Probing of cilia structure, function and disease mechanisms using soft X-Ray microscopy in C. elegans and RPE-1

The application of soft x-rays (SXRs) to image biological tissues remains relatively unexplored. Here, we propose to employ ALBA synchotron beamlines to explore SXR tomography (SXT) for imaging the nose tip of the nematode, Caenorhabditis elegans. Specifically, we are interested in the neuronal sensory cilia within openings (sensillae) of the worm’s cuticle. C. elegans is a leading model for investigating cilia structure and function, and probing ciliopathy disease mechanisms. As a CLEXM consortium partner that includes ALBA, we have developed SXR imaging workflows using the Sirius XT-100 benchtop instrument in University College Dublin. Now, we wish to benchmark and extend our findings with the higher energy SXT that is possible using synchotron beamlines. Our samples will be 1.5 um thick slabs of cryofixed nematode tissue, generated by FIB milling and polishing in a collaborator’s lab (Dr. Jorgen Plitzko; Max Planck). Beamtime will be used to image nose tip slabs from wild type worms, as well as several worm strains housing patient relevant-mutations in ciliopathy genes. To benchmark our C. elegans data, and extend our ongoing mammalian cell ciliogenesis project, we will also use beamtime to image primary cilia on plunge-frozen cultured cells (RPE-1) at various stages of cilium formation. Overall, this project will uncover the maximal potential of SXT for imaging primary cilia in cell and tissue context. The project can also reveal how patient mutations affect cilia structure and provide insight into the mechanisms underpinning cilium formation.