Structure and Function of Flagellar Central Pair Microtubule-Associated Complexes

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Cilia and flagella are slender cylindrical surface extensions of cells that wave or beat rhythmically, thereby either moving the cell through its liquid environment , as for example in the case of motile protozoa or sperm cells, or moving the liquid environment across the surface of the cell, as for example in the case of ciliated epithelia in the respiratory tract. The molecular machinery inside the cilia or flagella is collectively called the axoneme, and consists mainly of a highly ordered arrangement of specialized structural elements (microtubules) and molecular motors (dyneins) that the microtubules to slide lengthwise relative to each other in a highly regulated and orderly fashion. The work that will be performed with support from this award is directed toward understanding the mechanisms that regulate the microtubule-based motor dynein during motility of eukaryotic cilia and flagella. The approach focuses on mutations in a model organism, the photosynthetic protist Chlamydomonas reinhardtii, that alter dynein activity by disrupting assembly of a regulatory complex, the central pair apparatus. Based on many previous studies, projections from the two central pair microtubules interact with radial spokes, which in turn transmit regulatory signals to doublet microtubule-associated dyneins. A cascade of protein kinases has been implicated in this signaling process, but the role of central pair structures has not been determined. Although most mutations affecting central pair structure completely block motility, a mutation was recently described that allows altered motility. This mutation, cpc1, specifically prevents assembly of one row of central pair microtubule projections. The experiments that will be performed with support from this award will provide new information about the structures and functions that are missing in cpc1 flagella.The motility of isolated, reactivated cpc1 flagellar axonemes and the microtubule sliding rate of protease treated axonemes will be measured to characterize changes in beat frequency, waveform, and sliding velocity. The effects of specific protein kinase and protein phosphatase inhibitors on these parameters will be compared using cpc1 axonemes, wild type axonemes and axonemes from other central pair defective strains. Structures retained in axonemes from cpc1 and another central pair defective mutant, pf6, will be characterized by electron microscopy. Images from fixed, sectioned material will be compared with images from rapidly frozen, etched material to build three-dimensional models of central pair structure. This information will clarify possible interactions between central pair projections and radial spokes during flagellar motility. Molecular approaches will be used to clone the CPC1 gene, starting from available insertionally tagged alleles. Sequence of the corresponding cDNA will be used to predict the primary and secondary structure of the gene product and identify homology with proteins of known function. The results from this work will provide new insight into how flagellar motility is regulated.