Quantifying flow-induced transport of membrane-linked proteins in model and cell membranes.

Shear flow sensing is crucial in defining fundamental cell properties, including cell fate, self-renewal, motility, and homing behaviors. For instance, endothelial cells lining blood vessels translate blood flow into responses that in turn regulate the tension in blood vessels, and, in tumors, flow can regulate genes that promote cancer cell migration and metastasis. Recent studies suggest that glypican-1 (a lipid-anchored heparan sulfate proteoglycan at the surface of endothelial cells) may play a key role in the flow signaling pathway: its flow-mediated redistribution at the membrane appears to mediate the production of nitric oxide (NO), a key signaling molecule produced by the NO synthase eNOS. However, the molecular mechanisms that endothelial cells use to sense changes in flow and translate this mechanical stimulus into intracellular molecular responses (e.g., activation of eNOS by glypican-1) are currently unknown. We hypothesize that protein concentration gradients in the plasma membrane are formed by physical interactions with fluid flow. This is based on the premise that extracellular lipid-anchored proteoglycans like glypican-1 can be transported along the membrane by external flow, with the aqueous part of the protein acting as a molecular sail. To investigate the effect of flow on membrane-associated proteins and cell signaling, it is crucial to visualize and quantify the motility of membrane bound proteins in response to flow. Here, we present results in both reductionist model membrane systems and cell membranes aimed at determining the relationship between drift velocity and protein size, membrane lipid composition, and lipid anchor structure: (1) Our first approach measures the flow coupling and frictional coefficients of lipid-anchored model proteins (monomeric streptavidin fusions) of varying size reconstituted in glass-supported bilayers. (2) We also present the development of a unique labeling approach allowing the visualization of glypican-1 expressed at the membrane of living endothelial cells under flow. While glypican-1 is specific to endothelial cells, the principles of fluid mechanics are universal. We, therefore, anticipate that our methodology and results will apply to multiple cell lines and flow conditions.