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Visualization of caveolin‐1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras
Author(s) -
Volontè Daniela,
Galbiati Ferruccio,
Lisanti Michael P.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00164-7
Subject(s) - caveolae , caveolin , caveolin 1 , green fluorescent protein , microbiology and biotechnology , endogeny , fusion protein , biology , chemistry , signal transduction , biochemistry , recombinant dna , gene
Caveolin‐1, a suspected tumor suppressor, is a principal protein component of caveolae in vivo. Recently, we have shown that NIH 3T3 cells harboring anti‐sense caveolin‐1 exhibit a loss of contact inhibition and anchorage‐independent growth. These observations may be related to the ability of caveolin‐1 expression to positively regulate contact inhibition. In order to understand the postulated role of caveolin‐1 in contact inhibition, it will be necessary to follow the distribution of caveolins in living cells in response to a variety of stimuli, such as cell density. Here, we visualize the distribution of caveolin‐1 in living normal NIH 3T3 cells by creating GFP‐fusion proteins. In many respects, the behavior of these GFP‐caveolin‐1 fusion proteins is indistinguishable from endogenous caveolin‐1. These GFP‐caveolin‐1 fusion proteins co‐fractionated with endogenous caveolin‐1 using an established protocol that separates caveolae‐derived membranes from the bulk of cellular membranes and cytosolic proteins, and co‐localized with endogenous caveolin‐2 in vivo as seen by immunofluorescence microscopy. We show here that as NIH 3T3 cells become confluent, the distribution of GFP‐caveolin‐1 and endogenous caveolin‐1 shifts to areas of cell‐cell contact, coincident with contact inhibition. However, unlike endogenous caveolin‐1, the levels of GFP‐caveolin‐1 expression are unaffected by changes in cell density, serum starvation, or growth factor stimulation. These results are consistent with the idea that the levels of endogenous caveolin‐1 are modulated by either transcriptional or translational control, and that this modulation is separable from density‐dependent regulation of the distribution of caveolin‐1. These studies provide a new living‐model system for elucidating the dynamic mechanisms underlying the density‐dependent regulation of the distribution of caveolin‐1 and how this relates to contact inhibition.