A single-molecule view on intraflagellar transport in C. elegans chemosensory cilia.

Abstract

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have been focusing on intraflagellar transport (IFT), which is essential for the assembly and maintenance of cilia. In particular, we study the chemosensory cilia of C. elegans. To visualize IFT with live fluorescence microscopy, we generate mutant worms expressing fluorescent versions of the proteins of interest. Our fluorescence and image-analysis approaches allow us to visualize, track and quantify trains of IFT components moving together, as well as individual motors or IFT proteins. In C. elegans, IFT is driven by groups of tens of three different motor proteins: 2 kinesin-2's (the slow kinesin-II and the faster OSM-3), which drive transport of cargo trains from base to tip of the cilium, and IFT dynein, which drives transport back to the base. Cargo trains move in one go from base to tip and from tip to base. Speed and directionality appear to be regulated by motor proteins docking on and off the trains. Here I present new single-molecule data focusing on the entry of IFT components, including motor proteins, into the cilium. We observe that different IFT components attach to IFT trains in a sequential way, both in time and in location. Pooling the localization information of many individual molecules allows mapping of the 3D ultrastructure of the axoneme with a precision of several tens of nanometers, in wild-type as well as mutant strains lacking e.g. motors. These maps provide new insights in ciliary structure at the base, train-docking dynamics and the interplay of kinesin-II and OSM-3. Our results provide important new insights into how motors cooperate to drive intracellular transport.