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Endocytosis is involved in multiple cellular processes, including the uptake of transport proteins, uptake of "opportunistic ligands" such as bacterial and plant toxins and viruses, attenuation of signaling activity, receptor recycling and resensi-tization, down-regulation of cell contacts during epithelial-to-mesenchymal transition, antigen uptake and processing in MHC class Il-expressing cells, transcytosis and delivery of antibodies to the newborn baby, and synaptic function. About 15-20 years ago it was frequently - and persistently - claimed that only one endo-cytic mechanism existed, namely the one mediated by clathrin-coated pits and vesicles. However, in studies in which clathrin-dependent uptake was inhibited by potassium depletion and acidification of the cytosol, it became clear that clathrin-independent endocytic mechanisms also must be taken into consideration [1-6]. That different forms of clathrin-independent endocytosis exist is now well-established. Thus, although the clathrin-mediated endocytic machinery is by far the most well-studied type of endocytosis [7-9], a plethora of clathrin-independent endocytic mechanisms also appears in today's literature; however, the underlying molecular machinery is far from being clarified [10-16]. One such clathrin-independent endocytic mechanism can involve caveolae.

Caveolae are small, 50- to 70-nm membrane invaginations that are present on the plasma membrane of many different cell types [17]. Caveolae are often clustered within a short stretch of the plasma membrane, and they can even form large, sometimes branched invaginations deeply into the cytoplasm (Fig. 4.1). In vivo, caveolae are particularly abundant in adipocytes, endothelial cells, fibroblasts and smooth muscle cells, but they are also found in, for instance, the basal layer of epithelial cells in stratified epithelia [18]. In contrast, they are scarce or absent in hematopoietic cells and neurons, for example. In vitro, the presence of caveolae is more unpredictable because cultured cells do not always reflect the caveolin/cav-eolae status of the tissue of origin. In addition, the caveolin/caveolae status of the cells of origin is often unknown, not least when cells derive from cancers [17].

Lipid Rafts

Fig. 4.1 Electron micrographs of human myoepithelial cells grown in a chemically defined medium in the absence of insulin. Under these conditions the cells stop proliferating and express a differentiated phenotype including distinct bundles of actin filaments (shown as Ac in panel A) and numerous caveolae. In the presence of insulin, the cells do not show these characteristics but proliferate (see [19]). Note the deeply invagina-ted caveolae clusters in panels B and C (arrows). Scale bars = 200 nm.

Fig. 4.1 Electron micrographs of human myoepithelial cells grown in a chemically defined medium in the absence of insulin. Under these conditions the cells stop proliferating and express a differentiated phenotype including distinct bundles of actin filaments (shown as Ac in panel A) and numerous caveolae. In the presence of insulin, the cells do not show these characteristics but proliferate (see [19]). Note the deeply invagina-ted caveolae clusters in panels B and C (arrows). Scale bars = 200 nm.

Moreover, cells may express cav-1 without having structurally identifiable caveolae (our unpublished results). Also, the culture conditions may influence whether caveolae are frequent or absent in one and the same cell line [19] (Fig. 4.1).

Caveolae are a subset of lipid rafts which, in addition to an enrichment in cholesterol and sphingolipids, are characterized by the protein caveolin. There are three members of the caveolin family, caveolin-1 and -2, which are (co)expressed in many cell types, and caveolin-3 that is specific for muscle cells. Caveolin-1 and caveolin-2 exist in different isoforms. The caveolins form a characteristic hairpin loop into the membrane lipid bilayer, exposing both the N- and C-termini to the cytoplasm. In the N-terminal region the caveolin scaffolding domain is responsible for the interaction with numerous other proteins. The C-terminal region is attached to the membrane by palmitoyl anchors. Expression of caveolin-1 can be sufficient to generate caveolae, although other factors than caveolin may be involved. For instance, in polarized epithelial cells such as intestinal Caco-2 cells, expression of caveolin-1 results in formation of caveolae only at the basolateral surface [20].

Caveolae seem to represent a kind of multifunctional platform as they have been implicated in such different functions as cholesterol transport, calcium transport and homeostasis, signaling, regulation of endothelial nitric oxide synthase (eNOS) activity, and tumor suppression [17,21-27]. In addition, it has been debated over the years whether caveolae are also involved in endocytosis, although the many other functions of caveolae would contradict such a role. Historically, the reason for believing that caveolae could be involved in endocytosis comes from their characteristic, invaginated shape as seen in the electron microscope and, more recently, because dynamin, a GTP-binding protein involved in the formation of clathrin-coated vesicles, has been found in association with caveolae [28,29] (see Section 4.8). Since caveolae also bind a variety of ligands that become internalized (e.g.,

4.2 Caveolae are Largely Immobile, Nonendocytic Membrane Domains | 71

cholera toxin; see Section 4.5), it is tempting to conclude - by analogy to clathrin-coated pits - that they are endocytic, although the question of whether they are actually able to pinch off and deliver material to intracellular compartments has often been ignored. Recent studies, including electron microscopy (EM) and live cell digital imaging, strongly indicate that the answer to the question of whether caveolae are endocytic structures or not, is far from a simple "yes" or "no", but more complex [30]. Therefore, in the present chapter we will treat these aspects of caveolar biology and present a working model that includes the various findings and speculations (see Section 4.10).

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