The overall goal of my thesis is to define the mechanisms that regulate ciliary/flagellar motility. In particular, my focus is on the regulation of dynein-driven microtubule sliding by phosphorylation. The work is important because cilia are responsible for motile functions (e.g. movement of sperm, fluid movement along epithelia, such as in the respiratory system, oviducts, or brain ventricles) and sensory functions (e.g. the connecting cilia of photoreceptors, the mechano-sensitive primary cilia of the kidney, etc). Failure in assembly or function can lead to a wide range of diseases in humans including, polycystic kidney, blindness, infertility in males, and primary ciliary dyskinesia. Thus it is important to understand how cilia are assembled and how dynein-driven motility is regulated. My project is focused on the protein phosphatase PP2A, which based on previous studies and my new preliminary data, is located in the ciliary axoneme and controls dynein activity. PP2A is made up of three subunits, the anchoring A-subunit, the catalytic C-subunit, and the regulatory B-subunit. The B-subunit is responsible for providing the phosphatase with substrate specificity and sub-cellular localization. This proposal is designed to test the hypothesis that PP2A is localized and anchored in the axoneme by a regulatory B-subunit to modulate phosphatase activity. The following aims are designed to test this hypothesis:
In specific aim 1, 1 propose to use immuno-fluorescence and electron microscopy to test the hypothesis that the B-subunit of PP2A is localized to the outer doublet microtubules in position to regulate inner arm 11 dynein, the dynein that appears to be critical for control of microtubule sliding and ciliary bending.
For aim 2, using immunoprecipitation, chemical crosslinking, and mass spectrophotometry, I will test the hypothesis that the B-subunit, in addition to binding the A and C-subunits, interacts with other axonemal proteins that serve as a docking complex to anchor and localize it within the axoneme near 11 dynein. Finally, in aim 3,1 will address the function of the B-subunit by assessing motility and molecular phenotypes of mutants defective in B-subunit gene expression. Together, these data will comprehensively test the functional role of PP2A in the ciliary axoneme. Moreover, it is also possible that we will define a general role for PP2A B-subunits in targeting the phosphatase to microtubule dependent functions.
| Elam, Candice A; Wirschell, Maureen; Yamamoto, Ryosuke et al. (2011) An axonemal PP2A B-subunit is required for PP2A localization and flagellar motility. Cytoskeleton (Hoboken) 68:363-72 |
