I used cryo-electron tomography and subtomogram averaging of the isolated axonemes of different TT mutants. The protein complexes which were used for the study were selected by our colleagues from Nencki Institute in Warsaw based on the strong phenotype of mutants and therefore their potential importance for ciliary function. The workflow after obtaining the mutant and wild type cells was optimization of axonemal isolation, optimization of the sample preparation for the cryo-EM (plunge freezing), tomography acquisition, tomogram reconstruction and subtomogram averaging – data analysis.
The original specific tasks were:
a) Analyse as many as possible different positions of IFT trains within the cilia by cryo- electron microscopy and tomography
b) Determine the specific structure of IFT trains by cryo-electron tomography (cryoET) with the sub-tomogram averaging
c) Map the train formation, dispatching and cargo loading/unloading
Due to the fact that the proposal was being prepared almost two years before the particular work started, research objectives of my supervisor Gaia Pigino have changed accordingly. In particular, the Pigino lab members and especially PhD student Mareike Jordan made key findings of the IFT train structure. We therefore decided to use an identical methodology approach to map the location and potential function of different protein complexes present in cilia of another important model organism – Tetrahymena thermophila.
The modified specific tasks were as follows:
a) Analyze ciliary structure of different Tetrahymena thermophila mutants provided by our colleagues from Nencki Institute in Warsaw and compare them to the wild type structure
b) Determine the specific structure and location of these protein complexes within the axonemal (ciliary) structure by using cryo-electron tomography (cryoET) with the subtomogram averaging
c) Map the potential function of these protein complexes based on their connections to the known regulatory or motor domains and the mutants swimming phenotype
Overview of the results:
WP1: All of the planned objectives for this continuous work package were achieved: management, planning, design and dissemination.
WP2: All objectives accomplished: training on how to improve independent scientific thinking to get an exact approach to the complex biology problems as well as on how to set up an advanced experimental design as well as on how to perform a 3D reconstruction of tomogram datasets. I also had the opportunity to successfully develop other transferable skills such as advanced image processing, data analyses, 3D modelling and visualization. Furthermore, I developed in mentorship skills supervising master and PhD students of Dr Pigino; leadership, project management and administration of third-party funds and dissemination of results and public outreach which has a major impact on scientific communication.
WP3-5: With focus on different model organism (Tetrahymena thermophila): I successfully selected the protein complexes. We developed and optimized a protocol for the axoneme isolation and vitrification on the electron microscopy grids. I successfully localized an important protein complex with potential regulatory function and continuing with another three mutants, I localized another two previously unknown protein complexes.
Secondment: Objectives were achieved partially by the original partner Leica and partially by our new partner Zeiss. However, we had to cancel the onsite training in Vienna due to the organizational changes in Leica.
Our results will be published as original scientific papers and will be presented at different workshops and conferences (namely Meetings of Czechoslovak Microscopy Society in 2018 and 2019, The Scottish Consortium for Macromolecular Imaging in Glasgow on 10-11 September 2018, Dresden Science Night in 2018 and 2019).
