Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport

Abstract

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.

Figure 1.

Sec22b depletion and fusogenic function affects Weibel-Palade body elongation. (A) von Willebrand factor (VWF) immunostaining in endothelial cells (EC) transduced with shCTRL, shVAMP7, shYKT6 or shSec22b (green channel: mEGFP-expressing EC). (B) Quantification of Weibel-Palade body (WPB) length in shCTRL, shVAMP7, shYKT6 and shSec22b EC (n=3, one-way analysis of variance [ANOVA] with the Dunnett multiple comparisons test, ****P<0.0001). (C) WPB length in control and CRISPR SEC22B knockout EC (n=3, one-way ANOVA with the Dunnett multiple comparisons test, ****P<0.0001). (D) VWF immunostaining in mEGFP and mEGFP-Sec22b-DSNARE expressing EC (both in green). (E) WPB length in mEGFP and mEGFP-Sec22b-DSNARE expressing EC (n=3, t-test with Welch correction, ****P<0.0001).

Figure 2.

Sec22b depletion results in trans-Golgi network fragmentation. (A) Immunofluorescent staining of trans-Golgi network (TGN46) in control and Sec22b-depleted cells (blue channel: Hoechst nuclear staining). (B) Quantification of TGN dispersal in control and Sec22b knockdown (KD) endothelial cells (EC). (C) Quantification of TGN area coverage in shCTRL and shSec22b EC (n=5, ttest with Welch correction, ****P<0.001). (D) Weibel- Palade body (WPB) length in Sec22b KD EC with compact versus dispersed TGN (n=3, t-test with Welch correction, ****P<0.0001).

Figure 3.

Sec22b depletion results in retention of von Willebrand factor in the endoplasmic reticulum. (A) Western blot analysis of monomeric von Willebrand factor (VWF) under reducing conditions in control and Sec22b knockdown (KD) endothelial cells (EC). Uncleaved (proVWF) and cleaved (VWF) forms are indicated by arrows. α-tubulin was used as a loading control. Molecular weight standards are indicated on the left (kDa). (B) ProVWF:VWF ratio in control and shSec22b EC (n=8, paired t-test, *P<0.05). (C) Immunofluorescent staining of VWF in shCTRL and shSec22b human umbilical vein EC (boxed areas are shown magnified on the right, size bar corresponds to 10 mm for images or 5 mm for boxed areas). (D) VWF multimer blot (4 samples from 2 independent experiments) in control and Sec22b KD EC. (E) Line graph of the densitometry of VWF multimer bands. LMWM: low molecular weight multimers; HMWM: high molecular weight multimers

Figure 4.

von Willebrand factor accumulation in dilated rough endoplasmic reticulum in Sec22b-depleted endothelial cells. (A) Electron microscopy of control and Sec22b knockdown (KD) endothelium cells (EC) (dilated rough endoplasmic reticulum [ER] shown by white arrowheads, ribosome-studded dilated ER by white asterisks, and the connection of ER structures to rough ER sheets by a yellow arrowhead; scale bar corresponds to 2 mm). (B) Quantification of healthy versus dilated ER in control and Sec22b KD cells. (C) Quantification of ER width in control and Sec22b KD cells (t-test with Welch correction, ****P<0.0001). (D) von Willebrand factor immunogold staining (10 nm gold particles) in control and Sec22b KD EC (boxed regions are magnified on the right side with the corresponding color; scale bar represents 1 mm).

Figure 5.

Sec22b silencing results in reduced basal and stimulated secretion. (A) Intracellular von Willebrand factor (VWF) content in control and Sec22b-silenced endothelial cells measured by enzyme-linked immunosorbent assay (n=5, paired t-test, *P<0.05). (B) Basal VWF release presented as percentage of intracellular VWF content (n=5, paired t-test, *P<0.05) (C) Histamine-stimulated VWF release presented as percentage of intracellular VWF content (n=3 independent experiments, paired t-test, **P<0.01). (D) Proposed model of Sec22b-dependent VWF trafficking and Weibel-Palade body size control. SE: sorting endosome; WPB: Weibel- Palade body; iWPB: immature WPB; mWPB: mature WPB; ER: endoplasmic reticulum; rER: rough ER; TGN: trans-Golgi network.