4 b). We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential. Introduction LY3009120 Weibel-Palade bodies (WPBs) are the large secretory organelles, which can be up to 5 m long, of endothelial cells. They are uniquely cigar-shapedtypically cylindrical with hemispheric ends (Weibel and Palade, 1964; Wagner, 1990; Hannah et al., 2002; Michaux and Cutler, 2004). Their best understood components are the hemostatic protein von Willebrand factor (VWF; Sakariassen et al., 1979; Wagner et al., 1982; Sadler, 1998; Ruggeri, 1999) and the integral membrane protein P-selectin (Bonfanti et al., 1989; McEver et al., 1989). Regulated exocytosis of WPBs delivers VWF and P-selectin to the cell surface where they act in the recruitment of platelets and leukocytes. WPBs thus play a vital role in acute inflammation and in formation of the primary hemostatic plug. P-selectinCdeficient mice have delayed neutrophil extravasation upon injury (Mayadas et al., 1993; Subramaniam et al., 1997), whereas the commonest inherited human bleeding disorder, von Willebrand’s disease, largely results from mutations in VWF (Wagner, 1990; Nichols and Ginsburg, 1997; Sadler, 1998). The biogenesis of secretagogue-responsive WPBs is usually therefore of primary physiological importance, and their unusual shape has always implied that this process may be complex but little is known of the mechanisms involved. Protein content is thought to play an important part in secretory granule biogenesis, driving the formation of these organelles by selective aggregation at the TGN (for review see Thiele and Huttner, 1998) to form their dense proteinaceous core. One of the most dramatic demonstrations of cargo-driven biogenesis follows the heterologous expression of VWF, which triggers the formation of pseudo-WPBs in nonendothelial and, even, nonsecretory cells (for review see Michaux et al., 2003). Not only are the structures formed in this way indistinguishable from bona fide WPBs at the EM level, but they also recruit WPB-resident membrane proteins and undergo exocytosis in response to secretagogue stimulation. Cytoplasmic coat complexes act structurally during formation of vesicles and also help select membrane and content proteins for inclusion. Clathrin and the clathrin-associated adaptor protein complex AP-1 have been reported to act on immature secretory granules (ISGs) during granule maturation. In rat endocrine pancreatic cells, AP-1/clathrin-coated vesicles (CCVs) remove missorted mannose 6-phosphate receptors from ISGs (Klumperman et al., 1998). In neuroendocrine PC12 cells, the protein convertase furin and the mannose 6-phosphate receptor are also removed from ISGs via AP-1 CCVs (Dittie et al., 1999). The removal of missorted proteins and the gradual condensation of the protein content are major elements of the maturation of secretory granules (for reviews see Arvan and Castle, 1998; Tooze et al., 2001). Are AP-1 and clathrin involved in the formation of WPBs? EM analysis of human umbilical vein endothelial cells (HUVECs) revealed extensive coating of newly formed and forming perinuclear WPBs. Coverage of WPBs by visible coats along the long axis for up to 650 nm and complete coating of WPBs up to 150 nm in diameter have been observed. We used immunofluorescence to show that this coat contains clathrin and AP-1, the latter further confirmed by immuno-EM. Reducing the ability of AP-1 and clathrin to function by antiCAP-1 RNA interference (RNAi) or a dominant-negative AP180 construct, which prevents the inclusion LY3009120 of clathrin into forming coats, does not, as might be expected from the previous work on secretory granules mentioned in the previous paragraph, lead to the accumulation of missorted proteins within WPBs, but instead to a failure to form WPBs at all. Our results provide the first evidence that coat proteins can also play an essential role in the initial formation of a secretory granule rather than only during their maturation. Results WPBs are Rabbit Polyclonal to PAK3 coated By conventional transmission EM of ultrathin sections from epon-embedded HUVECs, WPBs appear as cigar-shaped membrane-bound organelles with internal VWF tubules (striations in longitudinal sections) that make up the bulk of their content. Interestingly, we found that some WPBs are extensively, possibly even completely, covered with a proteinaceous coat (Fig. 1, a and b). The scale of the coverage is remarkable, given the size of WPBs. At least part of the coat has a bristle-like structure, resembling clathrin. Fig. 1 c shows a similar coat surrounding the transverse section of a small immature WPB (arrowhead), which was identified based on its size and the characteristic LY3009120 granular appearance of the content protein..