Cilia are microtubule-based structures that are found on the surface of cells throughout the human body. Specialized cells that exhibit multiple (hundreds) of motile cilia that play key roles in transporting fluid are located on airway epithelial cells, brain ependymal cells and fallopian tube epithelial cells. For example, in the lung, multi- cilia play an important protective function by beating synchronously to generate mucociliary flow that expels inhaled irritants and pathogens. Defects in the formation or function of cilia contribute to numerous pathologies, collectively designated ciliopathies, and children born with ciliopathies frequently experience respiratory distress and are susceptible to lung infection. Defective cilia structures are also associated with, and contribute to, numerous other disorders, such as reproductive dysfunction, hydrocephaly, and cystic fibrosis. Despite the important nature of multi-cilia, little is known about their development or their regulation. The GOAL of this proposal is to describe novel molecular mechanisms contributing to multi/motile-cilia formation (i.e. multiciliogenesis) and maintenance of cilia function, using the multi-ciliated cells in the mammalian airway as a model. Our preliminary studies have identified an important role for the Hippo pathway effector Yap in the maturation of multi-ciliated cells. Our prior studies have shown that removal of Yap is required for airway epithelial cells to differentiate into a mature fate. Our recent observations indicate that non-nuclear Yap is playing an important function that impacts centrosome amplification, which is an early step in the generation of multi-cilia. Apical-localized centrosomes, known as basal bodies, serve as the organizing center for cilia, and the de novo amplification of these centrosomes is required for the formation of multiple cilia. Our observations suggest that Yap binds to and inhibits the activity of Trim32, a ciliopathy-associated ubiquitin ligase that we have identified to target a key effector of centrosome amplification. We HYPOTHESIZE that inhibition of Trim32 by cytoplasmic Yap induces centrosome amplification, which is an early requirement for multi-cilia formation. Our data further indicate that as the multi-ciliated cells mature, Yap is sequestered to the maturing apical domain away from Trim32, thereby shutting down centrosome amplification. Our RESEARCH STRATEGY includes defining in detail the role(s) of Yap in centrosome amplification and multi-cilia formation (AIM 1) and determining the roles and regulation of Trim32 in these processes (AIM 2). Notably, the roles of these proteins in multiciliogenesis have yet to be identified or studied, and non-nuclear roles for Yap are very poorly understood, particularly in any biological context. Our proposed experiments will therefore advance the current knowledge of multi-cilia formation, and contribute to a better understanding of disorders arising from cilia dysfunction that will hopefully guide future therapeutic development.
Cilia are apical localized microtubule-based organelles that play important roles in various tissues and organs. Multiple motile cilia that play key roles in transporting fluid are located on airway epithelial cells, brain ependymal cells and fallopian tube epithelial cells, and defects in the formation and action of these multi-cilia are associated with a range of diseases. Our proposed study focuses on understanding the molecular mechanisms by which multi-cilia develop with the hope that advancing this knowledge will offer novel therapeutic opportunity.