Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis.

IntroductionWeibel-Palade bodies (WPBs) are endothelial cell-specific storage organelles 1 that contain a number of hormones, chemokines, enzymes, and adhesive molecules that are rapidly released or recruited to the cell surface upon stimulation with specific agonists. Its main constituent, von Willebrand factor (VWF), 2 is also the driving force for the biogenesis of WPBs from the trans-Golgi network. [3][4][5] Current evidence suggests that VWF provides a platform for the cosegregation of a number of other bioactive compounds that include P-selectin, angiopoietin-2, and the chemokines interleukin-8 (IL-8) and eotaxin-3 into this intracellular storage compartment. 6 Release or surface presentation of WPB constituents enables the endothelium to control vascular homeostasis, by participating in diverse processes such as the arrest of bleeding, inflammatory responses, and angiogenesis.Regulation of WPB exocytosis by both Ca 2ϩ -and cAMPraising agonists involves signaling pathways initiated by agonist binding to G protein-coupled receptors (GPCRs) that ultimately result in fusion of WPBs with the plasma membrane. 7 Real-time imaging of the secretory behavior has shown that rapid release of WPBs is observed after stimulation of endothelial cells with Ca 2ϩ -raising agonists such as thrombin or histamine. 8,9 Stimulation of endothelial cells with agents that raise intracellular cAMP, such as epinephrine or vasopressin, promote a relatively slow release of WPBs, whereas a subpopulation of WPBs escapes cAMP-mediated exocytosis by clustering at the microtubule organizing center (MTOC). 8,10,11 We previously reported that the small GTP-binding protein RalA cosediments with WPBs on density gradients and multiple lines of evidence suggest that activation of Ral is a crucial step in both thrombin-and epinephrine-induced exocytosis of WPBs. [12][13][14] Ral has been implicated in regulated release of secretory granules of various origins, 15-18 playing a dual role in the process of exocytosis. Ral is involved in tethering secretory vesicles to specific sites on the plasma membrane through its GTP-dependent interaction with components of the exocyst complex. 16 In addition, Ral modulates exocytosis by enhancing ADP-ribosylation factor 6 (ARF6)-dependent phospholipase D1 activity, 19 resulting in the formation of fusogenic lipids that promote membrane fusion.The guanine exchange factor (GEF) involved in the activation of RalA in response to GPCR-mediated signaling in endothelial cells has not been identified. Recently, the activity of the RalGEF Ral-GDP dissociation stimulator (RalGDS) was found to be regulated by GPCR activation via receptor activation-mediated dissociation of RalGDS/␤-arrestin complexes. 20 In this study, we investigated whether RalGDS is involved in Ral-mediated exocytosis of WPBs. We report here that siRNA-mediated knockdown of RalGDS markedly inhibited thrombin-and epinephrine-induced WPB exocytosis. Furthermore, a RalGDS⌬382-597 mutant lacking The online version of this article contains a data supplement...