Reference : PhD Subject
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. Tri-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 of low cellular abundance or too small to be recognized by their shape need to be labelled with an electron dense marker to be detected. To address this challenge, we synthesized gold-based probes that can be delivered into living cells. The gold nanoparticles are coupled to antibody derivatives to selectively label the target proteins.
The objective of this thesis is reveal the gold particles in cell sections and in cryo-ET 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 in cell biology would benefit from an interdisciplinary training in biological sample preparation, FIB/SEM imaging, and data interpretation. The first part of the thesis will be devoted to study of the state of the art of in situ labelling approaches, and in getting familiar with the instrumentation. A second part of the thesis will be dedicated to the characterization of newly synthetized electron-dense markers and of their behavior in a cellular environment. In a third part the student will use the most promising electron dense nanoparticles conjugated to probes recognizing RNA polymerase II. He will experience data collection on the Titan Cryo-electron microscope both in standard imaging mode and in cryo electron tomography mode. The student will conduct his research on the Illkirch campus and will share his time between the structural biology group of the IGBMC, the chemobiology group of the ESBS and the high resolution imaging group of the IPCMS.
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).
Application Deadline : Nov. 30, 2020