Evidence for the polarized distribution of lipids and Piezo1 channel upon myoblast migration and first indications of their interplay

Details

Supervisors

Tyteca, Donatienne

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

The myoblast migration is an essential step for myogenesis. However, the spatiotemporal regulation of this process is poorly known. We suggest that lipid domains may contribute to this process. Indeed, in resting C2C12 murine myoblasts, the laboratory showed the coexistence of two types of domains in the outer leaflet of the plasma membrane: those co-enriched in sphingomyelin(SM)/cholesterol(chol)/GM1 ganglioside and those enriched in chol mainly. The goal of my master thesis was to evaluate if and how these domains could contribute to myoblast migration. We assessed whether lipids and lipid domains were able to polarize during myoblast migration. We showed that three types of domains stand out: chol-enriched domains and SM/chol-enriched domains at the front, and GM1-enriched domains at the rear. The fluorescence recovery after photobleaching (FRAP) technique was then used to show that only the lateral mobility of SM at the front, associated or not with domains, is restricted compared to the rear. This difference is abrogated upon actin depolymerization combined with membrane chol depletion, suggesting cytoskeleton and chol dependence of these domains. I then assessed by vital imaging of a calcium-specific fluorescent probe whether this lipid polarization was accompanied by calcium reorganization. While we could detect a faint cytosolic gradient decreasing from the rear to the front, small calcium flickers are present at the front. I next asked whether the mechanosensitive calcium channel Piezo1 was involved in myoblast migration. At rest, activation of Piezo1 induces a redistribution of focal adhesions to the cell periphery. During migration, Piezo1 polarizes at the front. Following its activation with the allosteric activator Yoda1, this polarization increases, and spontaneous migration is stimulated, while directed migration is decreased. Finally, I began to investigate the potential relationship between lipid domains, Piezo1 and focal adhesions. Activation of Piezo1 increases the abundance of lipid domains containing GM1 and GM1/chol on resting cells, but does not alter global lipid polarization in migration. In contrast, chol depletion prevents Piezo1 polarization during migration, abrogates the Yoda1-induced increase in Piezo1 polarization, and redistributes focal adhesions to the cell center. In conclusion, the results obtained show that the chol-, SM/chol- and GM1/chol- enriched domains polarize at the front, as does Piezo1. Moreover, activation of the latter stimulates its polarization and spontaneous migration of myoblasts, in dependence on membrane chol content.