INVESTIGATING THE FUNCTIONAL MORPHOLOGY OF IN SITU IFT TRAINS BY CORRELATIVE LIGHT-ELECTRON MICROSCOPY

Cilia and flagella are cellular organelles involved in several and crucial aspects for the cell, tissue and organs normal physiology. Cilia can be classified according to their ability to perform a movement or not. Eukaryotic unicellular organisms and particular cells of mostly evolved organisms, i. e. spermatozoa, have evolved motile cilia for the cell propulsion in liquid media. Ciliated epithelia direct the movement of fluids by the ciliary beating. The primary cilium is an immotile cilium present in all the so far studied metazoan. This organelle is ubiquitous in human somatic cells when they are in G0 and G1 phases. Primary cilium and motile cilia work as sensory antennae highly specialized in receiving and secreting molecular signals. Such signal pathways regulate many aspects of the cellular metabolism, the cellular life-cycle, and the eventual cellular differentiations. Thus, ciliary alterations trigger severe consequences in human health causing a group of diseases known as ciliopathies. Such alterations in ciliary integrity are generated also from mutations affecting peptides involved in the intra-flagellar transport system (IFT), a selective and specific system of flagellar proteins transport, delivery, and recycling in which are employed proteic complexes (IFT trains) able to move bi-directionally along the flagellar axoneme. IFT trains transport into the ciliary compartment all the components needed for a correct ciliary assembly and maintenance i.e. peptide and proteic complexes of the ciliary axoneme, and several membrane-associated receptors. Analysis directed to better understand all the IFT features are thus of primary importance. Many analyses have been performed by studying the green algae model organism Chlamydomonas reinhardtii. Studies on IFT dynamics were performed in vivo, lacking in high resolution imaging. Other studies oriented in the determination of IFT trains ultrastructural morphology, and 3D-modeling digital reconstruction were performed ex vivo by transmission electron microscopy (TEM) imaging. A very recent work by Stepanek and Pigino (2017) revealed new insights in IFT system by using the correlative light electron microscopy (CLEM), which combines in vivo dynamics observed by florescence microscopy with ex vivo high-resolution analysis by electron microscopy. This study tough providing interesting anew clues about IFT functioning, left other important questions open on the motility direction of the different IFT trains categories present in Chlamydomonas flagella. In the present thesis we aimed at providing deeper insights in IFT system by using CLEM, in order to correlate a specific function to a specific ultrastructural category of IFT train. The reported results highlighted “short narrow IFT trains” moving in both anterograde and retrograde directions along the axoneme, while “short wide IFT trains” and “long IFT trains” were observed to remain static during in vivo observation.