Endocytosis, clathrin-mediated

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... 

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. 

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

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... 

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.

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). 

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). 

As described above, mpCD is a commonly used inhibitor for both the clathrin and the caveolae pathway. See the section on Inhibiting Clathrin-Mediated Endocytosis for details. 

As described above (section Clathrin-Mediated Uptake ), several ligands for clathrin-mediated endocytosis (see section Clathrin-Mediated Endocytosis EGF, Tfn, LDL) can be used to highlight early endosomes or lysosomes, depending on different incubation times. 

Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE. Clathrin exchange during clathrin-mediated endocytosis. J Cell Biol 2001 155(2) 291-300. 

Sieczkarski SB, Whittaker GR. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol 2002 76(20) 10455-10464. 

Vieira AV, Lamaze C, Schmid SL (1996) Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274 2086-2089 Vijay-Kumar S, Bugg CE. Wilkinson KD, Cook WJ (1985) Three-dimensional structure of ubiquitin at 28 A resolution. Proc Natl Acad Sd USA 82 3582-3585 Wang HR, Kania M, Baumeister W. Nederlof PM (1997) Import of human and Thermoplasma 20S proteasomes into nudei of HeLa cells requires functional NLS sequences. Eur J Cell Biol 73 105-113... 

Higgins, M.K. McMahon, H.T. (2002) Snap-shots of clathrin-mediated endocytosis. Trends Biochem. Sci. 27, 257-263. 

Fig. 5 Synaptic vesicle recycling in the synapse. For synaptic vesicle recycling, several endocytic mechanisms appear to co-exist in synaptic nerve terminals. In the case of fast kiss-and-ran exo-cytosis/endocytosis, the fused vesicle does not collapse into the membrane but is retrieved directly by a fast process. The molecular machinery underlying this pathway is unknown. Vesicles that have fully collapsed into the membrane are recycled by clathrin-mediated endocytosis. Clathrin, along with other proteins, is involved in membrane invagination (see figure and text) and leads finally to the formation of a constricted pit. The GTPase dynamin (black ring) mediates membrane scission of the constricted pit. After removal of the clathrin coat, two pathways are possible (direct recycling and recycling via the early endosome). In all cases, before fusion the recycled vesicles have to be loaded with neurotransmitters (NT).

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