Caveolin Expression and Localization Varies Depending on the Physiological State of Cells in Culture

The localization of caveolin in cells has been shown to be dependent on culture conditions, growth state, and the degree of confluence. In studies in which caveolin has been expressed in cells lacking caveolae, such as lymphocytes [20] or cancer cells [21], caveolin-1 expression resulted in its localization to the plasma membrane, the formation of caveolae, and the redistribution of other caveolae-specific proteins to these newly formed caveolae. The level of expression and distribution of caveolin-1 in isolated versus confluent endothelial cells is dramatically different. As cells approach confluence, caveolin and cholesterol accumulate at the lateral borders of endothelial cells, along with other caveolin-associated proteins and signaling molecules, including annexin II and other caveolae-localized proteins [22]. The organization of caveolae and associated proteins in these lateral membranes has been well defined in venular endothelial cells (Fig. 8.2). In confluent venular endothelial cells and intact veins, a large tubular vesicular network forms from endosomes originating from caveolae. This system has been extensively characterized and identified as being critical in transcytosis and leukocyte extravasation, and is termed the vesicular vacuolar organelle (VVO). The VVO represents an extensive set of tubules and vesicles that are connected, dynamic, and extend to take up approximately 18% of the total volume of the cytoplasm [2325]. Several studies have linked caveolin and caveolae to macromolecule transport across the endothelium [26,27]. The structure and regulation of caveolae and its associated structures such as the VVO may be important in the functions of cav-eolin in vivo in control of vessel permeability and leukocyte migration. There is much that remains to be investigated using appropriate models, as well as studies in intact vessels and animals.

Both caveolin-1 and caveolin-2 exhibit widespread tissue distribution and extensive co-localization within the cell [28-31]. Both are found distributed in association with the plasma membrane and with intracellular membranes associated with several organelles in cells. Caveolin-1 and caveolin-2 also exhibit coordinate changes in their tissue-specific expression. For example, ablation of caveolin-1 in the mouse resulted in the widespread down-regulation of caveolin-2 in all of the tissues in which caveolin-1 was expressed [31]. Mutations in either caveolin-1 or caveolin-2 cause both proteins to mis-localize and accumulate with newly formed lipid bodies localized in the perinuclear region of the cell [32,33]. An intriguing study in cells lacking both caveolin-1 and -2 (LnCaP cells) showed that the formation of caveolae required expression of both caveolin-1 and caveolin-2, and that the localization of caveolin-1 to the cell surface required phosphorylation of caveolin-2 at specific sites [21]. However, the caveolin-2 null mouse shows normal caveolin-1 expression and normal caveolae in many tissues [34]. These data are intriguing, and suggest that much remains to be defined in the specific function, role and interactions between caveolin-1 and -2 in the regulation of caveolae organization and associated functions.

Fig. 8.2 Caveolin-1 is present in multiple subcellular locations. Top panel: a typical immu-noblot from subcellular fractions isolated from confluent human umbilical vein endothelial cells (HUVEC). Twelve fractions were collected and an aliquot, matched for recovery to compare the amount of caveolae in different fractions, loaded into lanes 1-12 (light to dense fractions). The first two fractions represent the cytosol (the fraction loaded). Caveolin was present in fractions 1-2 corresponding to the cytosol (Cyto), fractions 3-5 which contain the plasma membrane and caveolae (Cav), fractions 6-8 which contain the Golgi (Golgi), fractions 9-11 which contain the endoplasmic reticulum and microsomes (ER/Mc), fraction 12 which contains the nuclear pellet (not labeled), and the lane containing the homogenate (H). Bottom panel: a schematic of caveolin-1-positive membranes and vesicles in a "typical" HUVEC. Caveolin localizes to the apical plasma membrane in small clusters and large clusters of caveolae (1, 2), to vesicles near clusters of caveolae (3), to multiple clusters of vesicles within the cytosol which may correspond to early endosomes (4), a sorting or recycling compartment(s) (5), vesicles clustered in the perinuclear region (11), to the vesicular vacuolar organelle (VVO) which comprises a large volume in the cytosol adjacent to the lateral membrane, to regions within lateral membranes (6), regions of the ER (7) and Golgi (8), and domains within the basal membrane including focal adhesions (10). Inset: the inset in the bottom panel shows a single confocal "slice" obtained from confluent HUVEC following immunostaining with caveolin-1 antibody followed by Cy3 fluorophore conjugated to a second antibody. The location of the nucleus is indicated (N), and regions with intense staining are marked which correspond to caveolin-positive membranes and vesicles in large clusters adjacent to the plasma membrane (2,3,4), the lateral membrane and VVO (6,9), and the perinu-clear vesicles (11).

1821 8 Caveolin and its Role in Intracellular Chaperone Complexes 8.5

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