Endocytic pathway

Clathrin-coated vesicles mediate transport within the late secretory and the endocytic pathways. Their major coat constituents are clathrin and various adaptor complexes. 

Bannister L.H. and Dodson H.C. (1992). Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse ultrastructure and uptake of tracers. Micros Res Techn 23, 128-141. 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

THE ENDOCYTIC PATHWAY PLAYS MULTIPLE ROLES IN CELLS OF THE NERVOUS SYSTEM 151... 

The series of molecular events responsible for the uptake process constitutes the endocytic pathway, which enables cells to internalize macromolecules from the cell exterior, forming an endosome. The endosome is an intermediate organelle that serves as an essential component for many receptor-mediated signaling pathways and as a transport vector for eventual delivery to a specialized organelle known as the lysosome. Once in the lysosomal lumen, digestive enzymes provide essential metabolites from these macromolecules (i.e. free amino acids and lipids) directly to the cytosol for their use. 

Under some circumstances, lysosomal hydrolases may fail to be properly packaged in the TGN, so they enter the default pathway to the cell surface, where they are secreted. Although these hydrolases do little harm at the nearly neutral pH of most extracellular fluids, they can also be returned to lysosomes by a pathway known as receptor-mediated endocytosis. In this pathway, M6P receptors are sent to the plasma membrane, where they bind escaped lysosomal hydrolases and bring them back to lysosomes through the early and late endosomes. Receptor-mediated endocytosis is a major component of the endocytic pathways for trafficking of membrane proteins and merit more detailed consideration. 

In a simplified view, the total flow is as follows (Fig. 8). Both soluble and membrane proteins that are translated at the membrane-bound ribosome are first localized at the ER. Some of them are transported to the Golgi apparatus, whereas others remain at the ER. At the Golgi apparatus, including the trans Golgi network (TGN), the next selection occurs some are transported to the plasma membrane, others to the endosome and to the lysosome/vacuole finally, and still others remain there. The lysosome is also an important organelle for the other transport system, the endocytic pathway. In this pathway, proteins at the plasma membrane are internalized by endocytosis. The sorting to lysosomes is treated in the next section. 

Fig. 8. Secretory pathway and endocytic pathway a simplified view.

In endocytosis, vesicles are formed at the plasma membrane and then transported to an endosome. (More precisely, endosomes should at least be classified into early endosomes and late endosomes, but this fact is ignored here.) The endocytic pathway also includes the following routes from the endosome to the lysosome, from the endosome to the plasma... 

A nonubiquitous organelle, the melanosome, is specialized for melanin synthesis. It is somewhat similar to the lysosome, and the resident proteins are derived from the endocytic pathway. A sorting signal, the NQPLLT, was found in the cytoplasmic protein of a human membrane protein (Vijayasaradhi et al., 1995). 

Many cells have an asymmetric structure because of the necessity for function (Drubin and Nelson, 1996). For example, (the outer surface of) the plasma membrane of epithelial cells is fenced by a tight junction so that the lipids are separated between the apical part and the basolateral part (Fig. 9) (Eaton and Simons, 1995). Therefore, some molecular mechanisms must exist to sort the plasma membrane proteins into these two parts. Some signals related to the secretory/endocytic pathways have been found important (Matter and Mellman, 1994). Their details are not described here because the area is too specific for predictive purposes. 

Le Borgne, R., and Hoflack, B. (1998a). Mechanisms of protein sorting and coat assembly insights from the clathrin-coated vesicle pathway. Curr. Opin. Cell Biol. 10, 499-503. Le Borgne, R., and Hoflack, B. (1998b). Protein transport from the secretory to the endocytic pathway in mammalian cells. Biochim. Biophys. Acta 1404, 195-209. 

P. U. Le and I. Nabi. Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J. Cell Sci. 116 1059-1071 (2003). 

R. Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins crucial role of Doa4p ubiquitin isopeptidase, Mol Cell Biol, 2001, 21, 4482-94. 

Amerik, a. Y., Nowak, J., SwAMiNATHAN, S., and Hochstrasser, M. The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol Biol Cell, 2000, 11, 3365-80. 

TAT liposomes remain intact within one hour of translocation and slowly migrate through the cell, bypassing the endocytic pathway, to the perinuclear zone where they disintegrate (95). The mechanism utilized by TAT to migrate across the membrane was thought to be energy independent because it operates at similar rates at both 4°C and 37°C (95,96). Cell entry by TAT is also unhindered by metabolic inhibitors such as sodium azide or iodoacetamide (97). Peptides constructed of both the d and l amino acids of Antp can be detected intracellularly, the inference of which is that no specific receptor was required because both isomers had equal potential (98,99). 

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