Intraflagellar transport in the chemosensory cilia of C. Elegans tracked and captured with single-molecule fluorescence microscopy

Abstract

Cilia are protrusions present in most eukaryotic cells, with essential functions in motility and sensing. Development and maintenance of these microtubule-based organelles is crucially dependent on a specific transport process called intraflagellar transport (IFT). In the chemosensory cilia of C. elegans, two kinesin-2-family motors, heterotrimeric kinesin-II and homodimeric OSM-3-kinesin, act together in order to establish anterograde transport. Using quantitative fluorescence microscopy we show that kinesin-II gradually undocks from IFT trains that are initially formed out of tens of both motors allowing the OSM-3-kinesin train to reach terminal velocity already at the middle segment. The precise mechanism of how IFT trains are relayed between the two types of motors is, however, unknown and its unraveling requires assessing the dynamics of individual motor proteins. To this end we employed single-particle tracking in combination with photoactivated localization microscopy (sptPALM). We are able to follow single motor proteins deep inside the living organism and observe rich motor dynamics, such as transitions between processive walks, back-stepping, pausing and diffusion. Building superresolution images from single molecule localizations allows us to resolve the ultrastructure of cilia, onto which we can map single-motor trajectories. Our findings are the outset for a single-molecule view on IFT.