Friday, April 19, 2024
4/19/2024
Project Summary The primary cilium, present on most cell types of the human body, is required for a broad array of developmental and physiological functions. These include Hedgehog (Hh) signaling, which is used for the specification of cell types in the central nervous system, neuronal GPCR signaling, retinal photoreceptor homeostasis, olfaction, and cochlear hair cell function. However, the ways by which cilia are assembled and function in signal transduction are only partially understood. We are studying a kinase, called Cell Cycle-Related Kinase (CCRK), which is required for both ciliary length regulation, ciliary structure, and proper cilium-dependent Hh signaling. The mechanism by which CCRK functions is unclear but our published data indicates it functions to promote the import of Hh signaling components into the cilium. The only established substrate of CCRK is another kinase, ICK, which functions in ciliary length control, but is largely dispensable for early development and Hh signaling. We will hypothesize that, although phosphorylated ICK (in the TDY motif by CCRK) restricts ciliary length, unphosphorylated ICK functions in an overlapping manner with CCRK to promote ciliogenesis. We will test this hypothesis by analyzing ciliogenesis and Hh signaling in mouse mutants completely lacking both kinases. While CCRK appears to control ciliary import of Smo and Gli2, we hypothesize that it controls the import of other ciliary proteins that assemble the cilium and mediate other types of neuronal signaling. We will specifically test this hypothesis using fluorescent recovery after photobleaching of fluorophore-tagged ciliary cargo within the cilia or wild-type and Ccrk null cells. Finally, we propose that CCRK is continuously required to promote ciliary cargo import in the fully assembled cilium and that it does so in a dosage-dependent manner. Experiments aimed at acutely disrupting or increasing CCRK activity after ciliogenesis is complete are proposed to test this hypothesis. This combination of experiments should significantly clarify open questions regarding the mechanisms by which cilia are loaded with cargo that execute signaling functions critical for neural development and establish appropriate ciliary length.
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