Dysregulation of cilia during embryonic development causes a broad spectrum of debilitating birth defects. The mechanisms that control formation and function of embryonic cilia remain poorly understood. There is a need to open new research directions to advance our understanding of cilia biology and thereby create new opportunities to develop therapeutic interventions. The long-term goal of this research is to identify molecular targets that can be used to develop pharmacological approaches to prevent or treat cilia-associated defects. This pilot project is based on new results that indicate the vacuolar-type H+-ATPase (V-ATPase) proton (H+) pump localizes to embryonic cilia. This finding suggests V-ATPase pumps H+ out of cilia to regulate pH gradients that may control cilia development and/or function. The overall objective of this project is to measure pH in cilia for the first time and determine how V-ATPase functions at cilia. The central hypothesis is that V-ATPase is recruited to the ciliary membrane to establish and maintain pH gradients at the cilium. This hypothesis will be tested in two specific aims.
Aim 1 is to determine the role of V-ATPase activity in regulating pH at the cilium. Genetically encoded fluorescent pH sensors will be used to measure pH at cilia in vivo using zebrafish embryos. Genetic mutations and small molecule inhibitors that interfere with the V-ATPase will be used to visualize and quantify how V-ATPase activity modulates pH gradients in embryonic cilia. Immunogold labeling and electron microscopy will determine the localization of V-ATPase in cilia.
Aim 2 is to identify mechanisms that recruit V-ATPase to cilia. Analysis of mutants and expression of fluorescent fusion proteins in zebrafish embryos will determine the role of two subunits in guiding V-ATPase to cilia. This work is conceptually innovative since it introduces and tests the new theoretical concept that pH is tightly regulated at the cilium and may control cilia development and function. Technical innovations include establishing the first methods to quantify pH at cilia and visualize V-ATPase at cilia in living embryos. Development of these novel ideas and tools has the potential to move the field in new directions. This project is significant because it is expected to provide the first insights into V-ATPase activity at cilia, which may identify pH as a mechanism that can modulate cilia form or function. An important short-term impact of this project will be new results that can be used to formulate a working model for future research to investigate molecular mechanisms by which pH regulates cilia. An anticipated long-term impact is that the results from this study establish a new paradigm for how cilia are regulated, and that this opens new areas of research to pursue V-ATPase and/or pH regulation as molecular targets of therapeutic strategies to prevent or treat cilia-associated birth defects.
This research is relevant to public health because it seeks to advance our understanding of underlying causes of birth defects and identify new opportunities to develop therapeutic strategies to predict, prevent or treat birth defects. This project is therefore relevant to NIH's mission to develop foundational knowledge that may enhance health and reduce disability.