The goal of this project is to gain a better understanding of the mechanisms of microtubule-based movement in early embryos. We will identify and study the functions of all the microtubule motors present in early C. elegans embryos. This project is made feasible by key advances in the C. elegans system. First, the genome is almost completely sequenced and microtubule motor genes can be identified by database searches. Second, many of the mRNAs of C. elegans have been cloned as cDNAs, partially sequenced, and are available by mail. Third, virtually any single protein or combination of proteins can be eliminated during oogenesis via injection of inhibitory RNAs, an approach termed RNA-mediated interference (RNAi). The specific goals of the proposed research are as follows: 1) To identify all the microtubule motors in C. elegans. We have already identified 19 and expect to find a few more as the genome sequence is completed. 2) To use RNAi to screen the motors and identify those with unique functions in motility processes in early embryos. Of the 9 motors tested thus far, 3 have essential early functions. The RNAi phenotypes of those 3, and of other early motors we expect to identify in the remainder of the screen, will be studied in live embryos by video-DIC microscopy and in fixed embryos by immunolocalization of microtubules, centrosomes, DNA and other cell components. For further insights, we intend to develop new methods for fluorescence imaging of microtubule and chromosome behavior in live embryos. 3) To generate and use a panel of antisera that are specific for each of the C. elegans microtubule motors. These will be used to gain insight into the functions of motors through their distributions in fixed specimens. In addition they will identify motors that are present in embryos, but whose functions, as revealed by RNAi tests, may be obscured by redundancy. 4) To identify and study the functions of proteins that interact with the subset of microtubule motors that have important roles in early embryos. This will be done using immunoprecipitation, biochemistry, and yeast 2-hybrid analysis for identification of motor interacting proteins. Once identified, the functions of those proteins will be studied with the RNAi approach and immunolocalization. 5) To uncover functions for redundant embryonic motors by multiple motor knock-outs. Multiple knock-outs are straightforward with the RNAi approach.
