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Investigation of the Cell Membrane Architecture by Single-Molecule Tracking of Peptidic Toxins

Abstract : The cellular membrane is a vital part of the cell, which plays a crucial role in many cellular processes, such as, signaling and trafficking, and pathologies. This thesis aims to investigate the architecture of the cell membrane. The study uses the motion of two membrane receptors that are exploited by bacterial toxins to probe the architecture. Advances in light microscopy techniques have shown that many membrane receptors do not diffuse freely in the membrane, but undergo confined or anomalous diffusion. Currently a few models compete to explain the confinement of the receptors, such as the Picket-Fence model, lipid rafts and protein aggregates. To investigate the membrane, lanthanide doped nanoparticles (Y0.6Eu0.4VO4) are coupled to two different peptidic pore-forming toxins, the α-toxin of C. septicum and the ǫ-toxin of C. perfingens. Single molecule tracking of receptor bound labeled toxins in the apical membrane of MDCK cells in a wide-field microscope reveals the receptor motion with sub-diffraction resolution of down to 10 nm. The α & ǫ-toxin receptors both undergo confined diffusion with similar diffusion coefficients of 0.16 ± 0.14 µm2/s in temporaly stable domains of 0.5 µm2. To analyze the receptor trajectories, we intro- duced a novel approach based on an inference method. Our only assumption is that the receptor moves according to the Langevin equation of motion. This method exploits the information of the ensemble of the trajectory and the quality of the extracted values is verified through simulations. Both receptors are confined in a spring-like potential with a spring constant of 0.45 pN/µm. Tracking after cholesterol depletion by cholesterol ox- idase and cytoskeleton depolymerization by Latrunculin B, shows that confinement of single receptors is cholesterol dependent and actin depolymerization does not influence the confinement. Using the nanoparticle labels as a hydrodynamic force amplifier in a liquid flow, tests the response of the receptor to an external force and indicates attach- ment of the confining domains to the cytoskeleton. Finally, a model for the confinement of the receptor is proposed, based on the hydrophobic coupling of the receptor and the surrounding bilayer which can explain the spring-like potential of the confining domain.
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Contributor : Silvan Turkcan Connect in order to contact the contributor
Submitted on : Monday, July 25, 2011 - 3:57:29 PM
Last modification on : Friday, October 23, 2020 - 4:36:22 PM
Long-term archiving on: : Wednesday, October 26, 2011 - 2:22:47 AM


  • HAL Id : tel-00608124, version 2



Silvan Türkcan. Investigation of the Cell Membrane Architecture by Single-Molecule Tracking of Peptidic Toxins. Physics [physics]. Ecole Polytechnique X, 2010. English. ⟨tel-00608124v2⟩



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