Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies DOI Creative Commons
Elizaveta Korunova, Vitali Sikirzhytski, Jeffery L. Twiss

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 18, 2024

Abstract Single particle tracking (SPT) is a powerful technique for probing the diverse physical properties of cytoplasm. Genetically encoded nanoparticles provide an especially convenient tool such investigations, as they can be expressed and tracked in cells via fluorescence. Among these, 40-nm GEMs unique opportunity to explore Their size corresponds that ribosomes big protein complexes, allowing us investigate effects cytoplasm on diffusivity these objects while excluding influence chemical interactions during stressful events pathological conditions. However, it has been shown cytoplasmic viscosity tightly regulated plays crucial role maintaining homeostasis synthesis degradation. Despite this, GEM expression levels remain largely uncharacterized mammalian cells. To optimize estimate GEMs-expression we constructed dox-inducible system compare with previously reported constitutive system. The optimized level increases measured from 0.29 ± 0.02 μm 2 /sec GEMs-overexpressed 0.35 /sec; improve homogeneity throughout cell population; facilitates tracking. We also improved analyses by applying effective diffusion coefficient considering type motion assessing heterogeneity calculating standard deviations displacements. Statement significance Describing properties, environmental complex motion, essential understanding molecular-level changes function pathology. A recently developed approach uses self-assembling fluorescent probes, cells, through single-particle (SPT). One employs genetically multimeric (GEM) nanoparticles— scaffold structures similar ribosomes. This study addresses key limitation SPT examining how varying affect quality Our findings demonstrate controlled reduces overcrowding, diffusivity, enhances track detection. work contributes valuable insights into optimizing nanoparticle applications studying dynamics.

Language: Английский

How it feels in a cell DOI Creative Commons
Martina Bonucci, Tong Shu, Liam J. Holt

et al.

Trends in Cell Biology, Journal Year: 2023, Volume and Issue: 33(11), P. 924 - 938

Published: June 5, 2023

Language: Английский

Citations

26
Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation DOI
Yusheng Shen, Kassandra M. Ori‐McKenney

Developmental Cell, Journal Year: 2024, Volume and Issue: 59(12), P. 1553 - 1570.e7

Published: April 3, 2024

Language: Английский

Citations

11
A guide to single-particle tracking DOI
François Simon, Lucien E. Weiss, Sven van Teeffelen

et al.

Nature Reviews Methods Primers, Journal Year: 2024, Volume and Issue: 4(1)

Published: Sept. 12, 2024

Language: Английский

Citations

9
Viscosity-dependent control of protein synthesis and degradation DOI Creative Commons
Yuping Chen, Jo‐Hsi Huang,

Connie Phong

et al.

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: March 8, 2024

Abstract It has been proposed that the concentration of proteins in cytoplasm maximizes speed important biochemical reactions. Here we have used Xenopus egg extracts, which can be diluted or concentrated to yield a range cytoplasmic protein concentrations, test effect on mRNA translation and degradation. We find synthesis rates are maximal ~1x cytoplasm, whereas degradation continues rise higher optimal ~1.8x. show this difference optima attributed greater sensitivity viscosity. The different could produce negative feedback homeostatic system, where increasing above 1x physiological level increases viscosity selectively inhibits drives system back toward set point.

Language: Английский

Citations

7
Cytoplasmic fluidization contributes to breaking spore dormancy in fission yeast DOI Creative Commons
Keiichiro Sakai, Yohei Kondo, Yuhei Goto

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(26)

Published: June 18, 2024

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested changes in the biophysical properties of play key role homeostasis adaptation. However, it still remains unclear how cells control their cytoplasmic response to environmental cues. Here, we used fission yeast spores as model system dormant elucidate mechanisms underlying regulation properties. By tracking fluorescent tracer particles, found particle mobility decreased compared vegetative rapidly increased at onset dormancy breaking upon glucose addition. This fluidization depended on glucose-sensing via cyclic adenosine monophosphate-protein kinase A pathway. PKA activation led trehalose degradation through trehalase Ntp1, thereby increasing amount decreased. In contrast, rapid did not require de novo synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, measurement diffusion coefficients with particles different sizes suggests spore impedes movement larger complexes (40 150 nm) such ribosomes, while allowing free smaller molecules (~3 second messengers signaling proteins. Our experiments thus uncovered series events enable quickly fluidize breaking.

Language: Английский

Citations

7
Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes DOI Creative Commons
Abin Biswas,

Omar Muñoz,

Kyoohyun Kim

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 6, 2023

Abstract The packing and confinement of macromolecules in the cytoplasm nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary affect physiology remains largely unknown. Here, we show that nucleus is less dense than living systems establish maintain a constant ratio between these compartments. Using label-free biophotonics theory, nuclear set by pressure balance across envelope vitro , vivo during early development. Nuclear transport establishes specific proteome exerts colloid osmotic pressure, which, assisted entropic chromatin draws water into nucleus. C. elegans while nuclear-to-cytoplasmic (N/C) volume ratios change development, N/C robustly maintained. We propose maintenance biophysical driver one oldest tenets cell biology: ratio. In summary, this study reveals previously unidentified homeostatic coupling macromolecular densities drives organization with pathophysiologies such as senescence cancer.

Language: Английский

Citations

12
Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm DOI Creative Commons

Theodoros Pittas,

Weiyan Zuo,

Arnold J. Boersma

et al.

iScience, Journal Year: 2023, Volume and Issue: 26(4), P. 106367 - 106367

Published: March 9, 2023

The intracellular milieu is crowded with biomacromolecules. Macromolecular crowding changes the interactions, diffusion, and conformations of Changes in have been mostly ascribed to differences biomacromolecule concentration. However, spatial organization these molecules should play a significant role effects. Here, we find that cell wall damage causes increased effects Escherichia coli cytoplasm. Using genetically encoded macromolecular sensor, see spheroplasts penicillin-treated cells well surpass obtained using hyperosmotic stress. increase not because osmotic pressure, shape, or volume therefore crowder Instead, nucleic acid stain DNA show cytoplasmic mixing nucleoid expansion, which could cause Our data demonstrate alters biochemical cytoplasm induces conformational probe protein.

Language: Английский

Citations

11
Mesoscale molecular assembly is favored by the active, crowded cytoplasm DOI Creative Commons
Tong Shu, Gaurav Mitra,

Jonathan Alberts

et al.

PRX Life, Journal Year: 2024, Volume and Issue: 2(3)

Published: July 10, 2024

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism rapid spatiotemporal control in cells. Principles condensation have been revealed through vitro reconstitution. However, intracellular environments are much more complex than test-tube environments: they viscoelastic, highly crowded at the mesoscale, and far from thermodynamic equilibrium due to constant action energy-consuming processes. We developed synDrops, synthetic phase separation system, study how cellular environment affects condensate formation. Three features enable physical analysis: synDrops inducible, bioorthogonal, well-defined geometry. This design allows kinetic analysis synDrop assembly facilitates computational simulation process. compared experiments simulations determine that macromolecular crowding promotes nucleation but inhibits droplet growth coalescence. ATP-dependent activities help overcome frustration growth. In particular, stirring cytoplasm by actomyosin dynamics dominant potentiates mammalian reducing confinement elasticity. Our results demonstrate molecular favored combined effects active matter cytoplasm. These move toward better predictive understanding formation vivo.

Language: Английский

Citations

4
Dynamic structure of the cytoplasm DOI Creative Commons
Hyo-Jun Kim, Morgan Delarue

Current Opinion in Cell Biology, Journal Year: 2025, Volume and Issue: 94, P. 102507 - 102507

Published: April 6, 2025

The cytoplasm is a dense and complex milieu in which plethora of biochemical reactions occur. Its structure not understood so far, albeit being central to cellular functioning. In this review, we highlight novel perspective the physical properties are regulated space time actively contribute function. Furthermore, underscore recent findings that dynamic formation local assemblies within cytoplasm, such as condensates polysomes, serves key regulator mesoscale cytoplasmic dynamics.

Language: Английский

Citations

0
Giant KASH proteins and ribosomes synergistically establish cytoplasmic biophysical properties in vivo DOI Creative Commons
Xin Shun Ding, Hongyan Hao, Daniel Elnatan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 12, 2025

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While cytoplasmic macromolecular crowding affects multiple cellular processes in single cells, its regulation living animals poorly understood. Using genetically encoded multimeric nanoparticles for vivo rheology, we discovered that C. elegans tissues maintain distinct differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling diffusion: ribosome concentration, known regulator of crowding, works concert with previously unknown function the giant KASH protein ANC-1 scaffolding endoplasmic reticulum. These findings reveal by which establish properties, implications understanding organization species. Living unique intracellular under constraints crowding.

Language: Английский

Citations

0