Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad
| Autor: | Lexy von Diezmann, Ofer Rog |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Nucleoplasm
010304 chemical physics Chemistry Protein dynamics 010402 general chemistry 01 natural sciences Single Molecule Imaging 0104 chemical sciences Surfaces Coatings and Films Chromatin medicine.anatomical_structure Meiosis 0103 physical sciences Microscopy Materials Chemistry medicine Fluorescence microscope Biophysics Physical and Theoretical Chemistry Nucleus |
| Zdroj: | The Journal of Physical Chemistry B. 125:6162-6170 |
| ISSN: | 1520-5207 1520-6106 |
| DOI: | 10.1021/acs.jpcb.1c03040 |
| Popis: | 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 C. elegans 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. |
| Databáze: | OpenAIRE |
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