1. Exocytosis, endocytosis, vesicular transport
Anna L. Kiss
Deparment of Anatomy, Histology and Embryology,
Semmelweis University
Budapest
2017
Endocytosis: phagocytosis, pinocytosis; primary lysosomes, secondary lysosomes, phagosomes, autophagy, lyosomal desaeses, receptor-mediated endocytosis; clathrin coated vesivle, caveolae, endosomes (early. Late, recycling) transcytosis

2. Endocitózis/exocitózis

3. Endosome
Endosome: Content: gradually acidifying proton pump in their membrane.
Acidic pH: receptor-ligand disszociation
futher fate can be separated

4. Endosome
Endosome: cellular organelles with variable size and shape – close to the plasme membrane.
early or sorting endosome
recycling endosome
Multivesicular body /vs late endosome
lysosome

5. Multivesicular bodies

6. Lysosomes
EM: membrane surruonded vacuoles, fine granular structure inside.
Content: acidic hydrolytic enzymes (more than 40), pH optimum: pH 5.
Function: degradation of macromolecules by hydrolysis.

7. Lysosomes I. Primary lysosomes:contain ONLY lysosomal enzymes
Lysosomal enzymes: nucleases, proteases, glycosidases, lipases, phosphatases, sulphatases, …(more than 40). One lysosome can contain more enzymes!
Acidic pH : H+ pump (proton- ATPase, vacuolar type) in the membrane of the lysosomes.
Lysosomal membrane (and the neutral pH of the cytosol) protect the cell from the self-digestion.
Membrane protection: highly glycosylated proteins and lipids in the membrane ((sugar chains prevent direct contact of the enzymes with the membrane). Transport proteins and proton-ATPase in the membrane.
II. Secondary lysosomes

8. Exocytosis: transport from the cytoplasm to the cell membrane
Sorting from the Golgi network
To the plasma membrane
A. Membrane proteins released to the plasma membrane from the trans-Golgi network by vesicles, continuous, vesicles open by fusion
B. Secretory proteins containing vesicles, vacuoles pinch off from the trans-Golgi network, open by membrane fusion, their contant is released
Regulated exocytosis: exocytosis stimulated by signal (signal transduction) mast cell
constitutive exocytosis: continuous, without signal
cell membrane
secretion (regulated exocytosis)
constitutive exocytosis
trans-Golgi network
lysosome
2. To the lysosome (or late endosome)
Lysosomal proteins: localization signal: mannose-6-phosphate, receptors: man-6-P-receptors
rER 
nuclear membrane

9. Exocytosis
Membrane fusion: phospholipids of the membranes fuse with each other

10. On the way from Golgi to plasma membrane
along microtubulus

11. Exosomes, microvesicles

12. 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

13. Endocytosis of large particles (>0,1-0,5 μm)
Phagocytosis
Endocytosis of large particles (>0,1-0,5 μm)
Examples: macrophages and neutrophilic granulocytes (professional phagocytes)
bacterium
cytoplasmic process
macrophage
plasma
membrane
erythrocyte  
Phagocytosis of bacteria into a blood leukocyte (neutrophilic granulocyte). Electron micrograph.
A macrophage engulfs two erythrocytes. Arrow: margin of the cytoplasmic process. Scanning EM.

14. Fc-ends of IgG molecules
Phagocyted particles: debris of dead cells, bacteria, senescent erythrocytes, apoptotic cells and bodies, foreign particles, etc. …
Fc-ends of IgG molecules
Phases of phagocytosis:
Fc-receptor
bacterium
1. Adsorption: particles to be phagocytosed are bound to the cell surface (mostly to receptors in the plasma membrane).
Specific receptors: e.g. Fcγ-receptors, complement-receptors. Example: phagocytosis of bacteria covered with immunoglobulin molecules (IgG) into a macrophage cell.
Non-specific receptors: (scavanger receptors): receptors recognizing unusual carbohydrates or a changed phospholipid pattern on the surface
Living cells are not phagocytosed due to proteins on their cell surface which activate inhibiting receptors on the plasma membrane of the phagocytic cell (inhibition of phagocytosis).
plasma membrane 
actin microfilaments
2. Ingestion: the phagocytic cell with its collar-like cell process (pseudopodium, lamellipodium) surrounds the particle, the free margins of the process are closed over the particle.
Actin microfilaments are polymerised in the cell process. After complete closure, the particle is enclosed into a vacuole which becomes detached from the plasma membrane. Phagosome. 

16. If the acidification is not going on……

17. 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

18. Receptors

19. 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)

20. Macropinocytosis
Depends on ruffling

21. 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)

22. 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

24. 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.

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

26. 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)

27. Uncoating and fusion with early endosomes

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)

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

30. caveolae

31. 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

32. 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

33. 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

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

35. 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)

37. 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.

38. 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).

39. Lysosomes Primary lysosomes
Secondary lysosomes: primary lysosome+ phagocytotic vacuole
heterophage vacuole
autophage vacuole
(phagocytosed particles: dead cells, bacteria aged red blood cells, apoptotic cells and bodies, foreign particles, etc.)
autolysosome
heterolysosome

40. Autophagosome, electron micrograph
Autophagy
Focal digestion of cytoplasmic regions in unfavorable conditions („the cell eats itself”). Elimination of unnecessary and/or damaded cell components.
- A C-shaped sequestration cisterna surrounds a cytoplasmic region to be degraded. Two parallel membranes, the inner membrane disappears later.
- The cytoplasmic region, totally separated by the sequestration cisterna (autophagosome) is fused with a lysosome and degraded.
A widespread autophagy can lead to death of the cell, many regard it as a second form of programmed cell death.
Sequestration cisterna (arrows), electron micrograph.
Autophagosome, electron micrograph
Micrographs by Prof. J. Kovács

41. Autophagy

42. Types of autophagy

43. 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.

44. 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

45. Transcytosis
Transport through capillary endothel
Caveolae!!

46. 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

47. 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

48. Rab proteins
kihorgonyozás
dokkolás
fúzió

49. Vesicular transport

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

51. 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