The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling. SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylatedenriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling. \body

Background Sinus of Valsalva aneurysms (SOVAs) are infrequent findings and generally diagnosed incidentally. A SOVA may be at risk for rupture, which would lead to an aorto-cardiac shunt. These patients present similarly to decompensated heart failure. Case Presentation We present a case of a 44-year-old female with a ruptured non-coronary SOVA diagnosed by echocardiogram during evaluation for exertional dyspnea. A transesophageal echocardiogram (TEE) revealed a 2.1 cm non-coronary SOVA with windsock communication to the right atrium. The patient refused surgery, and two years later, presented with florid right heart failure with preserved left ventricular function. The right ventricle was severely dilated and hypokinetic with right atrial enlargement. After finally agreeing to surgery, a pre-operative catheterization revealed non-obstructive coronaries and a significant left to right shunt with elevated pulmonary pressure. The patient had suboptimal response to diuretic therapy and was sent for successful repair of the aneurysm with the closure of the aorto-atrial fistula via bovine pericardial patch and resolution of the left to right shunt as demonstrated by intraoperative TEE. Her right sided heart failure symptoms subsequently resolved. Discussion SOVA is a rare finding but should still be considered in the differential in young and middle-aged patients with symptoms of acute heart failure, hemodynamic compromise, and a new continuous heart murmur. Early surgical repair is highly recommended to prevent acute and long-term complications.