Analysis of the roles of kinesin and dynein motors in microtubule-based transport in the Caenorhabditis elegans nervous system

The heteromeric kinesins constitute a subfamily of kinesin-related motor co mplexes that function in several distinct intracellular transport events. T he founding member of this sub-family, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was show n using antibody perturbation to be required for the synthesis of motile ci lia, presumably by driving the anterograde transport of raft complexes. To further characterize heteromeric kinesin transport pathways, and to attempt to identify cargo molecules, we are using the model organism Caenorhabditi s elegans to exploit its well-characterized nervous system and simple genet ics. Here we describe methods for large-scale nematode growth and partial p urification of kinesin-related holoenzymes from C. elegans, and an in vivo transport assay that allows the direct labeling and visualization of motor complexes and putative cargo molecules moving in living C. elegans neurons. This transport assay is being used to characterize the in vivo transport p roperties of motor enzymes in living cells, and to exploit a number of exis ting mutations in C. elegans that may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagel lar transport motor CHE-3 dynein, as well as cargo molecules and/or regulat ory molecules. (C) 2000 Academic Press.