Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles.
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: α-toxin of Clostridium septicum and ε-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 nm for acquisition times of 51.3 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 ± 0.05 μm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm(2)/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 ± 0.03 pN/μm at 37°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.
Laboratoire d'Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U696, Palaiseau, France. silvan.turkcan@polytechnique.edu
Silvan
S
Türkcan
Jean-Baptiste
JB
Masson
Didier
D
Casanova
Geneviève
G
Mialon
Thierry
T
Gacoin
Jean-Pierre
JP
Boilot
Michel R
MR
Popoff
Antigoni
A
Alexandrou
D000199:Actins / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000494:pharmacology; D004285:Dogs; D005063:Europium / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry* / Q000187:drug effects; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D009038:Motion; D053758:Nanoparticles / Q000737:chemistry*; D010087:Oxides / Q000737:chemistry*; D058105:Polymerization / Q000187:drug effects; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013109:Sphingomyelins / Q000378:metabolism; D013816:Thermodynamics; D053778:Thiazolidines / Q000494:pharmacology
D000199:Actins; D001427:Bacterial Toxins; D019086:Bridged Bicyclo Compounds, Heterocyclic; C039811:Clostridium perfringens epsilon-toxin; D010087:Oxides; D052899:Pore Forming Cytotoxic Proteins; D011956:Receptors, Cell Surface; D013109:Sphingomyelins; D053778:Thiazolidines; C064270:hemolytic toxin, Clostridium septicum; D005063:Europium; D002784:Cholesterol; D002789:Cholesterol Oxidase; D013108:Sphingomyelin Phosphodiesterase; C037068:latrunculin B