Project Grant F31HD120012

THE MICROTUBULE LUMEN AS A NOVEL REGULATOR OF CILIA FUNCTION AND DEVELOPMENT - PROJECT SUMMARY CILIA HAVE DIVERSE ROLES IN CELL MOTILITY, SENSORY FUNCTION, AND SIGNALING. THE DISCOVERY OF GENES AND MECHANISMS ASSOCIATED WITH CILIA FUNCTION ARE CRITICAL FOR UNDERSTANDING AND TREATING A WIDE CLASS OF DISEASES KNOWN AS CILIOPATHIES, WHICH CAN IMPACT AIRWAY CLEARANCE, FERTILITY, AND VISCERAL ORGAN ASYMMETRY. RECENTLY, A NEW CLASS OF PROTEINS FOUND WITHIN THE LUMEN OF MICROTUBULES THAT COMPRISE THE CILIARY AXONEME, KNOWN AS MICROTUBULE INNER PROTEINS (MIPS), HAVE BEEN DISCOVERED THROUGH CRYO-EM AND STRUCTURAL STUDIES. HOWEVER, THE FUNCTIONAL SIGNIFICANCE OF THESE PROTEINS IN MOTILE CILIA HAS YET TO BE UNDERSTOOD EXCEPT FOR A HANDFUL OF GENES. FROM A PRELIMINARY SCREEN PERFORMED IN PREPARATION FOR THIS PROPOSAL, I ASCRIBED NEW ROLES IN MOTILE CILIA FUNCTION FOR 8 NOVEL MIPS. NOW, I AIM TO PURSUE TWO OF THESE MIPS, CIMIP5 AND TEX49, AND UNDERSTAND THEM IN DETAIL TO FURTHER OUR KNOWLEDGE OF THE ROLE OF MIPS IN CILIA FUNCTION AND DEVELOPMENT. THE OBJECTIVE OF THIS PROPOSAL IS TO DECIPHER THE ROLE OF THE MICROTUBULE LUMEN IN CILIA FUNCTION AND DEVELOPMENT USING THE TRACTABLE ZEBRAFISH SYSTEM. MY PRELIMINARY DATA ON CIMIP5 AND TEX49 LED ME TO HYPOTHESIZE THAT MIPS FORM AN INTERNAL SCAFFOLD THAT FINE-TUNES MOTILE CILIA BEATING, SUCH THAT LOSS OF SPECIFIC MIPS DISRUPTS THIS INTERNAL REINFORCEMENT IN A TISSUE-DEPENDENT MANNER, IMPAIRING FLOW GENERATING AND DEVELOPMENTAL PATTERNING. IN THE FIRST AIM, I WILL THOROUGHLY ASSESS THE REQUIREMENT FOR EACH MIP IN FIVE DISTINCT MOTILE-CILIATED TISSUES IN ADDITION TO DETERMINING THEIR DEVELOPMENTAL REQUIREMENTS USING CRISPR-GENERATED GERMLINE MUTANTS. IN MY SECOND AIM, I WILL UNCOVER THE UNDERLYING MECHANISMS THAT ALLOW MIPS TO CONTRIBUTE TO MOTILE CILIA FUNCTION BY DEVELOPING MY IMAGING SKILLSET FURTHER WITH EXPANSION MICROSCOPY AND TEM. OVERALL, MY WORK WILL DECIPHER THE ROLE OF THE MICROTUBULE LUMEN IN CILIA FUNCTION AND DEVELOPMENT USING A RANGE OF IMAGING AND GENETICS TECHNIQUES THAT WILL PROVIDE ME WITH STRONG TRAINING IN CELL AND DEVELOPMENTAL BIOLOGY. MOREOVER, MY WORK WILL HAVE IMPORTANT IMPLICATIONS FOR UNDERSTANDING THE GENETIC LANDSCAPE OF CILIOPATHIES AND COULD LEAD TO THE DEVELOPMENT OF TARGETED THERAPIES.