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Accueil > Pages personnelles > Aurélie Dupont

Research

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Interactions of living matter with its physical environment : from the single cell scale to fish schools

 

FRET as a tool to measure biochemistry in real time in living cells, quantitative FRET and biosensors

 
Quantitative FRET imaging and analysis :

The QuanTI-FRET method applied to FRET standards

Being able to measure the biochemical activity of a target protein in a living cell in a spatially and temporally resolved manner is quite a challenge. This is not possible with classical molecular biology methods, however, new tools have recently emerged, namely fluorescent biosensors. Most of them are based on the principle of "Förster Resonance Energy Transfer" (FRET), i.e. the transfer of energy between two fluorophores allowing to probe distances of the order of a few nanometers and thus changes in protein conformation. These biosensors have enormous potential but their development is hampered on the one hand by the difficulty to develop them and on the other hand by the difficulty of measuring FRET reliably in living cells. To unblock this last point, we developed a new method for measuring and quantitatively analysing FRET during Alexis Coullomb’s thesis, in collaboration with the teams of Don Lamb (LMU Munich) and Corinne Albigès-Rizo (IAB Grenoble). Starting again from the physical equations of fluorescence signal acquisition, we have refreshed the theoretical framework and proposed a new calibration method that allows the measurement of absolute FRET values and thus opening the way to new experiments. This work has been sponsored by the ANR (PDOC DeMeTr 2013).
 

  • "QuanTI-FRET : a framework for quantitative FRET measurements in living cells". Alexis Coullomb, Cécile M Bidan, Chen Qian, Fabian Wehnekamp, Christiane Oddou, Corinne Albigès-Rizo, Don C Lamb, Aurélie Dupont. Scientific Reports 10 (1), 1-11 (2020)
  • This method allowing quantitative FRET measurements in living cells is being transfered to an industrial partner thanks to a grant from the SATT Linksium.

     
     

Application to FRET-based biosensors, the AMPK example :
In collaboration with Uwe Schlattner’s team (LBFA, Grenoble), we are working on using the QuanTI-FRET method to measure the AMPfret biosensor in living cells. This is a biosensor based on the protein kinase AMPK that reports the AMP/ADP ratio within the cell. Hence, the energy state of living cells can be measured in a time and space resolved manner. The difficulty here is that AMPK consists of different subunits and the two fluorophores involved in FRET are not bound to the same subunit. The donor-acceptor stoichiometry is therefore not well defined and requires analytical developments allowed within the framework of the QuanTI-FRET method. This work is funded by the ANR (PRC betaFRET 2021).
 

  • “Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism”. Martin Pelosse, Cécile Cottet-Rousselle, Cécile M Bidan, Aurélie Dupont, Kapil Gupta, Imre Berger, Uwe Schlattner. Nature communications 10 (1), 1-13 (2019)
       

    B. Collective effects in complex environments.

     

A new research axis has started in 2020 with P Peyla (numerical simulation and theory in fluid mechanics, MoVE LIPhy team) and C Graff (ethology, LPNC, Grenoble) and then T Métivet (INRIA, Grenoble). We are interested in the spontaneous emergence of an ordered movement in a system composed of a large number of individuals. This intriguing and almost universal phenomenon can be found in bacteria on a sub-millimetre scale, in schools of fish stretching for kilometres, in human crowds or in flocks of birds. These collective movements result from local interactions between individuals from which large-scale patterns emerge. We approach this topic in an original way by seeking to understand the effect of a complex physical environment (flows, obstacles) on the collective swimming behaviour of small aquarium fish (Blue Neon, Paracheirodon innesi). In particular, we aim to investigate the coupling between their social interactions and their hydrodynamic interactions, which have so far mainly been studied separately. To this end, we combine an experimental approach in a controlled environment providing quantitative measurements with a numerical approach coupling the direct resolution of the 3D hydrodynamics with a cognitive model (P. Peyla, T. Métivet INRIA). In a first step, we wish to test the "faster-is-slower" effect of congestion at the passage of a constriction with this system in a liquid medium. This phenomenon of intermittent blockage is found both in the flow of granular and colloid materials and in the evacuation of crowds or herds. More complex experiments controlling the flow and the positioning of obstacles are being developed. This work is funded by the ANR (PRC FISHSIF 2021).
 
coming soon : we are organising a workshop in Cargèse in 2024 on collective movements in the living and in robots

  • "Collective orientation of an immobile fish school and effect on rheotaxis" Renaud Larrieu , Catherine Quilliet, Aurélie Dupont, and Philippe Peyla. Phys. Rev. E 103, 022137 (2021)

     


Past projects

 
Magneto-active substrates for mechanotransduction :

Magneto-active substrates : stimulation of living fibroblasts with concomitant force measurement.

Cells can sense the physical properties of their environment through a mechanism called mechanotransduction and accordingly modulate essential functions such as motility, adhesion or differentiation. Molecular interactions are now well described, showing very complex networks, however the coordination in space and time of mechanical and biochemical signals is not yet clear. The main goal is to understand this mechano-chemical coupling at the scale of an individual cell with an experimental approach. In order to be able to mechanically stimulate a living cell and simultaneously read its biochemical response, with a good spatio-temporal resolution (1µm , 0.1s), we have developed innovative methods : a quantitative method for measuring FRET-based biosensors and unique activable substrates.
 
The experimental objective was to impose a mechanical stress on the scale of a single cell in a local and dynamic way while trying to remain as physiological as possible, i.e. via a continuous substrate. To meet these specifications, we therefore invented a new experimental device : magneto-active substrates. In collaboration with Martial Balland (MicroTiss team) and Nora Dempsey (Institut Néel, Grenoble), Cécile Bidan (post-doc) succeeded in making a deformable substrate on which cells can adhere. It consists of magnetic inclusions, iron micro-pillars, in a thin layer of elastomer (PDMS). With a pair of electromagnets, we can apply a mechanical stress to the cells via their adhesions and also measure the traction forces exerted by the cells.
 

  • "Magneto-active substrates for local mechanical stimulation of living cells". Cécile M Bidan, Mario Fratzl, Alexis Coullomb, Philippe Moreau, Alain H Lombard, Irène Wang, Martial Balland, Thomas Boudou, Nora M Dempsey, Thibaut Devillers, Aurélie Dupont. Scientific Reports 8 (1), 1-13 (2018)

 

Collaborations

 

  • Philippe Peyla, LIPhy, Grenoble
  • Christian Graff, LPNC, Grenoble
  • Thibaut Métivet, INRIA, Grenoble
  • Uwe Schlattner, LBFA, Grenoble
  • Nora Dempsey et Thibaut Devillers, Institut Néel, Grenoble
  • Corinne Albigès-Rizo et Olivier Destaing, IAB, Grenoble
  • Martial Balland, Thomas Boudou, LIPhy, Grenoble
  • Alice Nicolas, LTM, Grenoble
  • Don C Lamb, LMU, Munich
  • Joachim Rädler, LMU, Munich
  • Erwin Frey, LMU, Munich
     
     

 

Current group

 

  • Renaud Larrieu, PhD candidate, supervised with Philippe Peyla, team MOVE, LIPhy
  • Océane Terral, PhD candidate, supervised with Cécile Delacour, Institut Néel
     
     

Alumni

 

  • Julien Leblanc, research engineer, 2020-2021
  • Alain Lombard : PhD candidate 2017-2020, now teacher
  • Alexis Coullomb : PhD candidate 2015-2018, now postdoc in Toulouse
  • Cécile Bidan : postdoc 2014-2017, now junior group leader MPI Potsdam, Germany
  • Sadequa Sultana : postdoc 2017, now biomedical research engineer