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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TRAIN SPOTTING (Mapping the Intraflagellar Transport - A High-resolution Study of Intraflagellar Transport Trains in Chlamydomonas Cells)

Teaser

Summary

I spent the whole project period at MPI-CBG Dresden by an intensive work and tight collaboration with the project host, Gaia Pigino, and her group. Even though the project objectives have slightly changed in comparison to the project proposal, we were able to collect results for two full original research papers which are now in preparation.

Objectives of the project TRAINSPOTTING were setup both for my career development (WP1 and WP2) and research (WP3-5). Even though the research work packages have partially changed, we substituted them with objectives which were using identical methodological setup and focused on the structural and functional analysis of a difference ciliary component than IFT. The new protein complex investigated is also a fundamental constituent of all motile cilia/flagella and is required for the assembly of functional cilia/flagella. Therefore, the newly identified objectives had similarly significant research impact to the one proposed in the original application.

These objectives were fully addressed. I was able to propose independently the scientific question and design the experiment. With a material science background, I am now fully capable to plan and carry out the biologically oriented research. I am fully independent at setting up and conducting
cryo-ET experiments and we were able to test the newest cryo-CLEM setup provided by Leica and Zeiss. I mastered the 3D reconstruction of tomogram datasets with advanced approaches, filtering, etc. I was fully capable of processing the data, model and visualize them. I proved to be able to manage the project and get everything done.

The project has achieved most of its objectives and milestones for the period, with relatively minor deviations. We fully succeeded to achieve the deliverables described in WP1 and WP2. The research work packages 3-5 were changed with regard to the fact that the project started almost two years after it was planned. We therefore revised the research objectives and formulated new goals.

Work performed

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 â€“ 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 â€“ 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).

Final results

Results of this project will be of particular interest to researchers working in connected fields, such as medicine or molecular biology. The exact location and function of more than 50% of axonemal proteins is still unclear. Highly progressive methods of cryo-EM coupled with advanced computational approaches is able to deliver valuable information for the ciliary research. This field combines findings from cellular and structural biology and is critically important for development in treatment of ciliopathies, further research in cellular transport and other related fields. In summary, the project complements ongoing projects in Europe, and significantly develops the field of cilia structure and function and the utilization of cryo-CLEM for high resolution structural studies. Because of the unique cutting-edge methods, obtained results of this research project will
also enhance the competitiveness of basic cell biology research in the EU.