Congential heart disease is the most common congenital malformation, which is the leading cause of infant mortality in the US and Europe. Heterotaxy, a left-right patterning disorder, is a common congenital heart disease and affected children have poor prognosis ever after extensive surgical intervention. While previous work in model systems has implicated cilia dysfunction in heterotaxy, the molecular mechanisms underlying the disease are not understood, as demonstrated by human genetic studies that have identified candidate heterotaxy genes with no known function in heart development, left-right patterning, or cilia biology. One such study, performed by our collaborators at Yale, identified Nup188, a gene encoding a component (nup) of the nuclear pore complex (NPC), as a potential Heterotaxy candidate gene. Our preliminary data show that knockdown of Nup188 or its binding partner Nup93 1) recapitulates a heterotaxy-like phenotype in a Xenopus tropicalis model and 2) leads to a loss of cilia in multiple Xenopus tissues and in RPE cells. Moreover, our data are consistent with these phenotypes being a reflection of a direct role for nups at the cilium base as both Nup188 and Nup93, but not other nups, localize to the basal body and daughter centriole in Xenopus epidermis and RPE cells. Based on these (and other) results, I hypothesize that Nup188 is critical for ciliogenesis. To directly test my hypothesis, I must first define the nup-cilia interactome and generate reagents that can specifically address the function of nups at bases of cilia without affecting their established roles in nuclear transport. Thus, in Specific Aim 1, I will use a proximity-based ?BioID? approach to identify Nup188 neighboring proteins and binding partners at cilia bases. I will then perform direct binding experiments with full length and truncated versions of Nup188 to identify a cilia binding domain. Using this mapping data, in Specific Aim 2, I will generate a cell line that facilitates the functional separation of Nup188 at the NPC from Nup188 at the cilia by generating an allele of Nup188 that lacks the cilium binding domain. Finally, I will utilize this cell line to probe the role of Nup188 in ciliogenesis. These experiments will elucidate the mechanism by which Nup188 contributes to cilia function and to heart position during development.
Cilia are essential organelles that, during embryonic development, contribute to left-right patterning and the proper positioning of organs including the heart. Therefore, understanding the molecular mechanisms that lead to cilia formation and function are critical for understanding the molecular etiology behind congenital heart diseases like heterotaxy.
