Single-Molecule Tracking of Chromatin-Associated Proteins in the Gonad.

Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multisecond trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromosomes within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this approach.