Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras. The subcellular distribution of caveolin-1 is modulated by cell-cell contact.
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.
Volonte
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Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Galbiati
F
F
Lisanti
M P
MP
D016475:3T3 Cells; D000818:Animals; D015415:Biomarkers; D051242:Caveolin 1; D022461:Caveolins; D002452:Cell Count; D005591:Chemical Fractionation; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins