Rab35 protein regulates evoked exocytosis of endothelial Weibel–Palade bodies

Weibel–Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exocytosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling hemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15, and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase–activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target Rab GTPases. Among these RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed that Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP-insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von Willebrand factor and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat, and pleckstrin homology domain–containing protein ACAP2 as an Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6, supported histamine-evoked WPB exocytosis, as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6.

basal medium or stimulated with histamine for 20 min. In both cases the extracellular medium contained FITC-labelled anti-VWF antibodies to capture released VWF at the cell surface. Subsequently, cells were fixed and analyzed by confocal microscopy. Note that histamine stimulated cells show a significant increase of surface complexes containing the FITC labelled anti-VWF antibodies. Scale bar: 10 µm. (C) Histogram depicting the fluorescence intensity of FITC-labelled anti-VWF antibodies at the cell surface of nonstimulated (basal) or histamine-stimulated HUVEC, as obtained by flow cytometry.

S2.
Depletion of TBC1D10A increases the histamine-evoked secretion of VWF.
(A) TBC1D10A and TBC1D10B knockdown in HUVEC. Cells were transfected twice for 48 h with siRNAs specific for TBC1D10A and TBC1D10B, respectively, as well as with an unspecific control siRNA. Shown are Immunoblot analyses of TBC1D10A/B depleted cells, which were co-transfected with TBC1D10A/B-GFP expression constructs. Blots were either probed with anti-GFP antibodies (upper panels) or with a TBC1D10A-specific antibody (lower panel, left) which recognizes the endogenous (arrow) as well as the GFP-tagged protein (asterisk). An antibody specifically recognizing the endogenous TBC1D10B was unfortunately not available. α-actin was used as loading control. (B) VWF secretion levels in TBC1D10A/B depleted cells. HUVEC were treated with siRNAs as described in (A) and stimulated with histamine for 20 min. Acute VWF secretion was then quantified by ELISA.
Results are expressed as the mean ±SEM of eight independent experiments (*** p<0.0001; paired t-test).
HUVEC were grown on coverslips and transfected with plasmids encoding VWF-RFP and TBC1D10A-GFP or TBC1D10B-GFP, respectively. Cells were fixed 24 hpt and analysed by confocal microscopy. The representative images display maximum-intensity projections.

S4. Live cell imaging of Rab35 in histamine stimulated HUVEC.
HUVEC expressing Rab35-wt-GFP and VWF-RFP as WPB marker were stimulated with histamine and subjected to confocal live cell microcopy. Shown are stills of a movie focusing on three WPB. Stimulation occurred at t = 0 sec. Note that the WPB in the center has fused with the plasma membrane at t = 16 sec (fusion is characterized by change of the elongated intracellular VWF-RFP signal into a diffuse round appearance), whereas the other two WPB did not fuse. Note also that Rab35-GFP accumulates at the site of WPB fusion. Scale bar: 10 µm.

S6. Live cell imaging of ACAP2 in histamine stimulated HUVEC.
HUVEC expressing ACAP2-GFP and VWF-RFP as WPB marker were stimulated with histamine and subjected to confocal live cell microcopy. Shown are stills of a movie focusing on one WPB that fuses with the plasma membrane between t = 0 sec (time of stimulation) and t = 10 sec. Note that the general ACAP2 distribution is similar to that seen in nonstimulated HUVEC and does not change upon WPB fusion (Fig. 5C). Scale bar: 10 µm.