The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis.
Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.
Medical Research Council National Institute for Medical Research, The Ridgeway, London, United Kingdom.
Ruben
R
Bierings
Nicola
N
Hellen
Nikolai
N
Kiskin
Laura
L
Knipe
Ana-Violeta
AV
Fonseca
Bijal
B
Patel
Athina
A
Meli
Marlene
M
Rose
Matthew J
MJ
Hannah
Tom
T
Carter
D015153:Blotting, Western; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006728:Hormones / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033921:Vesicular Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020302:Weibel-Palade Bodies / Q000187:drug effects / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D006728:Hormones; C501262:MYRIP protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C512779:SYTL4 protein, human; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D014841:von Willebrand Factor; C492131:RAB3B protein, human; C492134:RAB3D protein, human; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins