Movement requires energy which is translated into force generated at specific locations at the organismal and cellular level. Energy production and consumption are tightly regulated to ensure efficiency by metabolic reprogramming. However, the pathways that ensure frugal use of energy remain largely unexplored. I investigate this relationship between metabolism and motility in endothelial cells which line blood vessels, are highly motile, and dictate vascular health. Direct linking cell motility which is spatially heterogeneous to cell metabolism is technically challenging but biologically important. Here, I developed single-cell metabolic imaging to measure glycolysis in individual endothelial cells using genetically-encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. I show that cellular glycolysis fuels endothelial activation, migration and contraction and that the high lactate production sites co-localize with active cytoskeletal remodeling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Altogether, this method provided data that indicates that Rho-GTPase signaling coordinates energetic metabolism with cytoskeleton remodeling to regulate the motility of single endothelial cells.




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