Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC361

Poster Communications

Caveolae subpopulations in rat aortic smooth muscle

L. M. Davies1, R. Barrett-Jolley2, C. Dart1

1. Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom. 2. Institute of Aging and Chronic Disease, University of Liverpool, Liverpool, United Kingdom.

Caveolins act as coat proteins for caveolae, small flask-shaped pits that form in the plasma membrane of most cells. Aside from roles in caveolae formation, caveolins recruit, retain and regulate many caveolae-associated signalling molecules and are thus central to the function of these subcellular signalling compartments (1). Of the three isoforms, caveolin-1 and -2 form hetero-oligomers in most cell types whereas caveolin-3 forms homo-oligomers in striated muscle. The ability of caveolin-3 to interact with either caveolin-1 or -2 is controversial (1). Smooth muscle cells express all three isoforms and here we use immunogold electron microscopy and coimmunoprecipitation to investigate the caveolin profile of individual aortic caveolae. To assess the subcellular distribution of caveolin-1 and -3, plasma membrane sheets were ripped off rat aortic myocytes (2-4 days in culture) onto copper grids (2), dual-labelled with isoform-specific primary antibodies against caveolin-1 and -3 and visualised by addition of gold-conjugated secondary antibodies (gold particles of 5 nm and 15 nm respectively). No labelling was observed with secondary antibodies alone, and no cross-reactivity was seen between primary or secondary antibodies. Morphologically identifiable individual caveola labelled for both caveolin-1 and -3. Particle distributions were quantified by measuring the distance between gold particles corresponding to caveolin-3 and those corresponding to caveolin-1 using ImageJ (NIH, USA) software (3). Approximately 50 % of caveolin-3 particles were found within 100 nm of a caveolin-1 particle (51.7 ± 11.3%; mean ± SEM, 299 measurements over 6 separate images) compared to only 4.7 ± 3.8% of particles in simulated random distributions at equivalent density (199 measurements over 6 images, p<0.01, Student’s t test). Since caveolae are ~100 nm in diameter, this suggests that caveolin-1 and -3 coexist within a single caveola. Interestingly, some caveolae labelled exclusively for caveolin-1 (49.6 ± 12.4%; 6 images) or -3 (10.8 ± 5.3%; 6 images), indicating that caveolar subpopulations may exist containing distinct isoforms. In further experiments, antibodies against caveolin-1 coimmunoprecipitated both caveolin-2 and -3 from rat aortic homogenates, suggesting that caveolin-1 interacts with caveolin-2 and -3 in vivo (n=3). Similarly, antibodies against caveolin-3 coimmunoprecipitated both caveolin-1 and -2 (n=3). Our findings suggest that caveolin-1 and -3 form hetero-oligomeric complexes in aortic smooth muscle, but do not exclude the existence of subpopulations of caveolae containing either caveolin-1 or -3. This is consistent with studies that indicate that a proportion of smooth muscle caveolae are resistant to caveolin-1 gene ablation (4), and may mean that individual cells have multiple species of caveolae each containing distinct complements of signalling molecules.

Where applicable, experiments conform with Society ethical requirements