Identification and functional analysis of A-tubule MIPs

项目总结

PROJECT SUMMARY Juvenile myoclonic epilepsy (JME) is the most common form of inherited early-onset epilepsy, however, its molecular pathology is poorly understood. The most commonly mutated gene in children with JME encodes the motile cilia structural protein, Rib72. This discovery provided the first genetic link between epilepsy and motile cilia. A key function of motile cilia function is their ability to beat and move extracellular fluid. In addition to JME, defects in ciliary function result in diverse and devastating human disorders collectively known as ciliopathies. These disorders include hydrocephaly, respiratory distress, and both male and female infertility. How motile cilia are built and maintained to support persistent ciliary beating remains a key unanswered question in the field. The core of motile cilia — called the ciliary axoneme — is composed of two individual microtubules encircled by nine sets of doublet microtubules. As a requisite for the relentless bending that they endure, the doublet microtubules are uniquely stable relative to their cytoplasmic counterparts. Exquisite structural studies using single-particle cryo-electron microscopy and cryo-electron tomography have detailed the ciliary axoneme at sub-nanometer resolution and revealed that a novel set of microtubule binding proteins resides within the lumen of the doublet microtubules. These internal densities have been termed Microtubule Inner Proteins (MIPs), and their identities and functions remain largely unknown. During my postdoctoral training in the laboratory of Dr. Mark Winey (UC Davis), I discovered that the JME-associated protein Rib72 is required for the recruitment of many of the as-yet unidentified MIPs to the A-tubule of doublet microtubules in Tetrahymena thermophila ciliary axonemes. Loss of Rib72 results in motile cilia beating defects and impaired axoneme stability. These results suggest that A-tubule MIPs play an integral role in the establishment of functional motile cilia. To determine the identities of the Rib72-dependent A-tubule MIPs, we carried out a mass spectrometry screen comparing wild-type versus RIB72 knockout Tetrahymena ciliary axonemes and identified a set of candidate A-tubule MIPs. My lab at Santa Clara University has collaborated with the labs of Dr. Mark Winey and Dr. David Agard (UCSF) to establish a research pipeline for the identification and characterization of these candidate MIPs. To date, we have successfully localized and characterized the A-tubule MIP Fap115, and we have initiated studies of a second A-tubule MIP, Rib43. The long-term goals of this project are to identify A-tubule MIP components, to understand the functional roles of A-tubule MIPs in motile cilia and to determine the functions of A-tubule MIPs within axonemal doublet microtubules. To achieve these goals, we will: 1) identify and localize the A-tubule MIPs from our list of candidates, and 2) functionally characterize these A-tubule MIPs, including Rib43. In addition to answering key questions about motile cilia structure and function, our studies may provide novel therapeutic insights into ciliopathies, including JME.