Abstract
Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis.
Highlights
Weibel-Palade bodies (WPBs) are endothelial cell-specific regulated secretory granules containing polymers of VWF condensed into ordered helical tubules through a non-covalent association with the cleaved VWFpropolypeptide (Pro-VWF, Pro-) [1,2,3]
Because soluble WPB cargo proteins and P-selectin are stored in the mature organelle in an immobile or very slowly mobile state [9], the extent to which such cargo is re-mobilised within the WPB after plasma membrane fusion may determine the availability for diffusive release
Using epifluorescence time-lapse imaging, we followed the relationship between prefusion WPB length L (Fig. 2B) and the radius R0 of the rounded structure formed upon plasma membrane fusion of the same WPB
Summary
WPBs are endothelial cell-specific regulated secretory granules containing polymers of VWF condensed into ordered helical tubules through a non-covalent association with the cleaved VWFpropolypeptide (Pro-VWF, Pro-) [1,2,3]. In addition to VWF, WPBs may contain a cocktail of pro-inflammatory molecules including P-selectin, IL-8, IL-6, monocyte chemoattractant protein 1 (MCP-1), growth-regulated oncogene-a (GROa) or Eotaxin-3 [5,6] Because secretion of these molecules from WPBs may play a role in inflammatory processes within the vasculature, it is important to understand how such molecules gain entry to these organelles [6,7,8], how they are stored [9], mobilized and released during exocytosis. Because soluble WPB cargo proteins and P-selectin are stored in the mature organelle in an immobile or very slowly mobile state [9], the extent to which such cargo is re-mobilised within the WPB after plasma membrane fusion may determine the availability for diffusive release.
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