Abstract
The dynamic properties of molecules in living cells are attracting increasing interest. We propose a new method, moving subtrajectory analysis using single-molecule tracking, and demonstrate its utility in the spatiotemporal quantification of not only dynamics but also the kinetics of interactions using single-color images. Combining this technique with three-color simultaneous single-molecule imaging, we quantified the dynamics and kinetics of molecules in spatial relation to T cell receptor (TCR) microclusters, which trigger TCR signaling. CD3ε, a component of the TCR/CD3 complex, and CD45, a phosphatase positively and negatively regulating signaling, were each found in two mobility states: faster (associated) and slower (dissociated) states. Dynamics analysis suggests that the microclusters are loosely composed of heterogeneous nanoregions, possibly surrounded by a weak barrier. Kinetics analysis quantified the association and dissociation rates of interactions with the microclusters. The associations of both CD3ε and CD45 were single-step processes. In contrast, their dissociations were each composed of two components, indicating transient and stable associated states. Inside the microclusters, the association was accelerated, and the stable association was increased. Only CD45 showed acceleration of association at the microcluster boundary, suggesting specific affinity on the boundary. Thus, this method is an innovative and versatile tool for spatiotemporal quantification.
Highlights
Owing to technical developments and an increased number of commercially available instruments, remarkable progress has been made in the elucidation of biological macromolecule dynamics at the single-molecule level[1, 2], providing fundamental insight into the understanding of molecular functions in living cells[3, 4]
T cell receptor (TCR), composed of TCR subunits and CD3 subunits, recognizes antigenic peptides presented by major histocompatibility complex (MHC) molecules
We have introduced a new method of moving subtrajectory analysis to quantify both dynamics and kinetics spatiotemporally
Summary
Owing to technical developments and an increased number of commercially available instruments, remarkable progress has been made in the elucidation of biological macromolecule dynamics at the single-molecule level[1, 2], providing fundamental insight into the understanding of molecular functions in living cells[3, 4]. CD45 negatively regulates signaling by dephosphorylating TCR7, 8. TCR signaling proteins assemble into spatially segregated supramolecular activation clusters (SMAC) at the area of cell contact[9, 10]. A previous study using live cell imaging found that activation causes formation of www.nature.com/scientificreports/. Previous studies using single-molecule tracking on T cell surfaces revealed differences in diffusion coefficients between the inside and outside of lipid rafts[12,13,14]. We have introduced a new method of moving subtrajectory analysis to quantify both dynamics and kinetics spatiotemporally. We applied three-color single-molecule imaging to analyze different kinds of proteins simultaneously. Obtained images were analyzed using moving subtrajectory analysis, and we demonstrated that the new method quantifies dynamics and kinetics in spatial relation to the microclusters
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