Observing the Confinement Potential of Bacterial Pore-Forming Toxin Receptors Inside Rafts with Nonblinking Eu³⁺-Doped Oxide Nanoparticles

详细信息	查看全文 | 推荐本文 |

• 作者：Silvan Tü ; rkcan^&dagger ; ; ^{silvan.turkcan@polytechnique.edu} ; Jean-Baptiste Masson^&Dagger ; ; Didier Casanova^&dagger ; ; Geneviè ; ve Mialon^§ ; ; Thierry Gacoin^§ ; ; Jean-Pierre Boilot^§ ; ; Michel  ; R. Popoff^¶ ; ; Antigoni Alexandrou^&dagger ; ; ^{antigoni.alexandrou@polytechnique.edu}
• 刊名：Biophysical Journal
• 出版年：2012
• 期刊代码：P13_00063495
• 类别：bio
• 出版时间：16 May, 2012
• 卷：102
• 期：10
• 页码：2299-2308
• 文件大小：1184 K

摘要

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: 伪-toxin of Clostridium septicum and 蔚-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30聽nm for acquisition times of 51.3聽ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 卤 0.05 渭m². Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 卤 0.01 渭m²/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 卤 0.03 pN/渭m at 37掳C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.