The centrosome is a large eukaryotic organelle that organizes microtubules for cellular functions including setting up the mitotic spindle. Despite the important role of the centrosome, relatively little is known about its composition and regulatory mechanisms in metazoans. SPD-5 is a component of C. elegans centrosomes and contains multiple coiled-coil domains that may interact with other proteins thereby governing centrosomal structure (1). Because SPD-5 is thought to be a central component of centrosomes, it represents an excellent handle to pursue other genes and proteins that are also involved in centrosome function. We are taking two experimental routes to uncover additional centrosomal components and regulators.First, SPD-5 antibodies are being used to isolate protein complexes from embryonic extracts with the goal of purifying SPD-5-interacting proteins. Such proteins will be identified by mass spectrometry. To date, we have prepared embryonic extracts from N2 animals and we have successfully immunoprecipitated SPD-5 protein. Interestingly, two SPD-5 isoforms are apparent in our extracts, the significance of which remains to be determined. We are currently optimizing the purification procedure to obtain larger quantities of SPD-5 and interacting proteins. The interacting proteins we identify will be initially characterized by localization and RNAi inhibition studies.In the second route to understanding centrosome function, we are employing genome interaction screens to identify genes which, when knocked-down by feeding RNAi, alter the phenotypes of temperature-sensitive mutants. Building on the work of Jose Eduardo Gomes (see Gomes and Bowerman abstract), we have begun to use multiple-well plates for culturing worms and a robot to easily and accurately dispense the dsRNA-producing E. coli. We have performed a pilot semi-automated screen using the chromosome I library (2). Current results will be presented. In addition to examining
spd-5, we are planning to perform genome-wide interaction RNAi feeding screens with 20 different temperature-sensitive mutants defective in several different processes. We envision generating a genetic interaction map that will be useful for determining how many different processes require any given gene. We anticipate that our use of many different sensitized genetic backgrounds will enable us to identify gene requirements missed in previous classical and genomic screens.(1) Hamill, D. R. et al. Developmental Cell 3, 673-684 (2002).(2) Fraser, A. G. et al. Nature 408, 325-330 (2000).