In situ imaging of the protein complex Usher 2 involved in hearing and vision

Supervisor Organisation PhD Awarding Entity: phd location
Institut Pasteur
Sorbonne University
Institut Pasteur, Paris, France

Research Focus

The project aims to address the general structure in situ of the native protein complex Usher 2, needed for the development of the cochlea, the organ of hearing, and to elucidate the impact of deafness mutations in pathological processes. The Usher 2 syndrome is the most common form of hereditary hearing-vision loss in humans. We propose to combine cutting edge imaging approaches using cryo-electron tomography (cryoET), cryo-soft-X-ray tomography (cryoSXT) and super-resolution fluorescence microscopy (STED) on mouse cochlea samples to dissect the 3D organization of the Usher 2 complex in its native tissue environment. Combined with the atomic resolution structures already obtained in the lab by integrative approaches (Cryo-EM, NMR and X-ray diffraction), we will then be able to provide a comprehensive view of the Usher 2 complex in situ. Altogether, our structural results from the micro-microscale (cochlea) to the nano- and atomic scale (protein domains) will lead us to propose a model on how Usher 2 proteins and partners get organised in situ to form a protein network essential for the correct Cochlea development. This molecular characterization will have a potential impact on Usher 2 syndrome diagnosis and provide a deeper understanding of its physiopathology.


This project brings together a team of two accomplished researchers with highly complementary expertise to achieve the goals laid out in this proposal. A. Sartori-Rupp has a recognized expertise in experimental cryogenic electron microscopy and tomography acquired in world-class laboratories. She has developed and setup at Institut Pasteur original cryo-CLEM pipelines to address in situ structural biology questions. N. Wolff is expert in protein biochemistry and biophysics, is working since many years on the Usher syndrome and the molecular mechanisms of hearing, and has recently developed advanced workflow of cochlea sample preparations for in situ characterization by microscopies. Preliminary critical data have been already obtained concerning the two approaches of this project to ensure the smooth running of the PhD program (two color STED images, specific antibodies against Usher 2 proteins, dissection, chemical fixation, high-pressure freezing of cochlea and lift-out procedures).

The preparation of samples (cochlea dissection, immunofluorescence, HPF and preparation of tissue blocks), will be supervised by the Wolff’s team as well as the STED acquisition (in collaboration with G. Moneron and D. DiGregorio team), while the acquisition and the processing of the Cryo-electron tomograms will be done under the supervision of A. Sartori-Rupp and the NanoImaging Core Facility (NCF). The NCF – the cryo-EM facility of Institut Pasteur – is equipped with cutting edge systems from sample preparation to microscopy imaging that include all the advanced tools necessary to carry out the project (Glacios 200kV and Titan Krios 300kV cryo-electron microscopes). Therefore, the student will benefit from the experimental and theoretical expertises of the two teams and from the CLEXM consortium (soft-X-ray tomography, hard X-ray) that will work in strong synergy and complementarity to tackle this exciting and ambitious PhD project.

Aim 1

Preparation of samples; cochlea dissection, immunofluorescence, high pressure freezing and preparation of tissue blocks

Aim 2

In situ imaging of ankle links by high resolution fluorescence nanoscopy (STED)

Aim 3

Molecular organization of the Usher 2 protein network in the Cochlea using soft-Xray tomography

Aim 4

Ultrastructural organization of the Usher 2 protein network in the Cochlea using cryo-electron tomography in situ

Pictures Attached

Image description: The Molecular Organization of the Usher 2 complex involved in Hearing.


Role/Focus of PhD:

Ideally, the CLEXM candidate should seek solid foundation in protein chemistry, cell and molecular biology. Previous hands-on experience in EM and/or cryo-EM will be a plus. A genuine enthusiasm for advanced microscopy techniques will be essential for the successful development of the project. The trainee will also spend 1-3 months at laboratories of CLEXM partners for cryo-STX image recording.




Application Deadline: Accepting applications until 31st January 2024