Watching single molecules at work in living B cells

Authors

• J Xu
• M Dose
• EM Stevens
• Z Liu
• H Shroff
• D Mazza
• D Straveva
• GL Hager
• R Casellas

Abstract

Recent advances in high-resolution optical imaging make it possible to observe single molecules 'on the job' in living cells. For transcription factors (TFs), this not only enables direct measurements of residence time, but can also reveal other aspects of kinetic behavior. Here we describe an approach to apply this novel method to study B cell biology. To study single molecules of TFs during B cell activation, we generated knock-in mice with HaloTag fused to endogenous JUND or CTCF protein. With JF549-conjugated HaloTag ligand, single molecules were visualized and residence times were gleaned from tracking single molecules across many frames. Surprisingly, we found that the residence times of both JUND and CTCF were reduced by half in activated B cells compare to naive ones, and both proteins needed fewer non-specific trials before finding a specific binding site. On the other hand, the fast diffusion coefficient of both proteins significantly increased, indicating that TFs move faster (more “active”) in activated B cells. Indeed, 3D single molecule tracking revealed that the overall search time, i.e. the time between one specific binding event and the next, was significantly decreased upon B cell activation. We are now trying to explore the role of chromatin remodelers in TFs dynamics during B cell activation. B cells undergo class switch recombination (CSR) at the immunoglobulin (Ig) gene to generate antibodies of different isotypes, which is mediated by activation induced cytidine deaminase (AID). We are also interested in seeing single molecules of AID working during CSR in real time.

Scientific Focus Area: Immunology

This page was last updated on Friday, March 26, 2021