The implementation of the project has resulted in development of a new imaging technique and a new image analysis pipeline. The exploitation of the developed methods has resulted in several interesting research findings.
• Small-window illumination microscopy (SWIM) advances single molecule imaging in chemosensory neurons of C. elegans.
A new microscopy technique, SWIM was implemented to obtain detailed single molecule information in C. elegans chemosensory cilia (Figure). The essential idea is to control the epi-fluorescence illumination beam size to only illuminate the region of interest. Using a high laser power, one can bleach all molecules that are within the illuminated region, but any new molecule that enters the region is not bleached and can be imaged until it bleaches.
Dissemination: The methodology will be described in a book chapter in Springers Protocols, Single Molecule Analysis – Third Edition (accepted) and a methods paper will be submitted in a special issue of Optics Communications on Advances in Localization Microscopy
• Ultrastructure and IFT motor-entry dynamics at the ciliary base uncovered using single-molecule tracking microscopy.
Intraflagellar transport (IFT) along a microtubule-based axoneme orchestrates the growth and maintenance of sensory cilia, elongated signalling hubs protruding out of eukaryotic cells. Kinesin-2 motors drive anterograde IFT trains, assembled at the cilia base, into the cilium, importing cargo proteins hitchhiking on the trains. SWIM allows us to visualize and quantify the entry of individual anterograde kinesin-2 motors, kinesin-II and OSM-3, and cargo proteins associated with anterograde IFT trains, IFT dynein and tubulin, into cilia located at the tip of dendrites of chemosensory neurons in C. elegans.
Dissemination: Final analysis is ongoing and the manuscript is under preparation.
• Ciliary proteins utilize contrasting mechanisms to reach the ciliary base, assemble into trains and enter the cilia in C. elegans
The IFT proteins associated with cilia, have to be transported from the cell body to the cilia base, moving across the dendrites (several 10s of microns), where they assemble into IFT trains and cross the transition barrier to enter the cilia. By performing SWIM in the chemosensory neurons of C. elegans, we elucidate the different mechanistic strategies employed to orchestrate entry of proteins inside cilia of C. elegans. Further, we image changes in the dendritic transport of ciliary proteins in response to chemical repellants, to unravel the mechanism of regulation of IFT transport during chemotaxis.
Dissemination: Final experiments and analysis is ongoing.
• We have published a review article with focus on the regulation of intraflagellar transport motors in cilia and its relevance to ciliary structure, function and disease. Dissemination: Review titled ‘Mechanisms of Regulation in Intraflagellar Transport’ was published in the special issue of the Journal Cells: Primary Cilia in the Nervous System: Structure, Function and Disease Mechanisms. (
https://doi.org/10.3390/cells11172737(se abrirá en una nueva ventana)).
• We utilize SWIM to understand tubulin dynamics in chemosensory cilia of C. elegans. IFT trains travel bidirectionally along the microtubule-based backbone – the axoneme. Project is driven by a PhD student, E. Loseva, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Final experiments and analysis is ongoing.
• We utilize quantitative fluorescence microscopy to study the regulation of kinesin motors in the chemosensory cilia of C. elegans by kinases DYF-5 and DYF-18. Project is driven by a PhD student, W. Mul, with my role pertaining to supervising the project and providing analytical tools. Dissemination: Experiments and analysis is ongoing.
>> The felloship will be (has been) acknowledged for funding all the above discussed research works.
>> Invited talks, presentations and posters in conferences and online platforms
The results of this work have been disseminated in 3 invited talks, 1 virtual talk in a webinar series and 6 conferences (4 talks and 3 posters).
>> Public Outreach
A video including clay animation titled Worm Lab Tour (
https://youtu.be/T_ZjuqJLTr0(se abrirá en una nueva ventana)) was made to explain the research performed in my host laboratory, which is suitable for general audience. It has been and will be shared at numerous platforms.
