Subcellular fractionation, immunofluorescence microscopy and electron microscopy with immunogold labeling methods have each been used to examine the localization of caveolin-1 in different tissues. Early studies isolated caveolae and lipid rafts based on the resistance of these cholesterol- and sphingolipid-rich domains to solubilization in cold buffers containing Triton X-100 detergent and the light buoyancy of the fractions on sucrose gradients. A nondetergent methods was developed that is based on sequential density gradient centrifugation, in which the plasma membrane is first isolated, after which the caveolae and noncaveolae domains are separated on a second density gradient. An analysis of the purity and enrichment of the final caveolae fraction showed that this method yields a high-purity fraction, enriched in caveolae, and containing minimal contaminants (< 5%) from other organelle fractions . In contrast, when detergents such as 0.1% Triton X-100 are used with density gradient centrifugation to isolate membranes enriched in cholesterol and sphingolipids, the buoyant fractions have been shown to be a mixture of membranes from multiple organelles. Moreover, during the isolation procedure mixing of the lipids and artifactual associations of proteins has been shown to occur. A rigorous technique to isolate caveolae from lung tissue by using cationic colloidal silica particles showed that starting with the plasma membrane and avoiding the use of detergent eliminates many of the contaminants originating from intracellular membranes when detergent-based fractionation methods are used .
The analysis of caveolae distribution within cells has been carried out using subcellular fractionation with calculation of relative recoveries of material in differ ent fractions, and by immunomicroscopic methods and gene manipulation techniques. When the overall distribution of caveolin was examined to determine where caveolin is localized by either confocal microscopy or subcellular fractionation by these more rigorous methods, a substantial quantity of caveolin was found to be associated with the intracellular membranes, and caveolin-1 has been shown to be present at some level within most subcellular fractions.
Several studies have identified technical issues that must be considered in the immunolocalization of caveolin and caveolae-associated proteins. It is well known that epitopes in proteins can be masked by interactions of the protein with other proteins, macromolecules such as lipids and membrane surfaces. It appears that this technical issue is very important in studies on proteins that associate with cholesterol-/sphingolipid-rich membranes or surfaces rich in neutral lipids. Several published studies have reported apparently conflicting results with regard to the expression or localization of caveolae in cells or tissues. Some of the first commercially available antibodies against caveolin-1 were known to recognize epitopes that were easily hidden when caveolin localized to different intracellular sites. Methods such as brief treatment with SDS have been employed to allow for antigen retrieval , giving antibodies access to epitopes in caveolin on intracellular membranes in leukocytes. Some apparent discrepancies in these relative expression or localization studies can be explained by the differences in epitope accessibility dependent on fixation, permeabilization, and the antibody detection methods employed. A recent study expanded this point to show that a number of current commercially available antibodies that are in wide use differ in their abilities to bind to caveolin at distinct sites, and that individual antibodies show distinct localization patterns under different fixation and permeabilization conditions . Again, this study highlighted the importance of careful evaluation of negative data and the danger of making sweeping conclusions based on negative evidence. Confirmation of data interpretation by employment of two or more alternative approaches - for example, subcellular fractionation, combined with either immunomicroscopic methods or genetic studies with expression of mutant proteins in cells or animals - are critical when studies are carried out to evaluate the localization of caveolin and its relevance to regulation of signaling and trafficking pathways.
Because of the close association between caveolin and cholesterol- and sphingo-lipid-rich domains, the presence of caveolin in most subcellular fractions and organelles suggests that specialized membrane domains may be important functional units in many organelles and subcellular fractions. This is in addition to the prominent roles that have been identified for caveolae in the plasma membrane in the regulation of signal transduction and rapid endocytosis. Much remains to be investigated to define the functions of these domains within distinct organelles, or those involved in specialized trafficking between intracellular organelles based on either protein or lipid organization of these domains. The results of these studies clarify the point that negative data alone may be of little value, and that a combination of antibodies with several methods for fixation, antigen unmasking and detection of caveolins may be required to evaluate the total distribution of caveolin-1 within the cell.
1801 8 Caveolin and its Role in Intracellular Chaperone Complexes 8.4
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