Molecular sociology of virus-induced cellular condensates supporting reovirus assembly and replication DOI Creative Commons
X. Liu, Xian Xia,

Michael W. Martynowycz

et al.

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

Published: Dec. 6, 2024

Abstract Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) focused ion beam (FIB) milling-produced lamellae mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) host virus in 3D at two time points post-infection, enabling a detailed description these condensates mechanistic understanding MRV within them. Expanding over time, condensate fashions ribosomes its periphery, microtubules, lipid membranes, molecules interior, forming architecture that supports dynamic processes genome capsid assembly. A total six assembly intermediates identified inside condensate: star core, empty genome-containing cores, full virions, outer shell particle. Except for atomic resolution microscopy (cryoEM) extracts. The temporal sequence spatial rearrangement among choreograph life cycle condensates. Together, sociology MRV-induced highlights functional advantage transient enrichment right location replication.

Language: Английский

The dark side of fluorescent protein tagging – the impact of protein tags on biomolecular condensation DOI
Edoardo Fatti,

Sarah Khawaja,

Karsten Weis

et al.

Molecular Biology of the Cell, Journal Year: 2025, Volume and Issue: 36(3)

Published: Jan. 29, 2025

Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used study and properties condensates in vitro vivo, but there is evidence that may perturb proteins. In this study, we carefully assess effects on yeast DEAD-box ATPase Dhh1, a central regulator processing bodies (P-bodies), which are biomolecular involved mRNA metabolism. We show fluorescent well polyhistidine tag greatly affect Dhh1 lead with different dynamic properties. Tagging proteins vivo alters number P-bodies upon glucose starvation some even constitutive nonstressed cells. These data raise concerns about accuracy tagged experiments, highlighting need for caution when interpreting results.

Language: Английский

Citations

2
Scanning probe microscopy elucidates gelation and rejuvenation of biomolecular condensates DOI Creative Commons
Aida Naghilou, Oskar Armbruster, Alireza Mashaghi

et al.

Cell Reports Physical Science, Journal Year: 2025, Volume and Issue: unknown, P. 102430 - 102430

Published: Feb. 1, 2025

Language: Английский

Citations

1
Tag with Caution - How protein tagging influences the formation of condensates DOI Creative Commons
Kerstin Dörner, Michelle Gut, Daan Overwijn

et al.

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

Published: Oct. 5, 2024

Summary Fluorescent proteins and peptide tags are essential tools in cellular biology, but can alter the biochemical properties of target proteins. Biomolecular condensates, which have emerged as key principles organization, suggested to provide robustness cells, yet they also respond sensitively small changes environmental conditions—or tagging their components, our findings suggest. Here, we investigated effects sixteen widely used on condensate formation various model organisms, vitro , cells by computational modelling. We find that strongly influenced condensation for some proteins, while others remained unaffected. Effects varied, with enhancing decreasing condensation, depended protein being tagged. Coarse-grained simulations suggest charge fluorescent is a critical factor modulating behavior. Together, results underscore importance rigorous experimental design interpretation experiments.

Language: Английский

Citations

5
The dark side of fluorescent protein tagging: the impact of protein tags on biomolecular condensation DOI Open Access
Edoardo Fatti,

Sarah Khawaja,

Karsten Weis

et al.

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

Published: Nov. 24, 2024

Biomolecular condensation has emerged as an important mechanism to control various cellular processes through the formation of membraneless organelles. Fluorescent protein tags have been extensively used study and properties condensates in vitro vivo, but there is evidence that may perturb proteins. In this study, we carefully assess effects on yeast DEAD-box ATPase Dhh1, a central regulator processing bodies (P-bodies), which are biomolecular involved mRNA metabolism. We show fluorescent well poly-histidine tag greatly affect Dhh1 lead with different dynamic properties. Tagging proteins vivo alters number P-bodies upon glucose starvation some even constitutive non-stressed cells. These data raise concerns about accuracy tagged experiments, highlighting need for caution when interpreting results.

Language: Английский

Citations

2
Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates DOI

Leyla E. Fahim,

Joshua M. Marcus, Noah D. Powell

et al.

The Journal of Cell Biology, Journal Year: 2024, Volume and Issue: 224(1)

Published: Oct. 14, 2024

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering segmentation to resolve Condensate-specific lifetimes were used track protein–protein interactions by measuring FLIM–Förster resonance energy transfer (FRET). FLIM-FRET evaluate whether mRNA decapping complex subunits can form decapping-competent within P-bodies. Condensate revealed presence core subunit P-bodies under basal conditions disruption between enzyme (Dcp2) a critical cofactor (Dcp1A) during oxidative stress. Our results show context-dependent plasticity P-body interaction network, which be rewired minutes in response stimuli. Together, our FLIM-based approaches provide investigators an automated rigorous method uncover essential dynamics RNP live cells.

Language: Английский

Citations

1
Scanning probe microscopy elucidates gelation and rejuvenation of biomolecular condensates DOI Creative Commons
Aida Naghilou, Oskar Armbruster, Alireza Mashaghi

et al.

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

Published: Aug. 29, 2024

Abstract Comprehensive understanding of dynamics and disease-associated solidification biomolecular condensates is closely tied to analysis their mechanical characteristics. Despite recent technical advances in rheological studies condensates, these still vastly rely on methods restricted small forces, rendering measurements droplets with higher elasticities after transition solid challenging. Here, we develop assays for in-depth characterization by scanning probe microscopy. We demonstrate this technique measuring the behavior heterotypic poly-L-lysine heparin showcasing multi-route liquid gel transition, as well rejuvenation chemical alterations medium. Due wide-spread application microscopy biological fields, its capability rapid, high throughput, force range studies, integration nanoscale morphological measurements, our probe-based method a significant breakthrough investigating condensate behavior, leading accelerated development therapies.

Language: Английский

Citations

0
The reovirus μ2 protein, an enigmatic multifunctional protein with numerous secrets yet to be uncovered DOI
Guy Lemay, Simon Boudreault

Virology, Journal Year: 2024, Volume and Issue: 601, P. 110275 - 110275

Published: Oct. 24, 2024

Language: Английский

Citations

0
Molecular sociology of virus-induced cellular condensates supporting reovirus assembly and replication DOI Creative Commons
X. Liu, Xian Xia,

Michael W. Martynowycz

et al.

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

Published: Dec. 6, 2024

Abstract Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) focused ion beam (FIB) milling-produced lamellae mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) host virus in 3D at two time points post-infection, enabling a detailed description these condensates mechanistic understanding MRV within them. Expanding over time, condensate fashions ribosomes its periphery, microtubules, lipid membranes, molecules interior, forming architecture that supports dynamic processes genome capsid assembly. A total six assembly intermediates identified inside condensate: star core, empty genome-containing cores, full virions, outer shell particle. Except for atomic resolution microscopy (cryoEM) extracts. The temporal sequence spatial rearrangement among choreograph life cycle condensates. Together, sociology MRV-induced highlights functional advantage transient enrichment right location replication.

Language: Английский

Citations

0