Dahal, L. | bioRxiv Science

bioRxiv2023

Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging

Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

biophysics↗

bioRxiv2021

Recovering mixtures of fast diffusing states from short single particle trajectories

Single particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we developed methods to infer distributions of diffusion coefficients from SPT data with short trajectories, variable localization accuracy, and absence of prior knowledge about the number of underlying states. We discuss advantages and disadvantages of these approaches relative to other frameworks for SPT analysis. Significance statementSingle particle tracking (SPT) uses fluorescent probes to track the motions of individual molecules inside living cells, providing biologists with a close view of the cells inner machinery at work. Commonly used SPT imaging approaches, however, result in fragmentation of trajectories into small pieces as the probes move through the microscopes plane of focus. This makes it challenging to extract usable biological information. This paper describes a method to reconstruct an SPT targets dynamic profile from these trajectory fragments. The method builds on previous approaches to provide information about challenging SPT targets without discrete dynamic states while accounting for some known biases, enabling observation of previously hidden features in mammalian SPT experiments.

biophysics↗