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 complexes that function in several distinct intracellular transport events. The founding member of this subfamily, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was shown using antibody perturbation to be required for the synthesis of motile cilia, 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 Caenorhabditis elegans to exploit its well-characterized nervous system and simple genetics. Here we describe methods for large-scale nematode growth and partial purification 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 properties of motor enzymes in living cells, and to exploit a number of existing mutations in C. elegans that may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagellar transport motor CHE-3 dynein, as well as cargo molecules and/or regulatory molecules.