late endosomes: more deeply situated in the cytoplasm and having an acidic pH. Lysosomal enzymes begin to accumulate by a vesicular transport from the trans-Golgi network. Multivesicular bodies: the membrane of late endosomes is budding into the interior of the organelle to form vesicles (many vesicles in the lumen). This way integral membrane proteins can be degraded by lysosomal enzymes in the organelle. Lysosomes: mostly close to the Golgi-apparatus, surrounded by a membrane and having a pH of 4,5-5. Many hydrolytic enzymes destined for degradation of organic substances.

Fate of the receptors Fate of the ligands Recycling to the plasma membrane by a vesicular transport after releasing their ligands in the early endosome. Vesicles with empty receptors are budding off from the membrane of early (and partly also from late) endosomes and travel to the plasma membrane with which they fuse by exocytosis (receptor recycling). This way the membrane of vesicles (together with the receptors) is reintegrated into the plasma membrane. Example: LDL receptors. They reach the late endosome and are degraded in lysosomes (receptor downregulation). Example: EGF receptor (epithelial growth factor with its receptor). Fate of the ligands Degradation. The ligand is transported further from early endosome towards late endosome and lysosome where it is degraded. Or: Recirculation to the cell surface. In early endosome the ligand remains bound to its receptor and recycles back to the plasma membrane together with its receptor. Example: transferrin (an iron-transporting protein which after releasing iron in the endosome recirculates together with its receptor to the plasma membrane ready for a further cycle).

Uptake of LDL particles with receptor-mediated endocytosis LDL (low-density lipoprotein): Lipoprotein particles in the blood, transporting cholesterol and cholesterol esters. Partially covered by the apoprotein B. cholesterol cholesterol esters Apoprotein B LDL-binding site extracellular space plasma membrane adaptin binding clathrin-coated pit empty receptors recycle to the plasma membrane  adaptin-biding site is missing budding of vesicles fusion with endosome When the adaptin-binding site on the receptor is missing (mostly by genetic disorders), the receptor is unable to bind adaptin and clathrin. The uptake mechanism is not functioning, LDL remains in the blood. High cholesterol concentration in the blood leads to arteriosclerosis and heart attacks (familiar hypercholesterinaemy). early endosome free cholesterol in the cytosol  lysosomal enzymes

Transcytosis Transport of macromolecules in an intact form through the cell. Endocytosis → vesicular transport → early endosome→ vesicular transport→ exocytosis A special case of receptor-mediated endocytosis: the endocytosed molecules avoid lysosomal degradation.

Transcytosis Coated vesicles!!  Examples: Uptake and transport of maternal IgG through the gut epithelium in newborns. Uptake and transport of maternal IgG through the placental epithelium in the fetus. Uptake and transport of IgA molecules through surface epithelia or in a secretory epithelium from the connective tissue space into the lumen. apical membrane domain gut lumen recycling transport vesicles early endosome Coated vesicles!!  connective tissue space

Transcytosis Transport through capillary endothel Caveolae!!

Vesicular transport 2 main moments: - pinching off the vesicles: signals, coat proteins - vesicular transport to the target membrane and fusion: regcognition molecules and fusion proteins

Rab proteins kihorgonyozás dokkolás fúzió

Vesicular transport Formation of vesicles: coat proteins COPI, COPII: ER-Golgi clathrin: plasma membrane: vesicles transporting lysosomal emzymes caveolin signals: small G-proteins: GDP/GTP-binding proteins: Rab proteins: „identity” proteins ER-Golgi: ARP (donor membrane together with COP proteins) ARF: molekular switch (GTP-bound form: coat protein- membrane association GDP-bound form: uncoating, fusion can occur) Tethering and docking of the vesicles: fusion proteins: SNARE-s

Vesicular transport

Membrane fusion trans-SNARE complex Fusion complex: excluding water

Felhasznált illusztrációk forrása:  Röhlich: Szövettan, 3. kiadás, Semmelweis Kiadó Budapest  Alberts – Johnson – Lewis – Raff – Roberts – Walter: Molecular biology of the cell. 5. kiadás, Garland Science  Saját prep. és/vagy felvétel, ill. rajz Campbell – Reece: Biologie, Spektrum – Fischer Wikipedia