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Reference : UMR7104-GUEBUR-007
Workplace : ILLKIRCH GRAFFENSTADEN
Date of publication : Friday, June 26, 2020
Scientific Responsible name : Patrick SCHULTZ
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 September 2020
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
Characterization of nanometric probes for cryo electron tomography.
Important biological properties such as membrane-less compartmentalization, phase separation, condensate formation, weak or transient interaction are only occurring in the crowded environment of the intact cell. A current challenge is to identify key functional sites for transcription within the cell nucleus, and to determine their structure and molecular environment.
Cryo-Electron Microscopy approaches have been developed to visualize molecular assemblies in their cellular environment1. Three-dimensional (3-D) reconstruction of the cell is performed by cryo-Electron Tomography (cryo-ET) of thin cellular slices which are produced by cryo-sectioning the samples2 or by using a focused ion beam (FIB) to mill thin lamellae3. Cryo-ET reconstructions have revealed large molecular assemblies, such as mitochondria, ribosomes, nuclear pore complexes or cytoskeleton in molecular details1.
Molecular complexes with low cell abundance or too small to be recognized by their shape must be identified using a dense electron probe detectable by electron microscopy. We have synthesized gold-based probes that can be delivered into living cells. These gold nanoparticles are coupled with antibody derivatives to selectively label target proteins.
The objective of the thesis is to reveal gold particles in cell sections and tomograms to detect rare protein complexes such as RNA polymerase II molecules, to determine their spatial distribution and to identify their cellular interaction partners.
A graduate student with a background in physics or cell biology would benefit from interdisciplinary training in biological sample preparation, FIB/SEM imaging and data interpretation. The first part of the thesis will be devoted to study the state of the art of in situ labelling approaches, and to get familiar with the instruments. A second part of the thesis will be devoted to the characterization of newly synthesized electron dense markers and their behaviour in a cellular environment. In a third part, the student will use electron-dense nanoparticles conjugated with probes recognizing RNA polymerase II. He/she will experiment data collection on a Titan cryo-electron microscope in both standard imaging mode and cryo-electron tomography mode.
1 Mahamid, J. et al. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science 351, 969-972 (2016).
2 Al-Amoudi, A., Studer, D. & Dubochet, J. Cutting artefacts and cutting process in vitreous sections for cryo-EM. J Struct Biol 150, 109-121 (2005).
3 Marko, M. et al. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat Methods 4, 215-217 (2007).
The research project will take place under the sponsorship of the CNRS, the largest research institute in Europe, and more specifically within the framework of the interdisciplinary 80prime programme of the CNRS. This programme aims to develop innovative scientific research at the interface between different scientific fields in physics, chemistry and biological sciences. The candidate will preferably be affiliated to the Doctoral School of Life and Health Sciences of the University of Strasbourg (ED 414) but depending on his/her initial training, the student could also be affiliated to ED222 (Chemical Sciences) or ED182 (Physics and Chemistry-Physics).
The student will conduct his research on the Illkirch campus in the structural biology group of the IGBMC (UMR7104- http://www.igbmc.fr/) directed by Patrick Schultz. The Institute of Genetics and Molecular and Cellular Biology (IGBMC) is today one of the main research centers in Europe in this field. In France, it is the largest research unit that brings together Inserm, CNRS and the University of Strasbourg. The aim of the institute is to develop transdisciplinary research at the interface of biology, biochemistry, physics and medicine, but also to attract students from all over the world by offering very high-level training in the field of biomedical sciences. The IGBMC campus is located on the Illkirch Innovation Park in the suburbs of Strasbourg, an exceptional academic and industrial scientific environment that greatly favours collaborations and technology transfer.
Within the interdisciplinary framework of the project, the student will actively collaborate with the ESBS Chemistry-Biology group led by Guy Zuber (UMR7242, https://esbs.unistra.fr/recherche/) and the IPCMS High Resolution Imaging group led by Ovidiu Ersen (UMR 7504, http://www.ipcms.unistra.fr/). It will benefit from an exceptional scientific environment supported in particular by the national Frisbi structural biology infrastructure which deploys state-of-the-art instrumentation for the national and international community. For this project in particular, the candidate will have access to two cryo transmission electron microscopes, including a titan krios, a control transmission electron microscope and a scanning electron microscope coupled to a focused ion beam (FIB/SEM).
Constraints and risks
There are no particular constraints or specific risks related to the realization of this research project. The research institutes participating in the project are located in the Strasbourg conurbation and travel between the different sites is short.
Master scientific and technical English in the field, in written and spoken form.
To be able to communicate, pass on knowledge and present results.
Be able to work in a team.
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