Influence Of P-selectin Structure On Its Mobility In The Weibel-Palade Body And Plasma Membranes

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The leukocyte adhesion molecule P-selectin is stored in Weibel-Palade bodies (WPBs), a secretory organelle of endothelial cells. The extracellular domain of P-Selectin comprises 9 consensus repeats (CRs), an EGF domain (E) and a Lectin domain (L) at the N-terminus, forming a rod-like structure approximately 48nm in length. Although truncation of extracellular CRs of P-Selectin impairs leukocyte capture under flow conditions, how such modifications affect the mobility of P-Selectin in the WPB membrane and in the plasma membrane (PM) after exocytosis is not known. Using single WPB FRAP or TIRFM with single fluorophore (SF) detection and tracking the diffusion of P-Selectin-EGFP and N- and C-terminal truncations of P-Selectin-EGFP was investigated in WPB or PM during ionomycin (1 μM) -evoked WPB exocytosis at 37°C.P-Selectin-EGFP was immobile in the WPB membrane, but its N-terminal truncations rendered it mobile. On exocytosis SFs of P-Selectin-EGFP and its mutations were found to diffuse approximately freely in the PM in the vicinity of WPB fusion sites. The diffusion coefficient D for P-Selectin-EGFP was 0.14 μm2/s, (n=2890 SF). Deletion of 8 of the 9 CRs increased D to 0.18 μm2/s, (n=3907 SF). Removal of the L domain alone increased D to 0.24 μm2/s, (n=1716 SF). Deleting both L and 8CRs increased D to 0.29 μm2/s (n=1818 SF). Removing E had no effect. C-terminal truncation also altered D. The structure of P-Selectin influences its mobility in the WPB and PM.