Offre de stage M2 et de thèse financée
Formin conformations during actin filament assembly by cryo-electron microscopy
Formins are key regulators of actin assembly. They promote rapid actin elongation when they are bound to the polymerizing barbed end of actin leading to elongated structures within philopodia or the cytokenetic ring. From microfluidics biophysical assays on single actin filaments (Jegou et al., 2016, nature communication), a model has been proposed which indicates the coexistence of at least two conformations of formins at the end of actin. Two domains of formins are able to bind actin. The FH2 domain is believed to dimerize and encircle actin filaments while the FH1 domain is more flexible. A closed conformation of the FH2 domain would prevent actin elongation while an opened configuration (5 nm away) seems to favor a rapid polymerization of actin.
The purpose of our project (master 2 followed by a thesis) is to determine directly the different assumed conformations of formins bound to actin by cryo-electron microscopy structural studies.
Our purpose is to describe the conformations of formins (1) at the barbed end of actin, (2) throughout depolymerization and (3) alongside actin filaments.
Cryo-EM currently undergoes a resolution revolution that will enable the visualization of such conformational changes. Both the hardware (microscopes and detectors) and software (image processing) now allows the visualization of secondary structures in a physiological environment. We have access to facilities which provide such equipment.
As a first step, the sample preparation conditions will be optimized to guarantee the best possible imaging conditions. Subsequently, data will be collected with our in house microscope using negatively stained samples to get a first model.
Then cryo-EM vitrified samples will be prepared and data will be collected in facilities equipped with the state of the art microscopes.
The project will be carried out in close collaboration with colleagues (G. Romet-lemonne, A. Jegou, Institut Jacques Monod) who will provide the biological material and carry on biophysical assays (microfluidics, magnetic tweezers).
We are looking for candidates with a strong interest in structural biology, a curiosity for Cryo-EM and image processing methods.