1. 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

2. 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

3. II. Pinocytosis
Uptake of particles smaller than 0,1 μm, or macromolecules or dissolved substances by endocytosis. The resulting vesicles are 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

4. Receptors

5. 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, (06 March 2003); doi: /nature <> 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, (06 March 2003)

6. Macropinocytosis
Depends on ruffling

7. 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, (06 March 2003); doi: /nature <> 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, (06 March 2003)

8. 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

10. 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.

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

12. 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)

13. Uncoating and fusion with early endosomes

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

15. 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 

16. 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, (06 March 2003); doi: /nature <> 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, (06 March 2003)

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

18. caveolae

19. 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

20. 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

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

22. 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

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

24. Caveolae-mediated endocytosis:caveosome-like structures

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

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

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

28. 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, (06 March 2003); doi: /nature <> 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, (06 March 2003)

30. 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.

32. 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).

33. 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

34. 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.

35. 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

36. Transcytosis
Transport through capillary endothel
Caveolae!!

37. 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

38. Rab proteins
kihorgonyozás
dokkolás
fúzió

39. 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

40. Vesicular transport

41. Membrane fusion trans-SNARE complex Fusion complex: excluding water

42. 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