X-linked Opitz Syndrome protein, MID1, and its effects on cell adhesion force and protein interactions

X-linked Opitz Syndrome (XLOS), which is caused by loss of function of Midline-1 (MID1), is a disorder characterized by developmental defects along the ventral midline, including hypospadias and cleft lip with or without cleft palate. MID1 is highly expressed in tissues affected in Opitz syndrome, such as orofacial epithelia that are destined to contact and fuse to form the lip. The formation of the lip involves fusion of freely projecting epithelial-covered tissue. The epithelia covering these tissue projections must undergo a complex series of changes in adhesion to facilitate the intact closure of the lip. Disruptions to this process lead to persistence of disjointed tissue projections that manifests as cleft lip with or without cleft palate. Understanding the cellular mechanisms controlling this epithelial behavior will provide insight into the pathologies that arise in XLOS as well as how MID1 may impact cellular adhesion during development. Recent studies not only highlight a role for MID1 in the regulation of cell adhesion, they show that loss of MID1 function can lead to defects in cell adhesion complexes. To this end, this thesis explores the role of MID1 and its associated proteins in cell adhesion. Work presented here identifies that the MID1 paralog and binding partner, MID2, interacts with Plekstrin Homology Domain Family members 5 and 7 (PLEKHA5 and PLEKHA7). While PLEKHA7 is known to play a role in the regulation of cell-cell junctions, yeast-two hybrid and co-immunoprecipitation assays were used to show that PLEKHA5 also interacts with proteins involved in cell adhesion. To measure cell adhesion forces when MID1 function is disrupted, micropost arrays were used. In cells overexpressing a dominant negative MID1 mutation, cell-cell tugging forces and not cell-ECM traction forces contributed more significantly to decreased cell adhesion. Lastly, in this dissertation, to develop an in vitro model of XLOS, MID1 knockout epithelial cell lines were generated using CRISPR/Cas9 targeted against the N-terminal RING domain of MID1. MID1 wild type and MID1 knockout cell lines generated using this technique displayed phenotypic variability that depended on cell clone rather than specific genotype. However, this work identifies future strategies that will help to generate an in vitro model of XLOS that could be used to study the cellular functions of MID1.