Mapping the Planar Cell Polarity Interactome in Myoblasts

Abstract

Wnt7a/Fzd7 signaling drives the symmetric division and motility of satellite stem cells through the planar cell polarity (PCP) pathway, stimulating skeletal muscle repair and growth. Moreover, stimulating Wnt7a/Fzd7 signaling through Wnt7a treatment ameliorates symptoms of Duchenne Muscular Dystrophy (DMD), a disease marked by degenerative muscle wasting. Wnt7a thus serves as a promising therapeutic for DMD; however, little is known concerning the signaling effectors downstream of Wnt7a that impinge on the PCP pathway to mediate cell division and motility. In this study we attempt to identify key transducers of Wnt7a signaling. To this end we have developed an array of TurboID fusion proteins that includes known and suspected PCP signaling components. Stable primary myoblast cell lines expressing each TurboID construct were developed. Biotin labeling experiments using the TurboID cell lines have revealed an extensive list of novel PCP protein candidates that are responsive to stimulation by Wnt7a. Moreover, the intersecting interactome between known PCP components has shed light on the spatial relationship of these novel PCP candidates within the Wnt-PCP signaling cascade. Novel PCP-interacting proteins have been validated through siRNA knockdowns to test their role in Wnt7a-mediated cell motility. Subsequent TurboID experiments were conducted with a panel of novel proteins identified in the initial labelling experiments to expand the interactome and validate these hits. Based on our combined results, CTGF gene expression was identified as a novel Wnt7a activity readout. Additionally, we demonstrate that p38γ phosphorylates the PCP protein Dvl2, inducing Dvl2 to interact with the spindle apparatus protein NuMA1 in a Wnt7a-dependent manner. The findings from this study have yielded critical information regarding Wnt7a's mechanism of action and will aid in the development of Wnt7a-based therapeutics.