Weibel-Palade bodies recruit Rab27 by a content-driven, maturation-dependent mechanism that is independent of cell type

Hannah MJ, Hume AN, Arribas M, Williams R, Hewlett LJ, Seabra MC and Cutler DF
Source: J Cell Sci
Publication Date: (2003)
Issue: 116 (19): 3939-3948
The presence of many cytoplasmic organelles creates a challenge for the cell in correctly maintaining and controlling each compartment, which in turn requires their correct identification. The authors studied the role of the small GTPase Rab27a in organelle identity in Weibel-Palade bodies (WPBs), specialized storage vesicles in endothelial cells. WPBs contain proteins such as von Willebrand Factor (VWF), P-selectin and IL8. To test the temporal association of Rab27a with WPBs, HUVECs were nucleofected with a VWF-EGFP fusion protein, a chimera that is effectively incorporated into newly synthesized WPBs. VWF-EGFP expression and immunostained Rab27a was visualized in confocals section 5 and 24 hours post transfection. The results reveal that newly formed WBPs are Rab27a-negative and then undergo a maturation process that involves the acquisition of Rab27a.
The identification of organelles is crucial for efficient cellular function, yet the basic underlying mechanisms by which this might occur have not been established. One group of proteins likely to be central to organelle identity is the Rab family of small GTPases. We have thus investigated Rab recruitment to membranes using endothelial cells as a model system. We report that Weibel-Palade bodies, the Von Willebrand Factor storage compartment of human umbilical vein endothelial cells, contain Rab27a. We have also found that Weibel-Palade body-like structures induced in HEK-293 cells by the expression of von Willebrand factor can recruit endogenous Rab27a. In the absence of von Willebrand Factor, Rab27a is not lysosome associated, indicating that it can distinguish between the Weibel-Palade-body-like organelle and a classical lysosome. Finally, a time course of Weibel-Palade-body formation was established using a green-fluorescent version of von Willebrand factor. Newly formed Weibel-Palade bodies lack Rab27a, which is acquired some hours after initial appearance of the cigar-shaped organelle. We conclude that a lumenal cargo protein drives the recruitment of Rab27a to the organelle membrane by a novel mechanism that is indirect, maturation-dependent and cell-type independent.