Marc Schwartz

LFA-1 activation plays a major role in reducing the velocity of rolling leukocytes and causing firm adhesion to the endothelium. The signaling cascades that cause LFA-1 activation during this process have been described through mostly correlative experiments, but the precise occurrence and location of LFA-1 activation in response to specific signals has not been directly shown. Tim Springer’s group has developed a method to visualize LFA-1 activation using FRET microscopy by linking fluorophores to cytoplasmic domains of αL and β2 subunits that separate upon activation of LFA-1. We propose this method as an experimental means to directly examine the activation of LFA1 in rolling leukocytes. Jurkat cells were transfected with constructs obtained from Tim Springer’s lab, and imaged using both confocal and spectral systems. TNF-α and PMA were both tested for their ability to activate LFA-1 in Jurkat cells, using the decrease in FRET signal as a measure of activation. TNF-α was unable to activate LFA-1, whereas PMA produced a measurable increase in CFP emission, indicating a decrease in FRET. Many challenges remain before this method can be successfully implemented in the dynamic system of rolling leukocytes.