1. Endocytosis of Gene Delivery Vectors: From Clathrin-dependent to Lipid Raft-mediated Endocytosis 
Ayman El-Sayed, Hideyoshi Harashima 
Molecular Therapy 
Volume 21, Issue 6, Pages (June 2013)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Schematic presentation of the cell membrane domains. The cell membrane contains clusters of lipids, lipid rafts, which exist within the non-raft region, a generally disordered lipid milieu. These lipid rafts are small (10–200 nm), heterogeneous, highly dynamic domains. Sphingolipids with long, unsaturated hydrocarbon chains associate with cholesterol within such domains. In addition, these rafts include: (i) proteins attached to glycosylphosphatidylinositol anchors (GPI-AP) that are inserted in the outer leaflet of the membrane, (ii) proteins attached to the inner leaflet of the membrane, (iii) transmembrane proteins that have a cytoplasmic domain in addition to an outer domain that is exposed at the cell surface.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Classification of the endocytic pathways based on the membrane regions that make up the primary endocytic vesicles. (i) Clathrin-mediated endocytosis takes place in non-lipid raft regions through the recruitment of clathrin from the cytosol into the inner leaflet of the cell membrane to stabilize the formed vesicle. (ii) Phagocytosis and macropinocytosis are endocytic mechanisms that transport large particles, in phagocytosis, or large amounts of extracellular fluids, in macropinocytosis, into uncoated vesicles. These vesicles formation involves vast areas of the cell membrane and the resulting phagosome or macropinosomes would be expected to contain both lipid raft and non-raft regions of the membrane. (iii) Endocytic pathways that take place in lipid raft regions can occur in two ways: (a) stabilization of the endocytic vesicles formed in lipid rafts by enrichment of certain proteins, such as caveolin for caveolae-mediated endocytosis, or flotillin for flotillin-dependent endocytosis, (b) formation of vesicles through the action of small guanosine triphosphatases (GTPases), such as Cdc42 and Arf1 for GRAF1-dependent endocytosis, Arf6 for Arf6-dependent endocytosis, or RhoA for RhoA-dependent endocytosis.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Intracellular fate of clathrin-mediated endocytosis. In clathrin-mediated endocytosis, clathrin is recruited from the cytosol into the inner leaflet of the cell membrane to form the clathrin-coated pit. The pit then is pinched off, to form a clathrin-coated vesicle that then begins to lose its clathrin coat in the cytosol. This is followed by the fusion of these uncoated vesicles with each other and with various vesicles from other endocytic pathways to form an early endosome. The early endosome becomes acidified to a pH in the range 6.1–6.8 and during acidification, the cargos inside the endosome are sorted and some of the cargos are recycled back to the cell exterior through recycling endosomes. The endosome then matures into a late endosome, with the pH reaching 4.8–6. This is followed by fusion between the late endosome and a lysosome to form an endolysosome where digestive degradation of the cargo takes place at a pH of ∼4.5.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Schematic presentation of the stages of ruffling during macropinocytosis. Macropinocytosis starts with an increase in actin polymerization which results in cell membrane ruffling. The planar membrane ruffles may fold and fuse with the plasma membrane to form macropinosomes. In the cytosol, the macropinosome loses the actin filaments on its surface. Actin polymerization is inhibited in the presence of cytochalasin D while ruffling is inhibited by amiloride. Amiloride inhibits Na+/H+ exchanger protein resulting in protons being accumulated in the forming ruffle. The accumulating protons inhibit Cdc42 and Rac1 via acidification resulting in the suppression of ruffling. The closure of macropinosome can be inhibited by wortmannin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Schematic presentation of the endocytic pathways that take place in lipid raft domains of the cell membrane and some of their key regulators. Caveolae-mediated endocytosis takes place in lipid rafts that are enriched in caveolin and the resulting vesicles are stabilized by cavin. Scission of the vesicles from the cell membrane takes place via the action of dynamin. Similarly, flotillin-dependent endocytosis results in the uptake of the cargo into vesicles enriched in flotillins shortly after flotillins become phosphorylated by Fyn kinase. GRAF1-dependent endocytosis is mediated by the complementary role of Cdc42 with Arf1 in meditating actin polymerization. Actin and GRAF1 then drive the formation of the endocytic vesicles. Similarly, Arf6 mediates the formation of the Arf6-dependent endocytic vesicles in addition to Rac1 which plays a role in scission of these vesicles. RhoA-dependent endocytic vesicles are formed via the function of RhoA and actin and vesicles formed are pinched off through the action of dynamin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions