Ciliary Growth by Intraciliary Trafficking

To visualize the movement of microtubule motors and putative cargo molecules in living C. elegans cells, heritable lines of transgenic worms are isolated that express these proteins labeled with the green fluorescence protein (GFP) (26). The in vivo transport of these fusion proteins is then monitored using time-lapse fluorescence microscopy (Fig. 1 A). Briefly, genomic sequence encoding microtu­bule motors or cargo molecules and their upstream regulatory sequences isolated using standard molecular biology techniques. These sequences are placed up­stream and in frame with the GFP gene in the C. elegans transformation vector

pPd 95.77 (kindly provided by the Fire Lab, Carnegie Institute of Washington, Baltimore, MD). This vector drives the expression of a motor/cargo::GFP fusion protein. Once constructed, the transformation vector is microinjected into the adult hermaphrodite gonad with a coinjection marker, and heritable lines express­ing the fusion protein of interest are isolated and maintained by self-fertilization. To monitor transport, worms are anesthetized in 1 or 10 mM levamisole, mounted on poly-L-lysine-coated slides, and time-lapse images of moving fluorescent par­ticles are collected at 100X magnification every 0.5-1.0 seconds. To investigate the involvement of other genes in this transport pathway, these constructs were introduced into various mutant background by either microinjection or genetic crosses.