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Sec22b controls VWF trafficking and WPB size
tubules when exposed to the internal milieu of the TGN.13-
15 Quantitative or qualitative defects of VWF, for instance
due to mutations in VWF, cause von Willebrand disease
(VWD), the most common inherited bleeding disorder.16
VWF mutations that affect the synthesis or processing of
the protein often result in altered WPB morphology, with
WPB being either round or short.17 Upon regulated, explo-
sive release from WPB, VWF unfurls into strings up to 1 mm
long that are anchored on the apical side of the endotheli-
um.18-20 VWF strings create an adhesive platform for platelets
to initiate the formation of the initial platelet plug at the site
of vascular damage.21,22 The adhesive capacity of VWF
towards platelets and self-associating plasma VWF is pro-
portional to WPB size.23 In turn, WPB size is determined
before budding from the TGN by incorporation of so-called
“VWF quanta” and it was previously shown that reduced
VWF synthesis or unlinking of Golgi stacks affects WPB
24
length. However, how endothelial cells control WPB size
and thus hemostatic activity of VWF is largely unknown. Due to the distinctive shape of their WPB, endothelial cells are an excellent model system for elucidating how cells manage formation and morphology of lysosome- related organelles. As WPB formation is driven by VWF, monitoring intracellular VWF trafficking can be used as a tool to study the complex mechanisms involved. VWF undergoes extensive post-translational modification during its journey through the endothelial secretory pathway.25 VWF enters the ER as a single pre-pro-polypeptide chain that forms tail-to-tail dimers by formation of disulfide bonds between the C-terminal cysteine knot (CTCK) domains of two proVWF monomers.26 After dimerization- dependent exit from the ER, proVWF dimers are transport- ed to the Golgi, where VWF propeptide is cleaved from the proVWF chain. Inter-dimer disulfide bonds between cys- teines in the D3 domains lead to formation of head-to- head VWF multimers.27 VWF multimers are then con- densed into tubules that are packaged into newly forming
WPB that emerge from the TGN.28,29
Trafficking of proteins during formation and maturation
of subcellular organelles, such as WPB, is dependent on membrane fusion, which is universally controlled by SNARE proteins.30 The SNARE complex consists of a v- SNARE on the vesicle membrane and t-SNARE on the acceptor membrane which together form a four-helix bun- dle that allows the membranes to fuse. Although several SNARE have been associated with WPB exocytosis,7 the SNARE that take part in the biogenesis of lysosome-related organelles and WPB are not known. The subfamily of lon- gin-SNARE (VAMP7, YKT6 and Sec22b), which derives its name from an N-terminal self-inhibitory longin-domain that can fold back on the SNARE domain, controls mem- brane fusion events that traffic proteins to and from the Golgi.31
In this study we addressed the role of longin-SNARE in the formation of WPB. Using a targeted short hairpin (sh)RNA screen of longin-SNARE in primary endothelial cells we identified Sec22b as a novel determinant of WPB morphology. Sec22b silencing resulted in short WPB, disin- tegration of the Golgi complex, reduced proVWF process- ing and retention of proVWF in a dilated ER. Our data sug- gest that the distinctive morphology of WPB and thus the adhesive activity of their main cargo VWF is determined by the rate of membrane fusion between ER and Golgi, which is dependent on Sec22b-containing SNARE com- plexes.
Methods
Antibodies
The antibodies used in this study are listed in Online Supplementary Table S1.
Cell culture, lentiviral transfection and transduction
Pooled, cryopreserved primary human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany). HUVEC were cultured in EGM-18 medium, i.e., EGM-2 medium (CC-3162, Lonza, Basel, Switzerland) supplemented with 18% fetal calf serum (Bodinco, Alkmaar, the Netherlands). Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Cell Culture (Wessel, Germany) and were grown in Dulbecco modi- fied Eagle medium containing D-glucose and L-glutamine (Lonza, Basel, Switzeland) supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. HEK293T cells were seeded on collagen-coated plates or flasks and were transfected with third-generation lentiviral packaging plasmids pMD2.G, pRSV-REV and pMDLg/pRRE (Addgene, Cambridge, MA, USA) using transit-LT1 (Mirus Bio LLC, Madison, WI, USA) following the supplier’s protocol. After incu- bation for 6-8 h, the medium was exchanged for EGM-18. Virus particles were collected 24 and 48 h following transfection and were filtered through 0.45 mm pore filters in EGM-18. Two batches of virus were used to transduce HUVEC, cord blood outgrowth endothelial cells or HEK293T cells for a period of 48 h. Transduced endothelial cells were selected by puromycin (0.5 mg/mL), which was added to the medium for 72 h after the sec- ond virus installment.
DNA constructs for shRNA silencing of longin-SNARE, CRISPR editing and mEGFP-Sec22b-DSNARE
The LKO.1-puro-CMV-mEGFP-U6-shC002 vector, which simultaneously expresses monomeric enhanced green fluores- cent protein (mEGFP) and a non-targeting control shRNA from the cytomegalovirus (CMV) and U6 promoter, respectively, was described previously.32 shRNA targeting Sec22b, VAMP7 and YKT6 were obtained from the MISSION® shRNA library devel- oped by TRC at the Broad Institute of MIT and Harvard and dis- tributed by Sigma-Aldrich (Online Supplementary Table S2). Fragments containing the shRNA expression cassette from the shRNA library were transferred to the LKO.1-puro-CMV- mEGFP-U6 vector by SphI-EcoRI subcloning. CRISPR-mediated depletion of Sec22b in HUVEC was performed essentially as described previously.33 LentiCRISPR_v2 (a gift from Dr. Feng Zhang; Addgene #52961), a lentiviral vector which simultane- ously expresses Cas9 endonuclease and guide (g)RNA has been described previously.34 gRNA were designed to target exon 1 of the SEC22B gene using the CRISPOR Design tool (http://crispor.tefor.net/)35 by submitting the DNA sequence of SEC22B exon 1 flanked by 100 bp up- and downstream (chro- mosome 1: 120,1501898-120,176,515 reverse strand) (Online Supplementary Figure 2A). gRNA sequences that have a high pre- dicted efficiency with limited off-target effects were selected. The gRNA used in this study are shown in Online Supplementary Table S3 and were cloned as hybridized complementary oligos (with BsmBI restriction site-compatible overhangs on either side) into BsmBI-digested LentiCRISPR_v2 plasmid. LVX-mEGFP-LIC has been described previously.36 To construct a human Sec22b variant that lacks its SNARE domain (Gly135-Lys174), a synthet- ic Sec22b fragment was generated by gene synthesis in which codons 135-174 were removed from the 214-codon Sec22b cod- ing sequence and was flanked by BsrGI and NotI sites. The resulting Sec22b-DSNARE fragment was cloned in frame behind
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