A fundamentally important challenge is to understand the extent to which the products of multi-gene families are functionally specialized, and the many dynein isoforms co-existing in the same cell present an important opportunity to understand dynein specialization. The present project focuses on the interplay of the two non-axonemal or cytoplasmic dyneins-1 and -2 in cilia formation. Because of the essential roles of cilia/flagella, intraflagellar transport (IFT) has evolved to ensure the proper assembly of these organelles. The ciliated protozoan Tetrahymena thermophila is an excellent system in which to study IFT, and promises to yield new insights into IFT and cytoplasmic dyneins. In contrast to what has been observed in other systems, the knockout of dynein-2 components in Tetrahymena results in only a mild reduction in the number and length of cilia. In these knockout cells, the dynein-2 is catalytically inert, but there is evidence that the Dyh2 tail domain continues to be expressed. Thus, either Tetrahymena does not require IFT to form functioning cilia, or Tetrahymena IFT can be powered by alternative motors other than dynein-2. An intriguing possibility is that the remaining Dyh2 tail domain can recruit other motors to substitute for the missing Dyh2 catalytic domain. Further, here is the opportunity to visualize both cytoplasmic dyneins in the same cell undergoing different processes, thus providing insights about the spatial coordination of the two dyneins. Combinations of genetics, immunochemistry, and fluorescence microscopy will be used to determine how the intracellular location and the level of expression of one cytoplasmic dynein is affected by the depletion of the other cytoplasmic dynein.
Broader Impact. The project focuses on the function of a protein motor in the regulation of cilia formation in a model organism. In a wide spectrum of organisms, cilia/flagella play essential roles, including the coordinated movements of fluids across a tissue surface, the movement of gametes, and the establishment of the left-right axis in embryos. In Tetrahymena, active cilia are required for cell motility, feeding, and cell division. A central objective of this RUI project is to integrate research into education while enabling undergraduate students to engage in complex, open-ended experiments that provide each student the opportunity to employ strategies in molecular biology, genetics, cell biology, and serology. Since 2003 at Harvey Mudd College, the laboratory has mentored 32 students who were from 7 different academic majors. Of the 21 who have now graduated, ten are in graduate school. To date, undergraduates have co-authored two peer-reviewed papers and three abstracts presented at national meetings.
