Light-based Observation and Control of Cellular Organization

Dahan

Maxime Dahan Team Leader Tel:

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Our group aims at deciphering the interplay of physical and biological mechanisms that underlie the establishment and maintenance of the multiscale architecture of living cells. To this end, our belief is that it is critical to analyze at a molecular scale the mobility of proteins and to properly account for the stochastic mechanisms that govern the behavior of individual molecules. Yet, it must be also recognized that the transfer of information, from the molecular to the cellular scale, is mediated by networks of interactions whose dynamics and organization need to be described and are largely influenced by the properties of their individual molecular constituents.

In this context, our team develop and apply imaging assays using two complimentary approaches:

  1. with single molecule imaging tools, we are probing the structural, stoechiometric and dynamic properties of macromolecular assemblies in live cells,

  2. with magnetic and optogenetic manipulation, we are studying the cell response to controlled perturbations, in order to decipher the molecular circuits ensuring the processing of information and decision-making events.

3D imaging for super-resolution imaging and single-molecule tracking

Figure 1: 3D super-resolution image of actin filaments labeled with a fluorescent actin binding peptide.
Figure 1: 3D super-resolution image of actin filaments labeled with a fluorescent actin binding peptide.

Our group develops innovative optical methods for biological imaging. In the last years, we have focused on techniques for fast and sensitive 3D imaging. We have in particular implemented novel approaches based on adaptive optics and multifocus microscopy for super-resolution imaging and single-molecule tracking in living cells. These optical methods are complemented with efficient computational techniques for the processing and visualization of single molecule data.

Target search of DNA-binding proteins

Figure 2: left: each spot represents a single fluorescently labelled transcription factor in the nucleus of a human cell. Right: reconstructed individual trajectories.
Figure 2: left: each spot represents a single fluorescently labelled transcription factor in the nucleus of a human cell. Right: reconstructed individual trajectories.

DNA-binding proteins need to bind short specific sequences within a large competing pool of non-specific sequences. It is thus necessary to identify the mechanisms by which they rapidly find their target. We have developed Imaging assays to directly track the motion of individual transcription factors in the nucleus of live eukaryotic cells.

Magnetic and optogenetic manipulation of cell polarity

The functional response of a cell rely on the coordinated dynamics of a network of interacting molecules that is able to process the information of internal or environmental cues. It is thus essential to develop experimental methods to probe at a systems level the spatial organization, the connectivity and the kinetics of these networks. In contrast to biochemical, pharmacological or genetic tools that permanently and uniformly affect the biological systems, our goal is to perturb the cell in a temporally – and spatially- controlled manner and to optically record its response. To this end, we are implementing novel assays to manipulate intracellular signaling using functionalized nanoparticles or optogenetic stimulation. These methods are used to probe mechanisms governing cell polarization during migration or division.

 

Selected list of publications

  1. Spencer C. Knight, Liangqi Xie, Wulan Deng, Benjamin Guglielmi, Lana Bosanac, Lea B. Witkowsky, Elisa T. Zhang, Mohamed El Beheiry, Maxime Dahan, Zhe Liu, Jennifer A. Doudna and Robert Tjian, “Dynamics of CRISPR-Cas9 Genome Interrogation in Living Cells”, Science (2015) 350:823-6.
  2. Normanno, L. Boudarene, C. Dugast-Darzacq, J. Chen, C. Richter, F. Proux, O. Benichou, R. Voituriez, X. Darzacq, and M. Dahan, « Probing the target search of single DNA-binding proteins in mamallian cells », Nat. Commun. 6:7357 (2015).
  3. Mohamed El Beheiry, Maxime Dahan and Jean-Baptiste Masson, « InferenceMAP: Whole-cell Mapping of Single-Molecule Dynamics with Bayesian inference », Nature Methods 12, 594–595 (2015).
  4. Bassam Hajj, Jan Wisniewski, Mohamed El Beheiry, Jiji Chen, Andrey Revyakin, Carl Wu, Maxime Dahan, « Whole-cell, multicolor, super-resolution imaging using volumetric, multifocus microscopy », PNAS (2014) 111(49):17480-5.
  5. Jiji Chen, Zhengjian Zhang, Li Li, Bi-Chang Chen, Andrey Revyakin, Bassam Hajj, Wesley Legant, Maxime Dahan, Timothee Lionnet, Eric Betzig, Robert Tjian, and Zhe Liu, “Single-molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells”, Cell (2014) 156, 1274–1285.
  6. Ibrahim Cisse, Ignacio Izeddin, Sebastien Causse, Lydia Boudarene, Adrien Senecal, Leila Muresan, Claire Dugast-Darzacq, Bassam Hajj, Maxime Dahan, Xavier Darzacq, « Real time dynamics of RNA Polymerase II clustering in live human cells », Science (2013) 341, 664-7.
  7. El Beheiry and M. Dahan, “ViSP: tool for visualizing 3D super-resolution data”, Nature Methods (2013) 10, 689–690.
  8. Christian G. Specht, Ignacio Izeddin, Pamela C. Rodriguez, Mohamed El Beheiry, Philippe Rostaing, Xavier Darzacq, Maxime Dahan and Antoine Triller ”Quantitative nanoscopy at inhibitory synapses: counting gephyrin molecules and receptor binding sites”, Neuron (2013) 79, 308–321.
  9. Etoc, D. Lisse, Y. Bellaiche, J. Piehler, M. Coppey, M. Dahan, « Subcellular control of Rac signalling by magnetogenetic manipulation in living cells», Nature Nanotechnology (2013) 8, 193–198.
  10. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Katsov, J. Wisniewski, G. Mizuguchi, P. Soulle, F. Mueller, C. Dugast Darzacq, X. Darzacq, C.Wu, C. I. Bargmann, D. A. Agard, M. Dahan and M.G.L. Gustafsson, “Fast multi-color 3D imaging using aberration corrected multi-focus microscopy”, Nature Methods (2013) 10(1):60-3.
  11. Studer, J. Bobin, M. Chahid, H. Moussavi, E. Candes, M. Dahan, « Compressive Fluorescence Microscopy for Biological and hyperspectral imaging» Proc. Natl. Acad. Sci. USA (2012) 109(26):E1679-87
  12. Pinaud, S. Clarke, A. Sittner, and M. Dahan, “Probing cellular events, one quantum dot a time”, Nature Methods 7, 275-85 (2010).
  13. Bouzigues, M. Morel, A. Triller, M. Dahan, “Asymmetric redistribution of GABAA receptors during GABA gradient sensing by nerve growth cones”, Proc. Natl. Acad. Sci. USA 104, 11251 (2007).
  14. Courty, C. Luccardini, Y. Bellaiche, G. Cappello, M. Dahan, “Tracking individual kinesin motors in living cells using single quantum dot imaging”, Nanolett. 6, 1491-1495 (2006).
  15. Brokmann, J.P. Hermier, G. Messin, P. Desbiolles, J.P. Bouchaud, et M. Dahan, « Statistical Aging and Non-ergodicity in the fluorescence of single nanocrystals », Phys. Rev. Lett. 90, 120601-1 (2003).
  16. Dahan, S. Lévi, C. Luccardini, P. Rostaing, B. Riveau and A. Triller, « Diffusion dynamics of glycine receptors revealed by single quantum dot tracking » Science 302, 442 (2003).
  17. Deniz A., Laurence T., Beligere G., Dahan M., Martin A., Chemla D., Dawson P., Schultz P., and Weiss S., « Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2 »,  Natl. Acad. Sci. USA 97, 5179-5184 (2000).