UC San Diego

We recently demonstrated that migrating human leukocytes respond to small physiologic fluid stresses by active control in the projection and retraction of local cytoplasmic extensions (pseudopods). However, the regulatory mechanisms involved in this type of fluid shear response are currently unknown. Recent observations in our lab showed that the formyl peptide receptor, a G-protein coupled membrane receptor, is important for the pseudopod retraction response of neutrophils under fluid shear. We hypothesized that the surface distributions of these membrane receptors determines how the cell responds in a laminar flow field. In the first part of this work I developed a method to compute the fluid stress distributions on the membrane of a single migrating leukocyte. We found that the shear stresses are highest at the top of an adherent cell and is lowest in the contact region with its substrate. However, the cell retracts its pseudopods more in the vicinity of the attachment sites close to the substrate adhesion region than at its top. Membrane protrusions, such as pseudopods, also experience enhanced shear stresses but tend to be lower than that at the top surface. This evidence shows a non-linear relation between the shear magnitude and degree of pseudopod retraction. The second part of the dissertation focuses on the location of FPR before and after flow application. Using FPR-GFP-transfected cells, we observed that there is an increased aggregation of receptors in a perinuclear compartment of the cell. There is a very small decrease in GFP-derived fluorescence intensity in the cell membrane and cell cytoplasm, suggesting that internalization of the receptor is not limited to a single region on the surface of the cell. The prolonged FPR detention in an intracellular compartment suggests that fluid shear stress may prevent receptors being recycled back to the membrane. This feature may serve as a desensitization mechanism of the cell to prevent activation on the cell surface. The observation is in line with previous observations suggesting that laminar fluid flow may deactivate the cell even in the presence of low concentrations (up to 10nM fMLP) of inflammatory mediators