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Characterizing the effect of caveolin-1 mutations and age-related changes in the caveolin-1 signalling interaction with endothelial nitric oxide synthase Lim, John Elpis


Caveolin-1 (Cav1) is a membrane protein that plays important structural and signalling roles in several mammalian cell types. While best known for the formation of flask-shaped membrane structures known as caveolae, which have been implicated in endocytic, mechanoprotective, and signalling roles, Cav1 also readily oligomerizes within lipid rafts to form Cav1 domains known as scaffolds. Disrupting Cav1 expression promotes tumour survival and migration, and Cav1 knockout animals suffer from pulmonary and cardiovascular conditions. While Cav1 is well-known to interact with multiple cell signalling partners, notably its inhibition of endothelial nitric oxide synthase (eNOS), it remains unclear if this interaction is caveolae- or scaffold-dependent, of if it involves the caveolin scaffolding domain or the tyrosine-14 residue of Cav1. Here, we used confocal and single molecule localization microscopy (SMLM) network analysis of Cav1 and eNOS localization in a transiently transfected Cav1 knockout MDA-MB-231 cell line, as well as whole-mount labeling of Cav1 and eNOS in mouse pulmonary arteries to study Cav1 interaction with eNOS. We found that phosphomimetic Cav1-Y14D mutant caveolae and scaffolds had similar features to caveolin scaffolding domain (CSD) mutants, while phosphorylation-deficient Cav1-Y14F mutant Cav1 structures closely resembled Cav1-WT blobs. Using binary mask analyses of Cav1 and eNOS, we identified that eNOS interacts with caveolae and scaffolds approximately equally, and that CSD mutant and Y14D mutant Cav1 had a slight but non-significant increase in Cav1-eNOS co-occurrence across all Cav1 domains. Additionally, to investigate changes in Cav1 and eNOS localization and behaviour with age, we used confocal microscopy to image Cav1, eNOS, and the Golgi marker GM130 in the pulmonary artery endothelium young and old mice. We observed that eNOS expression decreases with age (p < 0.001), especially outside the Golgi apparatus, that Golgi flatten with age while length remains constant (p < 0.0001), and that Cav1-eNOS co-localization decreases slightly with age (p = 0.03) despite decreased eNOS expression. Altogether, these results provide insights into the properties and characteristics of Cav1 nano-domains, their interactions with eNOS, and the effects of Cav1 CSD and Y14 mutation and age on its signalling activity.

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