The nutrient transporting enterocytes of the intestine and sensory hair cells of the inner ear perform their physiological functions using specialized membrane protrusions found on their apical surfaces. These protrusions must be organized into elaborate arrays with exquisite precision during their creation in order to function correctly. Remarkably, enterocytes and hair cells use homologous adhesion complexes to control the organization of their apical protrusions. Multiple lines of evidence suggest that the entire complement of proteins that compose these homologous adhesion complexes remains to be elucidated. This represents a significant gap in knowledge of how epithelial cells create their functional apical domains. The continued existence of this gap impedes the diagnosis and development of treatments for patients suffering from epithelial disease related to these homologous adhesion complexes, as exemplified by Usher syndrome. The long-term goal is to understand how specialized epithelia use adhesion to create their apical domains by identifying and functionally characterizing all the components of these homologous apical adhesion complexes. The current objective here is to determine the role of Calmodulin-like protein 4 (CALML4) in epithelial apical assembly. CALML4 is a newly identified component, discovered by the PI, found in both homologous adhesion complexes. Excitingly, CALML4 is one of 27 candidate genes for Usher syndrome Type 1H (USH1H), a subtype of Usher syndrome whose genetic cause is currently unknown. Preliminary data shows that CALML4 is critical for proper enterocyte apical assembly and is a direct binding partner for Myosin-7b (Myo7b), a myosin found in the enterocyte adhesion complex. Furthermore, CALML4 directly associates with the homologous myosin found in the hair cell system, Myosin-7a (Myo7a), suggesting CALML4 plays an identical role in sensory epithelia. Both Myo7a and Myo7b are essential for proper function of their respective adhesion complexes. The central hypothesis is that CALML4 acts as a myosin light chain that is critical for myosin-dependent apical adhesion complex function in specialized epithelia. This hypothesis will be tested through two specific aims: (i) investigating the role of CALML4 in force production by these myosins using in vitro actin-sliding filament assays and examining the motility of chimeric myosins that contain the light chain-binding domains of Myo7a/Myo7b, and (ii) defining the role of CALML4 in epithelial cells through genetic manipulation of an enterocyte cell culture model. Within these aims, we will pioneer novel tools to explore the cellular pathology of mutations associated with Usher syndrome. The approach is innovative, since it identifies CALML4 as a novel component of these homologous adhesion complexes, and challenges the existing viewpoint that Usher syndrome is exclusively a neurosensory disease. The proposed research is significant because defining the role of CALML4 in these homologous adhesion complexes will expand the current understanding of how epithelial cells from diverse tissues use adhesion as a mechanism to assemble their apical domains, and may provide insight into the genetic cause of USH1H.
The proposed research is relevant to public health because apical domain dysfunction of transporting and sensory epithelial cells results in serious human disease. The discovery of CALML4 as a novel component of the homologous adhesion complexes that control apical domain formation of these specialized epithelia is a clear vertical step forward for the field, and extends the knowledgebase of how functionally divergent epithelia use adhesion as a common mechanism to promote apical morphogenesis. The importance of this discovery is further underscored by the fact that CALML4 is the leading candidate gene for Usher syndrome Type 1H, suggesting that knowledge from this proposal may aid in diagnosis and treatment of this disease in the future.