Caveolae-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. 

Rejman J, Oberle V, Zuhom IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377 159-169... 

Rejman J, Bragonzi A, Conese M (2005) Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 12 468 174... 

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... 

J. E. Schnitzer, P. Oh, E. Pinney, and J. Allard. Filipin-sensitive caveolae-mediated transport in endothelium reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J. Cell Biol. 127 1217-1232 (1994). 

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). 

Ferrari A, Pellegrini V, Arcangeli C, et al. Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 2003 ... 

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). 

In addition to the well characterized roles of clathrin-caveolae-mediated endocytosis and macropinocytosis/phagocytosis, an ill-defined route of nonclathrin-noncaveolae mediated endocytosis still exists (31,32). It seems that all of the until now poorly understood mechanisms of internalization can be summarized in this topic. 

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). 

Caveolae-Mediated Endocytosis and Caveolae-Like Endocytosis Pharmacological Inhibitors... 

Staurosporine (2-5 pM, 60 minutes), a general kinase inhibitor, has been used to discriminate caveolae-mediated uptake from clathrin-mediated uptake (63). 

In many studies, folate or folic acid (5nM) is applied to study caveolae-mediated endocytosis resp. potocytosis (27,119,120). 

However, the initial step of uptake is a branch of the endocytic uptake and corresponds to one of the mechanisms described in the section on Initial Mode of Internalization. In most studies, clathrin-mediated or caveolae-mediated mechanisms are discussed. 

Different incubation times and concentrations of applied marker might lead to different patterns of distribution. For example, LysoTracker Red is only selective for lysosomes when applied in low concentrations for a short time (10 minutes prior to imaging). The same has been described for other markers such as EGF and Tfn (as described in the section Caveolae-Mediated Endocytosis ). 

Keep in mind that exocytosis might occur during sample preparation (e.g., preparing cells for flow cytometric analysis). After the experiment (and prior to analysis) cells should be kept below 4°C to block all active processes (such as exocytosis). Exocytosis might also be blocked with NEM (see section Caveolae-Mediated Endocytosis and Caveolae-Like Endocytosis Pharmacological Inhibitors ). 

Gene delivery systems can distribute plasmids to the desired target cells, after which the plasmid is internalized into the cell by a number of mechanisms, such as adsorptive endocytosis, receptor-mediated endocytosis, micropinocytosis, caveolae-mediated endocytosis and phagocytosis (see Section 1.3.3.2). The intracellular fate of plasmids depends on the means by which they are internalized and translocated to the cytoplasms and then to the nucleus. In coated-pit endocytosis, DNA complexes first bind to the cell surface, then migrate to clathrin-coated pits about 150 ran in diameter and are internalized from the plasma membrane to form coated vesicles. 

Cell-surface receptors are involved in both phagocytosis and pinocytosis. At least four distinct mechanisms of pinocytosis have been characterized macropinocytosis, clathrin-mediated endocytosis, raft/caveolae-mediated endocytosis, and clathrin-and caveolae-independent endocytosis (1). Selected receptor-mediated aspects of these mechanisms are outlined below. 

Neurotrophin-Trk receptor complexes are internalized and retrogradely transported from distal axons to the neuronal cell body where they signal to the soma to mediate target-dependent survival, growth and gene expression. The neurotrophin-Trk receptor complex is internalized by four mechanistically diverse and highly regulated pathways macropinocytosis clathrin-mediated endocytosis caveolae-mediated endocyto-sis and Pincher-mediated endocytosis. The kinase activity of Trk is probably required for receptor internalization. 

AR endocytocsis was only partially blocked by inhibiting clathrin-coated pit endocytosis with hyperosmotic sucrose. A complete blockade of internalization was accomplished by treatment with filipin, an agent that inhibits caveolae-mediated internalization. These data suggest that the a2A-AR is internalized via both clathrin- and caveolae-mediated pathways and could account for the observations of DeGraff et al. (70) that noted only a modest effect of arrestin overexpression on a2A-AR endocytosis. 

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