Caveolae endocytosis

Pelkmans L, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M (2005) Genomewide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436 78-86... 

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... 

Caveolae-Linked Endocytosis and Non-Caveolae, Clathrin-Independent Endocytotsis Offer Delivery of Drugs to Novel Intracellular Targets... 

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). 

M. Kirkham and R. G. Parton. Clathrin-independent endocytosis New insights into caveolae and non-caveolar lipid raft carriers. Biochim. Biophys. Acta 1745 273-286 (2005). 

Figure 2 Proposed pathways for liposomal entry into the cell enhanced by peptides. These include direct cell entry suggested as the mechanism of entry by cell-penetrating peptides and receptor-mediated endocytosis by caveolae- and clathrin-dependent endocytosis.

Caveolae-dependent endocytosis occurs in cholesterol- and sphingomyelin-rich flask-shaped invaginations of the cell membrane known as caveolae (67). The shape is determined by a framework constructed by the protein caveolin. The protein binds plasma cholesterol, inserting into... 

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.

Since their discovery in the early 1950s (14,15), caveolae had been considered to be uninteresting static organelles that have no capability for the uptake of particles. However, in the past two decades, caveolae have moved into the focus of many researchers because they seem to play an important role in the uptake of various agents, but this is not without controversy. In 2002, Thomsen et al. published strong evidence that caveolae are static fixed domains that are not involved in endocytosis (16), but it has also been reported that caveolae and caveolin can clearly be internalized—at least after specific stimuli, as shown with simian virus 40 (SV40) (17), or by treatment with okadaic acid (18). 

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. 

Cholera Toxin Subunit B. a marker for caveolae-dependent endocytosis. partly co-localizes with liposomal FITC>dextran indicating that pH-sensitive liposomes are taken up-at least to a certain extend— via caveotae pathway... 

Cave olae -mediated and caveolae-like endocytosis... 

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... 

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. 

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

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 ). 

Parton RG, Richards AA. Lipid rafts and caveolae as portals for endocytosis new insights and common mechanisms. Traffic 2003 4(11) 724. 

Damm EM, Pelkmans L, Kartenbeck J, Mezzacasa A, Kurzchalia T, Helenius A. Clathrin- and caveolin-1-independent endocytosis entry of simian virus 40 into cells devoid of caveolae. J Cell Biol 2005 168(3) 477-488. 

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