Primary Cilium Submicron Organization and Dynamics

Abstract

The aim of this work is to contribute to the characterization of the biophysical properties of the ciliary microenvironment in order to understand central processes of the organelle's biology such as protein import, movement and crowding organization. First, we analyzed the movement of a small soluble protein (EGFP) to establish its pattern of diffusion in different regions of the ciliary compartment (body and distal tip). Using advanced fluorescence fluctuation correlation spectroscopy (Comprehensive correlation analysis) with the Zeiss Airyscan Detector, we characterized the organization and dynamic fingerprint of these subcompartments. Preliminary results showed that both ciliary body and distal tip have permeable microdomains explaining why EGFP presents a pattern of diffusion compatible with dynamic partition rather than free diffusion. However, at the ciliary body diffusion is more confined than at the ciliary tip. Measurements on cytoplasm and nucleus were made to compare with cilia crowding organization. Moreover, to study ciliary structural properties, we used environment-sensitive probes (ACDAN for the intra-ciliary compartment and LAURDAN for the ciliary membrane) combined with spectral phasor analysis to study molecular crowding and membrane's lipid order. Molecular crowding seems similar along cilia (base, body and distal tip). However, the ciliary base membrane was more ordered (less fluid) than the rest of the organelle. In conclusion, we put together a comprehensive protocol to study biophysical properties of primary cilium using state-of-the-art fluorescence techniques. This protocol will allow the evaluation of how different treatments or conditions affect the structure and dynamics of this organelle. (Acknowledgements: Work supported in part by NIH P41-GM103540 and Agencia Nacional de Investigación e Innovación Uruguay.)