Clathrin-mediated

Traub LM (2005) Common principles in clathrin-mediated sorting at the Golgi and the plasma membrane. Biochim Biophys Acta 1744(3) 415-37... 

LDH-FITC is well overlapped with red fluorescent clathrin-TR, but not with caveolin-1-TR (Figure 13.10). This is dear evidence that clathrin-mediated endocytosis is the prindpal mechanism for the cellular internalization of LDH particles. Caveolae-mediated endocytosis, if any, seems not to be responsible for LDH uptake. 

Fig. 13.11 Effects of clathrin-mediated endocytosis inhibitors on the internalization of LDH-FITC. Cellular uptake (%) of LDH was calculated by comparison with that in the absence of inhibitor (100%).

The bulk of pinocytosis in the nervous system is mediated by clathrin-mediated endocytosis (CME) [55] and this is the best-characterized pathway. More detail about clathrin-mediated pathways will be given when receptor-mediated endocytosis and the synaptic vesicle cycle pathways are considered. Pinocytosis through CME is responsible for uptake of essential nutrients such as cholesterol bound to low density lipoprotein (LDL) and transferring, but also plays a role in regulating the levels of membrane pumps and channels in neurons. Finally, CME is critical for normal synaptic vesicle recycling. 

Extracellular ligands (hormones, neurotrophins, carrier protein, adhesion molecules, small molecules, etc.) will bind to specific transmembrane receptors. This binding of specific ligand induces the concentration of the receptor in coated pits and internalization via clathrin-coated vesicles. One of the best studied and characterized examples of RME is the internalization of cholesterol by mammalian cells [69]. In the nervous system, there are a plethora of different membrane receptors that bind extracellular molecules, including neurotrophins, hormones and other cell modulators, being the best studied examples. This type of clathrin-mediated endocytosis is an amazingly efficient process, capable of concentrating... 

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... 

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. 

However, an alternative pathway that bypasses clathrin-mediated endocytosis and EEs appears to be available as well. This model of endocytosis known as kiss and run or its variant kiss and stay have attracted increasing interest in recent years [74] (Fig. 9-9B). Kiss and run has been directly demonstrated with dense-core granules in neuroendocrine cells [84, 85], and this model would explain some observations that are not readily accommodated by the classical pathway. The kiss and run model proposes that neurotransmitters are released by a transient fusion pore, rather than by a complete fusion with integration of the synaptic vesicle components into the plasma membrane. Synaptic membrane proteins never lose their association and the vesicle reforms when the pore closes. As a result, the empty vesicle can be refilled and reused without going through clathrin-mediated endocytosis and sorting in the EEs. 

Brodin, L., Low, P. and Shupliakov, O. Sequential steps in clathrin-mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10 312-320, 2000. 

Verstreken, P., Kjaerulff, O., Lloyd, T. E. et al. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109 101-112,2002. 

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]. 

ASIC acid-sensing ion channels CME clathrin-mediated endocytosis... 

Hussey SL (2002) Efficient delivery of streptavidin to mammalian cells clathrin-mediated endocytosis regulated by a synthetic ligand. J Am Chem Soc 124 6265-6273... 

Fig. 2.2 Cellular internalisation pathways proposed for carbon nanotubes (CNTs) (A) phagocytosis (B) membrane piercing by passive diffusion (C) caveolae-mediated endocytosis and (D) clathrin-mediated endocytosis... 

The GAT domain (GGA and Toml) has recently joined the ranks of ubiquitin-binding domains [91]. As the name implies, this domain is found in the GGA-and Toml-like proteins, two regulator classes of clathrin-mediated vesicular traffic. All proteins harboring the GAT domain also contain an N-terminal VHS domain, which is named after the Vps27, Hrs, and STAM proteins. Interestingly, these latter proteins are known to contain a ubiquitin-binding UIM motif, which appears to be replaced by the GAT domain in the GGA and Toml-like proteins (Figure 12.6). 

Clathrin-mediated endocytosis involves the internalization of transmembrane receptor-ligand complexes stimulating the formation of a coated pit that eventually buds off the membrane to form an intracellular endocy-totic vesicle. This process is dependent on the protein clathrin that is recruited to the membrane and forms a cage-like structure around the forming pit. Internalization via clathrin-dependent pathway allows the uptake of particles approximately 120nm in size (63-65). Once internalized, the clathrin coating disassociates from the endosome to be recycled and to allow the endosome to fuse with an intracellular compartment, usually a... 

Previous work has shown that the majority of cells internalize liposomes through an endocytic pathway (4,5). There are multiple pathways for internalization involving vesicles of 50 300 nm in diameter. These include clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis, macropinocytosis, and nonclathrin- noncaveolae-dependent endocytosis (6). 

Figure 1 Endocytosis of liposomes five different routes into the cell. Multiple pathways can be used by the cell to internalize liposomes. Besides the well-characterized clathrin-mediated endocytosis, other pathways can be applied by the cell. Possible alternative pathways include phagocytosis or macropinocytosis—two pathways that internalize by an actin-driven protuberance of the plasma membrane. Other routes include the involvement of caveolae where substances are taken up into the cell bypass the traditional endosome/lysosome system (particles might escape from being degraded in lysosomes). Finally there exists an ill-defined mechanism that is neither mediated by caveolae nor by clathrin. In a single cell type, two or more of these mechanisms can coexist. Source Adapted from Ref 8.

Clathrin-mediated (or clathrin-dependent) endocytosis normally occurs at specialized sites, where complex structures called coated pits are assembled in order to concentrate surface proteins for internalization. The coat consists of many different proteins that are needed for stabilization of both the pit and the forming of the clathrin-coated vesicle. The two most abundant proteins found within these structures are clathrin and the adaptor protein AP-2 (9). 

Acidification of the cytoplasm has been described as blocking clathrin-mediated endocytosis, but might affect cell morphology and viability (43,44). The mechanism of inhibition is still ill defined but it is clear that the pH of the medium has no effect on the intralysosomal pH (45). It should be noted that cytosolic acidification shows cell-type variations and might not be highly specific. 

The water-soluble methyl-P-cyclodextrin (mpCD) is known to form soluble inclusion complexes with cholesterol, leading to depletion of cholesterol from the plasma membrane (16,46,47). As a result, cholesterol-rich microdomains, which are involved in caveolae-mediated as well as clathrin-mediated endocytosis, are destroyed. mpCD therefore decreases both clathrin- and caveolae-mediated uptake. The two other well-known cyclodextrins [a-, and y-cyclodextrin (6 and 8 units of a-1,4 glucose)] do not bind cholesterol effectively (both are not specific for cholesterol, but might remove phospholipids from the plasma membrane) and have no significant effect (46). 

Phenylarsine oxide (20 [rM 30 minutes) is used as an inhibitor for receptor-mediated (clathrin-mediated) endocytosis (48). 

The calcineurin inhibitors tacrolimus (FK 506) and cyclosporin A block the function of dynamin and are thought to be specific for clathrin-mediated uptake (50). The smal guanosine triphosphate (GTP)ase dynamin is also involved in other processes and is therefore described in section Dynamin Dependence on Liposome Uptake. ... 

Epidermal growth factor (EGF), low-density lipoprotein (LDL), and transferrin (Tfn) are often used as model peptides because all three are taken up via clathrin-mediated endocytosis, but they are sorted into different pathways after endocytosis. 

The human LDL complex delivers cholesterol to cells by receptor-mediated, clathrin-mediated endocytosis. LDL is used to follow the lysosomal directed pathway. Once internalized, LDL dissociates from its receptor and ultimately accumulates in the lysosomes (111). 

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