Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells

• 成果类型: Article
• 署名作者: Valverde-Mendez, Diana; Sunol, Alp M.; Bratton, Benjamin P.; Delarue, Morgan; Hofmann, Jennifer L.; Sheehan, Joseph P.; Gitai, Zemer; Holt, Liam J.; Shaevitz, Joshua W.; Zia, Roseanna N.
• 署名单位: Princeton University; Princeton University; Stanford University; Princeton University; Vanderbilt University; Vanderbilt University; Universite de Toulouse; New York University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13862
• DOI: 10.1073/pnas.2406340121
• 发表日期: 2025-01-28
• 关键词: anomalous subdiffusion; chromosomal loci; rna-polymerase; protein; localization; cytoplasm; ORGANIZATION; dna; transcription; translation

摘要

The crowded bacterial cytoplasm is composed of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial genetically encoded multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-3,240 to +2,700 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears subdiffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent subdiffusion arises from geometrical confinement within the nucleoid and by the cell boundary.