Investigation des Interactions Toxine-Cellule et du Confinement des Récepteurs dans la Membrane Cellulaire

Abstract : The cell membrane is the interface of communication and exchange between the cell and the outside world. As such, its structure and composition is of integral importance to the cell’s continued survival. The proteins within the membrane provide the necessary functionalities to the membrane for successfully acting out its role. The membrane receptors experience a highly heterogeneous environment in the cell membrane that enhances their efficiency. We studied this environment via single particle tracking of cell membrane receptors tagged with luminescent lanthanide-doped nanoparticles. The nanoparticles provided a continuous, uninterrupted signal of the movements, yielding trajectories of several minutes. We then used a method based on statistical Bayesian inference to analyse and compare the trajectories obtained and, hence, extract a confinement potential of arbitrary shape. We first validated the Bayesian inference approach by demonstrating that this method can also be used to calibrate an optical tweezers setup. Furthermore, we showed that this method outperforms established calibration methods for optical traps. We then applied this approach to the confined trajectories of epsilon-toxin (produced by Clostridium perfringens) receptors in Madin-Darby canine kidney cells. In particular, we studied the evolution of the confinement potential and diffusivity within the domains upon addition of domain-destabilizing agents, as well as the occasional ‘hopping’ events, during which receptors are seen to hop into an adjacent confinement domain, and the associated 'hopping' energies. Additionally, we inquired into the effect of an externally applied force, implemented via a hydrodynamic flow on the receptors, and discovered an actindependent confinement of the microdomains in addition to the lipid-dependent confinement. To further investigate the nature of membrane receptor confinement, we classified the potentials obtained from raft and non-raft proteins using a decisiontree method and a clustering algorithm. The results showed that non-raft proteins reside in domains that produce a steeper potential boundary with a flatter potential in the centre of the domain as compared to raft proteins. Finally, we extended the single-particle tracking of toxins to three dimensions by registering the width of the point-spread function of the nanoparticle signal. In this way, we were able to observe the internalization trajectory of a heavy-chain segment of the botulinum toxin A of Clostridium botulinum in cells of the intestinal mouse cell line m-ICcl2.
Complete list of metadatas

https://pastel.archives-ouvertes.fr/tel-01179545
Contributor : Maximilian Richly <>
Submitted on : Wednesday, July 22, 2015 - 6:24:08 PM
Last modification on : Wednesday, January 23, 2019 - 10:28:59 AM
Long-term archiving on : Friday, October 23, 2015 - 11:31:53 AM

Identifiers

  • HAL Id : tel-01179545, version 1

Citation

Maximilian U. Richly. Investigation des Interactions Toxine-Cellule et du Confinement des Récepteurs dans la Membrane Cellulaire. Biophysique [physics.bio-ph]. Ecole Doctorale Polytechnique, 2015. Français. ⟨tel-01179545⟩

Share

Metrics

Record views

592

Files downloads

1230