Sunday, November 24, 2024
11/24/2024
PROJECT SUMMARY A remarkable feature of lipid membranes is their fluidity: they can self-heal, bend, and circulate. Individual cells also experience and respond to the flows in their environment. Flow responses regulate diverse processes such as blood pressure, bone density, and neural growth. This is particularly apparent in blood vessels, where a monolayer of endothelial cells forms the interface between flowing blood and stationary tissue. Correlation between regions of low flow and atherosclerotic plaques was observed a century ago, leading to the hypothesis that shear flow impacts endothelial cell function. Understanding how cells accomplish mechanotransduction of shear stress into cellular signals is of wide interest. However, the molecular determinants behind flow mechanotransduction remain unclear. Particularly, we lack information on the lateral movement of extracellular membrane proteins located at the cell-fluid interface. While flow has been observed to transport membrane proteins, how this transport affects protein function and cell responses remains unknown. The goal of the proposed studies is to quantitatively measure the physical interactions specific to lipid membranes that determine how lipids and proteins move in response to flow and test whether flow transport of a membrane protein activates intracellular signaling in endothelial cells. Our central hypothesis is that physiologically significant protein and lipid concentration gradients arise from physical interactions between fluid flow and complex membranes. This hypothesis is based on the premise that extracellular lipid-anchored proteoglycans like glypican-1 can be transported along the plasma membrane by external flow, with the aqueous part of the protein acting as a molecular sail. We will accomplish three specific aims: Our first aim is to identify the fundamental properties and principles that govern flow transport of membrane-linked proteins in model membranes and to build a model to predict protein motion in physiological contexts. In the second aim, we will determine how the flow-mediated lateral transport of a physiologically important membrane protein (glypican-1) initiates the short-term flow response in endothelial cells. In our third aim, we will investigate how lipid sorting by flow contributes to flow signaling in our model system and living cell membranes. Our approach is to conduct parallel experiments in model membranes and living cells, allowing us to directly relate physiological function to molecular biophysics. The experiments rely on the PI's expertise using experimental microfluidics and confocal microscopy to determine fundamental membrane properties. While the model protein studied here is specific to endothelial cells, the principles of fluid mechanics that we will uncover are universal. We, therefore, anticipate that our models will apply to multiple cell lines and flow conditions, and will lay the groundwork for future research directions.
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