Cytoplasmic dynein is a high molecular weight, multisubunit complex that functions as a microtubule-associated motor. Cytoplasmic dynein is proposed to be required for the endocytic pathway, retrograde transport of organelles in axons, organization of the Golgi, and various mitotic processes including spindle assembly. The ability of cytoplasmic dynein to mediate microtubule-dependent transport of vesicles in vitro requires the addition of dynactin, a large multisubunit complex. Until recently, analyses of cytoplasmic dynein and the associated dynactin complex have relied mainly on biochemical approaches, and this study presents a complementary genetic approach. A genetic screen has been developed, using the filamentous fungus Neurospora crassa, that allows the isolation of hundreds of mutants defective for cytoplasmic dynein or dynactin. These mutants, designated ropy (ro), have curled hyphal growth and asymmetric nuclear distribution. Approximately 1200 ro mutants have been isolated, defining 23 complementation groups. ro-1 encodes the heavy chain of cytoplasmic dynein, while ro-3 and ro-4 encode the dynactin subunits p150(Glued) and Arp1, respectively. The goals of this project are to utilize this genetic system to identified genes required for cytoplasmic dynein or dynactin activity, to examine the domain structure of cytoplasmic dynein heavy chain and p150(Glued) and determine which ro gene products interact to form or regulate these complexes, and to investigate the relationship between ro genes encoding novel proteins and cytoplasmic dynein and dynactin. The major experimental approaches employed in this study are: 1) cloning and characterization of ro genes; 2) immunolocalization of cytoplasmic dynein, dynactin, and novel Ro proteins in a wild-type strain and in various ro mutants; and 3) immunoprecipitation analysis to examine cytoplasmic dynein and dynactin subunit interactions.
