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

Diana Valverde-Mendez; Alp M. Sunol; Benjamin P. Bratton; Morgan Delarue; Jennifer L. Hofmann; Joseph P. Sheehan; Zemer Gitai; Liam J. Holt; Joshua W. Shaevitz; Roseanna N. Zia

doi:10.1073/pnas.2406340121

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

Diana Valverde-Mendez, Alp M. Sunol, Benjamin P. Bratton, Morgan Delarue, Jennifer L. Hofmann, Joseph P. Sheehan, Zemer Gitai, Liam J. Holt, Joshua W. Shaevitz, Roseanna N. Zia

Princeton University

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	American English
Article number	e2406340121
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	122
Issue number	4
DOIs	https://doi.org/10.1073/pnas.2406340121
State	Published - Jan 28 2025

ASJC Scopus subject areas

General

Keywords

anomalous diffusion
bacterial cytoplasm
biophysics
microbiology

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10.1073/pnas.2406340121

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Valverde-Mendez, D., Sunol, A. M., Bratton, B. P., Delarue, M., Hofmann, J. L., Sheehan, J. P., Gitai, Z., Holt, L. J., Shaevitz, J. W., & Zia, R. N. (2025). Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells. Proceedings of the National Academy of Sciences of the United States of America, 122(4), Article e2406340121. https://doi.org/10.1073/pnas.2406340121

@article{11afc568de2549a798aae73ac176be12,

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

abstract = "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.",

keywords = "anomalous diffusion, bacterial cytoplasm, biophysics, microbiology",

author = "Diana Valverde-Mendez and Sunol, {Alp M.} and Bratton, {Benjamin P.} and Morgan Delarue and Hofmann, {Jennifer L.} and Sheehan, {Joseph P.} and Zemer Gitai and Holt, {Liam J.} and Shaevitz, {Joshua W.} and Zia, {Roseanna N.}",

note = "Publisher Copyright: Copyright {\textcopyright} 2025 the Author(s).",

year = "2025",

month = jan,

day = "28",

doi = "10.1073/pnas.2406340121",

language = "American English",

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Valverde-Mendez, D, Sunol, AM, Bratton, BP, Delarue, M, Hofmann, JL, Sheehan, JP, Gitai, Z, Holt, LJ, Shaevitz, JW & Zia, RN 2025, 'Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells', Proceedings of the National Academy of Sciences of the United States of America, vol. 122, no. 4, e2406340121. https://doi.org/10.1073/pnas.2406340121

Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells. / Valverde-Mendez, Diana; Sunol, Alp M.; Bratton, Benjamin P. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 122, No. 4, e2406340121, 28.01.2025.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells

AU - Valverde-Mendez, Diana

AU - Sunol, Alp M.

AU - Bratton, Benjamin P.

AU - Delarue, Morgan

AU - Hofmann, Jennifer L.

AU - Sheehan, Joseph P.

AU - Gitai, Zemer

AU - Holt, Liam J.

AU - Shaevitz, Joshua W.

AU - Zia, Roseanna N.

PY - 2025/1/28

Y1 - 2025/1/28

N2 - 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.

AB - 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.

KW - anomalous diffusion

KW - bacterial cytoplasm

KW - biophysics

KW - microbiology

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U2 - 10.1073/pnas.2406340121

DO - 10.1073/pnas.2406340121

M3 - Article

C2 - 39854229

SN - 0027-8424

VL - 122

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 4

M1 - e2406340121

ER -

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

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