In the discussion of whether caveolae are endocytic structures, or not, it is important to distinguish between constitutive endocytosis and stimulated endocyto-sis. Similarly, frequency and time are also important parameters in relation to endocytic processes. Hence, if a certain endocytic mechanism operates rarely and very slowly it does not add much to the cell's total internalization of membrane, receptors and ligands. Moreover, such a mechanism can be very difficult to document.
It was shown recently that caveolae, in contrast to clathrin-coated pits, are not involved in efficient, constitutive endocytosis in nonstimulated cells . This was in part documented by using photobleaching approaches on different cell lines expressing N- and C-terminally green fluorescent protein (GFP)-tagged caveolin-1 (GFP-tagged caveolin). Caveolin which was found to be incorporated into caveolae at the plasma membrane was highly immobile, whilst some intracellular, caveolin-1-associated structures were dynamic.
The basic idea of using GFP-tagged caveolin fusion proteins and photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) to evaluate whether caveolae are involved in efficient, constitutive endocytosis operating as a parallel internalization mechanism to that of clathrin-coated pits is as follows. Fluorescent caveolin becomes incorporated in caveolae at the plasma membrane, as well as in presumptive caveolar endocytic and recycling vesicles. If caveolae are involved in efficient, constitutive endocytosis, then there will be a rapid turnover of caveolae at the plasma membrane. If a group of fluorescent caveolae at the plasma membrane is bleached, then it will be rapidly replaced by new fluorescent caveolae, a process that is reflected in a rapid recovery of fluorescence intensity in the bleached field of interest. Alternatively, if caveolae are immobile structures at the plasma membrane - that is, there is no internalization/recycling or lateral mobility of caveolin/caveolae taking place - then the bleached field of interest will remain bleached over time. A prerequisite indeed is that the expressed fluorescent cav-eolin behaves as endogenous caveolin, namely that it becomes associated with caveolae and that it does not change the amount of caveolae at the plasma membrane. This was tested by using quantitative immunogold labeling electron micros copy . Moreover, no differences were found between results obtained with N-and C-terminally tagged caveolin-1.
FRAP experiments applying bleaching fields over both the cell periphery/plasma membrane and the interior of HeLa cells, A431 cells, and Madin-Darby canine kidney (MDCK) cells revealed that the fluorescence recovery at the plasma membrane was very low compared to that of the interior of the cells . This means that caveolin-associated membrane inside the cell (for instance, associated with the trans-Golgi network (TGN) or caveosomes; see Section 4.7) is mobile, whereas caveolae at the plasma membrane are highly immobile. Thus, the experiments revealed that the mobile fraction of fluorescent caveolin associated with internal structures was about 80%, which is quite comparable to the value obtained for other membrane proteins moving without constraints . However, the mobile fraction of fluorescent caveolin associated with caveolae at the plasma membrane was as low as about 5-20%, a value comparable to that of E-cadherin after this adhesion molecule has become trapped by the actin cytoskeleton to form immobile structures . Also, the low diffusion coefficient obtained for caveolae-associated fluorescent caveolin was in the same order of magnitude as that of E-cadherin.
An alternative approach to FRAP in the study of the mobility of a fluorescent fusion protein is that of fluorescence loss in photobleaching (FLIP). Here, a certain region of interest is exposed to several bleaching cycles, and the fluorescence intensity in the region of interest as well as of the entire cell is measured. When FLIP is applied to a fluorescent-caveolin-expressing cell it is striking that there is basically no fluorescence recovery in the region after the first bleaching cycle, and the fluorescence intensity of the entire cell falls very slowly . Importantly, in particular the fluorescence signal deriving from caveolae at the plasma membrane (seen as strongly fluorescent dots or elongated structures at the rim of the cells; the resolution of the confocal microscope does not allow distinction to be made between single caveolae and clusters of caveolae) was hardly influenced by the bleaching cycles. This confirms the FRAP data, and further stresses that caveolin/ caveolae are highly immobile. When, for comparison, FLIP was applied to GFP-Rab7-expressing cells, a completely different result was obtained. Thus, within each bleaching cycle large amounts of GFP-Rab7 diffused into the bleaching region, and the fluorescence level of the entire cell fell continuously. In fact, the GFP-Rab7-expressing cells could be completely fluorescence-depleted by FLIP . This is in agreement with Rab7's properties as a small, mobile GTPase that rapidly switches between being membrane-associated and, after GTP hydrolysis, cytoso-lic .
Like noncaveolar rafts, caveolae are cholesterol-based structures, and cholesterol depletion with methyl-b-cyclodextrin (mbCD) leads to the disappearance of caveolae [35-37]. When cholesterol is removed from fluorescent caveolin-expressing cells, FLIP revealed a gradual disappearance of the distinct caveolin fluorescence at the plasma membrane. Thus, caveolin exhibits an increased mobility after cholesterol depletion, possibly due to a loss of caveolae. Also, a much more efficient removal of fluorescence from the bleaching region was obtained compared to control cells . These findings support the notion that cholesterol plays an important role in the maintenance of caveolar integrity.
4.3 Caveolae May Show Local, Short-Range Motility: A Role in Transendothelial Transport? | 73
Moreover, the actin cytoskeleton may help to stabilize caveolae at the plasma membrane [38,39]. Thus, we have shown previously by using a yeast two-hybrid system that the actin-binding protein filamin is a binding partner for caveolin-1. Activation of Rho leads to reorganization of the actin cytoskeleton and subsequently to the reorganization of caveolae . This was further substantiated in FLIP studies of fluorescent caveolin-expressing cells treated with the actin-depoly-merizing drug cytochalasin D. Here, the normal fluorescent staining of caveolae at the plasma membrane was perturbed and caveolae began to move laterally and to cluster in the plasma membrane .
Taken together, the results of these studies show that plasma membrane-associated caveolin-1 is a highly immobile molecule, and that caveolae are not involved in efficient constitutive endocytosis. Furthermore, the actin cytoskeleton plays an important role in keeping caveolae immobilized. However, it should be emphasized that this immobility of caveolae in the normal steady-state situation is not incompatible with a role of caveolin and caveolae in endocytosis under special conditions where profound changes of the actin cytoskeleton takes place (see Section 4.4). This makes the unraveling of caveolar function even more intriguing.
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