Single Fluorophore Detection And Tracking Of Fluorescent Weibel-Palade Body Membrane Proteins During Exocytosis

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The membranes of mature Weibel-Palade bodies (WPBs), an endothelial cell-specific secretory organelle, contain the leukocyte adhesion molecule P-selectin, the tetraspanin CD63 and the small GTPase Rab27a. Exocytosis delivers these proteins to the cell surface, however, little is known about their mobility in the WPB membrane and how this changes after insertion into the plasma membrane. P-selectin-EGFP, EGFP-CD63 and EGFP-hRab27a were transiently expressed in HUVECs by Nucleofection to label the membrane of WPBs, and WPB exocytosis was evoked by ionomycin (1μM) at 37°C. Using TIRFM and single fluorophore (SF) detection and tracking the diffusion of these proteins in the plasma membrane following exocytotic release was investigated. These data were compared to mobilities for each protein, determined previously by confocal FRAP, in the membranes of individual mature WPBs (Kiskin et al 2007).We have shown, by FRAP analysis, that P-selectin-EGFP is immobile, EGFP-CD63 mobile (0.12μm2/s, (n=31 WPBs) and EGFP-hRab27a highly mobile (0.68μm2/s, (n=43 WPBs) in the limiting membrane of individual WPBs (Kiskin et al 2007). During exocytosis P-selectin-EGFP, EGFP-CD63 or EGFP-hRab27a was released and EGFP-SFs detected and automatically tracked. In each case the SFs were found to diffuse freely in the vicinity of the fusion site with diffusion coefficients of 0.14μm2/s, (P-selectin-EGFP; n=2890 SF), 0.2μm2/s, (EGFP-CD63; n=3134 SF) and ∼0.50μm2/s, (EGFP-hRab27a; n=1023 SF). These data reveal changes in membrane mobility of some but not other WPB membrane proteins following exocytosis.Kiskin et al (2007). Eur. Biophys. J. 36, S141.