Figure 1 Visualization of intraflagellar transport (IFT), the motion of heterotrimeric kinesin-II motors and OSM-6 subunits of rafts from the transition zone to the distal tip of sensory cilia. a. Schematic of C. elegans chemosensory cilia. Upper panel shows differential interference contrast light microgragh of head of an adult worm oriented to the left. Boxed region shows position of an amphid channel containing sensory cilia. This region is represented in the schematic (lower panel) which shows a close-up of sensory cilia oriented with their distal endings facing left, and contacting the external environment through openings in the cuticle. Green represents fluorescent kinesin-II or OSM-6, which accumulate at the transition zone at the base of the cilia (orange arrowhead) and move (black arrow) as puncta (orange arrow) to the distal endings of the cilia. b. Fluorescence micrographs of sensory cilia in GFP transgenic worms as represented by the boxed region and the schematic (shown in panel a). Fluorescent kinesin-II motors and fluorescent OSM-6 subunits of IFT rafts accumulate at the base of the transition zones where they appear as large puncta (arrowheads). Arrows indicate position of puncta of fluorescent kinesin-II and fluorescent OSM-6 as they travel towards the distal tip of the sensory cilium which is oriented to the left (as in part a). Bar, 5.2mm.
Methods Fluorescent kinesin-II was produced by creating transgenic worms expressing KAP::GFP. To this end the entire coding sequence of the kap gene and 2kb of associated upstream promoter sequence from genomic cosmid F56C9 were inserted into the GFP vector pPD95.75, which was acquired from Dr. A. Fire (Carnegie Institute of Washington). Strain N2 worms were cotransformed with the resulting construct and plasmid pRF4 containing a semidominant marker mutation, rol-6(su1006). Stably transformed lines expressing KAP::GFP were recovered from resulting roller hermaphrodites. Fluorescent rafts were visualized using a transgenic line expressing OSM-6::GFP, a generous gift of Dr R.K. Herman (U of Minnesota). To control for the velocities of KAP::GFP and OSM-6::GFP in cilia, we watched ODR-10::GFP vesicles moving in dendrites; see Noelle Dwyer, Ph.D. thesis, UCSF. This transgenic line expressing ODR10::GFP was a generous gift of Dr C. Bargmann (UCSF). The transgenic worms were anesthetized with 100mM levamisole to immobilize them, and IFT of the GFP fusion proteins was observed using fluorescence microscopy on a Nikon E600 microscope. To determine the velocity of KAP::GFP and OSM-6::GFP motility, time lapse images were captured at one second intervals and processed using a Photometrics SenSys KAFO400 camera equipped with a Uniblitz shutter drive and Metamorph image analysis software. Relative positions of fluorescent particles in the captured images were determined using an objective micrometer.