Cleft lip with or without cleft plate (CLP) is a common birth defect with multifactorial etiologies. X-linked Opitz Syndrome, which is caused by loss of function of Midline-1 (MID1), is a disorder characterized by multiple developmental defects, including hypospadias and CLP. Both the formation of the lip and the urethra involve fusion of freely projecting epithelial-covered tissue. MID1 is highly expressed in tissues affected in Opitz syndrome, and in particular the orofacial epithelia that are destined to contact and fuse to form the lip. These epithelia undergo a complex series of changes in adhesion to facilitate formation of the lip. Understanding the cellular mechanisms controlling this epithelial behavior will provide insight into the causes of CLP and possibly highlight avenues by which to prevent the disorder. To this end, we are studying the role that MID1 and its associated proteins play in lip development. Several studies highlight the role of MID1 as an E3 ubiquitin ligase in the regulation of PP2Ac, the catalytic subunit of the major serine threonine phosphatase, PP2A (Protein phosphatase 2A), which has roles in modifying cell behaviors such as motility, cytoskeletal dynamics, and growth. However, little is known about the close paralog of MID1, known as MID2, which functions as a binding partner of MID1. In Opitz syndrome, mutated MID1 forms aggregates within the cytosol, which bind MID2, directing it from its normal microtubular distribution to the aggregates. The downstream effects of this mislocalization remain unclear. Through yeast-two hybrid screens, our lab has found novel MID2 binding partners, PLEKHA5 and PLEKHA7 (pleckstrin homology domain containing, family A members 5 and 7), which interact directly with MID2, but not MID1. Interestingly, PLEKHA7 has recently been found to participate in the microtubule-dependent regulation of E-cadherin endocytosis and thus the maintenance and dynamic control of apical cell-cell junctions including adherens junctions. In this proposal plan, intercellular forces in epithelial cells will be quantified in cels expressing dominant negative MID1 constructs, as well as in cells in which MID1 and MID2 expression are knocked down. Furthermore, the ability of specific mutants in MID1 and MID2 to rescue these defects in cell adhesion will be investigated. The specific mutants to be tested include those which are unable to regulate PP2Ac levels and those unable to bind the PLEKHA proteins. In addition, these constructs will be tested by transfection into chick embryonic facial epithelia in ovo. This project will provide insight into whether the MID1 and MID2 control of epithelial adhesion involves both localized regulation of PP2A activity and the interaction with the PLEKHA proteins, as well as the importance of both MID proteins in the orchestration of lip morphogenesis.

Public Health Relevance

This project will investigate the role of MID1 and MID2 in epithelial maintenance and adhesion. While it is known MID2 forms strong heterodimers with MID1, a protein where aberrations in function result in cleft lip and palate formation, little is known about how MID2 protein interactions affect cell behavior. This project will highlight the MID interactions that regulate epithelial adhesion, a dynamic and important process during formation of the lip.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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NIDR Special Grants Review Committee (DSR)
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Frieden, Leslie A
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University of Washington
Schools of Dentistry/Oral Hygn
United States
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