Uncoated vesicles

As noted above, synaptic vesicles are not typically generated at the level of the TGN. Instead, they are assembled from endocytosed material retrieved from the synaptic plasma membrane. Synaptic vesicle and plasma membrane lipids and proteins are synthesized in the endoplasmic reticulum and modified in the Golgi apparatus, where they are then packaged in secretory vesicles. These synaptic precursors are delivered to the plasma membrane from the cell body by the constitutive secretory pathway. Synaptic vesicle proteins must be retrieved by clathrin-mediated synaptic vesicle endocytosis, a variant of RME with some neuron-specific components. Once the vesicle sheds its clathrin coat, the uncoated vesicle fuses with a... 

Fig. 5.3 Immunogold labeling of human A i ARs on ultrathin sections of transfected CHO cells, (a) Small aggregates of gold particles (arrow) on the plasma membrane after a 60-min incubation with 10 nM agonist NECA at 4°C. (b) After a 15-min incubation with agonist at 37°C A3 ARs are visible in uncoated vesicle at level of the cortical cytoplasm (Trincavelli et al. 2000)...

LDL have bound to LDL receptors. The coated region surrounding the bound receptor, referred to as a coated pit, pinches off and becomes a coated vesicle. Subsequently, uncoated vesicles are formed as clathrin depolymerizes. Before uncoated vesicles fuse with lysosomes, LDL are uncoupled from LDL receptors as the pH changes from 7 to 5. (This change is created by ATP-driven proton pumps in the vesicle membrane.) LDL receptors are recycled to the plasma membrane, and LDL-containing vesicles fuse with lysosomes. Subsequently, LDL proteins are degraded to amino acids, and cholesteryl esters are hydrolyzed to cholesterol and fatty acids. 

The best-documented way of endocytosis is receptor-mediated uptake of ligands via clathrin-coated vesicles (reviewed in Schmid 1997). Receptors are recruited and concentrated into clathrin-coated pits at the plasma membrane. After coated vesicle formation, the clathrin coat is removed by the concerted action of auxihn and heat shock protein 70 (Ungewickell et al. 1995). The uncoated vesicles fuse with early endosomes in a rab5-regulated manner (Rubino et al. 2000). 

A pre-requisite for clathrin-coat assembly is the recruitment to the membrane of an adaptor complex. Similar to what has been observed for the recruitment of coatomer to Golgi membranes, adaptor binding is dependent on the presence of ARF-GTP. However, in contrast to COPI vesicle formation, ARF-GTP is suggested to act in a process before budding and not as a stoichiometric coat component. Other differences between COP-coated and clathrin-coated vesicles concern their uncoating mechanism. Disassembly of clathrin-coated vesicles is believed to depend on the chaperoneHSC 70 and on auxilin. 

Phospholipid vesicles, uncoated or polyethylenglycol-coated. They can be used to vehicle dtugs, antibodies or nucleic acids to target cells. 

Ungewickell, E. (1985). The 70-kd mammalian heat shock proteins are structurally and functionally related to the uncoating protein that releases clathrin triskelia from coated vesicles. EM BO Journal, 4, 3385-91. 

In the classic model of synaptic vesicle recycling in nerve terminals, synaptic vesicles fuse completely with the plasma membrane and the integrated vesicle proteins move away from the active zone to adjacent membrane regions (Fig. 9-9A). In these regions, clathrin-mediated synaptic vesicle endocytosis takes place rapidly after neurotransmitter release (within seconds) [64]. The process starts with the formation of a clathrin-coated pit that invaginates toward the interior of the cell and pinches off to form a clathrin-coated vesicle [83]. Coated vesicles are transient organelles that rapidly shed their coats in an ATP/chaperone dependent process. Once uncoated, the recycled vesicle fuses with a local EE for reconstitution as a synaptic vesicle. Subsequently, the recycled synaptic vesicle is filled with neurotransmitter and it returns to the release site ready for use. This may be the normal pathway when neurotransmitter release rates are modest. Clathrin/ EE-based pathways become essential when synaptic proteins have been incorporated into the presynaptic plasma membrane. 

FIGURE 2 3-7 Schematic diagram of the synaptic vesicle cycle. Neurotransmitter-filled vesicles held in the reserve pool are trafficked to a readily releasable pool where they are docked, primed and fused with the plasmalemma at the synaptic cleft. Also depicted is the clathrin-mediated endocy-tosis of the fused vesicles, which is followed by their uncoating and recycling via early endosomal fusion and budding of vesicles. This returns the vesicles to the reserve pool. Some of the phosphoproteins which regulate these steps are shown. For a more detailed description of this process and the phosphoproteins involved the reader is directed to the excellent text by Cowen et al. [67]. 

Attempts to achieve a tissue specific transport of liposomes have recently been described. Cohen et al. coated liposomes with aggregated immunoglobulin ( 7). The take up rate of these vesicles into phagocytes could be increased by a factor of 60 compared to uncoated liposomes. 3 to 25 times more liposomes are taken up by the corresponding cells, if they are loaded with the appropriate antibodies (58). Similar attempts to achieve a "homing" have been carried out using lipid fixed antibodies (59). 

M. Kirkham, A. Fujita, R. Chadda, S. J. Nixon, T. V. Kurzchalia, D. K. Sharma, R. E. Pagaon, J. F. Hancock, S. Mayor, and R. G. Parton. Ultrastructural identification of uncoated caveolin-independent early endocytic vesicles. J Cell Biol. 168 465-476 (2005). 

Uncoated fused-silica capillaries have similarly been applied to elec-trokinetic chromatography (EKC) analysis of solute interactions with both liposomes (33,34) and surfactant vesicles (59,60). 

Uncoating requires an interaction with the uncoating ATPase Hsc70. Apparently, however, hydrolysis of phosphatidylinositol (4,5)-bisphosphate is required, which is carried out by the protein synaptojanin. Synaptojanin has two phosphatase domains, and in its absence clathrin-coated vesicles accumulate. Furthermore, the... 

Non-clathrin-coated pit internalization can occur through smooth imagination of 150-300 nm vesicles or via potocytosis. This pathway has been shown to be involved in the transport of folate and other small molecules into the cytoplasm. Plasmids are taken up by muscles through the T-tubules system and caveolae via potocytosis. Muscle cells appear to take up plasmids through the T-tubule system and caveolae via potocytosis. Apart from coated or uncoated pit pathways, cells may also take up plasmid/cationic carrier complexes via plasma membrane destabilization. Particles greater than 200 nm in diameter are not... 

Greene, L. E., and Eisenberg, E. (1990), Dissociation of clathrin from coated vesicles by the uncoating ATPase. J. Biol. Chem. 265, 6682-6687. 

Heuser, J., and Steer, C. J. (1989). Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia A candidate intermediate in the vesicle uncoating reaction. J. Cell Biol. 109, 1457-1466. 

As noted above, iron-loaded serum transferrin has the role of transporting Fe(III) to cells requiring it. Once it reaches a target cell, it binds to the transferrin receptor (TfR) on the cell outer surface. TfR is a disulfide-linked dimer that binds two transferrin molecules. At neutral pH, apo transferrin does not bind to the transferrin receptor. Once formed, the transferrin receptor complex becomes detached from the cell membrane and enters the cell enclosed in a clathrin-coated vesicle. Uncoating of the vesicle generates an endosome in which the pH is lowered to 5.5, promoting release of iron. At this pH, the transferrin remains bound to its receptor, and... 

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