Identification of a novel Cripto/MyosinII interaction that promotes stem cell-mediated tissue regeneration

Stem cells are necessary for proper development, tissue homeostasis and regeneration while dysregulation of their activity leads to diseases such as diabetes, neurodegeneration and cancer. Yet, the molecular and cellular mechanisms that drive stem cell mediated regeneration remain to be fully elucidated. We hypothesized that the stem cell marker, Cripto (or TDGF1), is a regulator of tissue regeneration. Using the zebrafish model of caudal fin wound healing, we show that expression of the zebrafish Cripto homolog, one-eyed pinhead (or oep), is increased at 96 hours post amputation within a previously established window of stem cell enrichment in this model. We further demonstrate that Cripto is necessary and sufficient for stem cell mediated regeneration in this in vivo model as well as in an in vitro wound healing assay. We identified non-muscle myosin II's (MYH9/10) as novel Cripto binding proteins using co-immunoprecipitation (Co-IP) and mass spectrometry and confirmed the interaction between these proteins via Co-IP/Western blot from mammary epithelial cells and endogenous immunofluorescence co-localization in mesenchymal stem cells, a key cell type that contributes to wound healing in zebrafish and mammals. Notably, the effects of Cripto and MYH9/10 inhibitors on regeneration of wounded zebrafish caudal fins were not additive suggesting Cripto and MYH9/10 have overlapping functions and mechanisms of action. Cripto and its homologs are cell-surface GPI-anchored glycoproteins that can be released via GPI anchor cleavage by phospholipases. We previously demonstrated that tissue-specific stem cell functions of Cripto depend upon its soluble secreted form and this led us to test whether MYH9/10 function may be required for transporting Cripto to the cell surface or mediating release of soluble Cripto from the cell surface. Indeed, we find that cell-surface localization of Cripto in and its release as a soluble factor from mammalian cells requires MYH9/10 function. Using Bio-Grid proteomics, we discovered Rab11A, an endosomal and exosomal marker, to be a co-binder of GRP78 and Myosin IIs. We then pharmacologically inhibited Myosin IIs using Blebbistatin or Rho Kinase (ROCK) inhibitors in mesenchymal stem cells transfected with wild-type, constitutively active, and dominant negative Rab11A constructs. Our results show that the expression of Rab11A regulates Cripto and Myosin II binding interactions, suggesting a possible role for Rab11A in the endosomal and exosomal trafficking of Cripto. Based on our findings, we propose a new model whereby MYH9/10/Rab11A-mediated Cripto transport enables cells to generate a pool of extracellular Cripto that can subsequently function in autocrine and/or paracrine pathways to promote stem cell mediated regeneration and wound healing.