Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

Niewidok, Benedikt; Igaev, Maxim; Graca, Abel Pereira da; Strassner, Andre; Lenzen, Christine; Richter, Christian; Piehler, Jacob; Kurre, Rainer; Brandt, Roland

doi:10.1083/jcb.201709007

Cited by 120 publications

(115 citation statements)

References 46 publications

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“…Fortunately, single-molecule tracking has recently emerged as a powerful tool for examining LLPS in vivo (51,111,112). Indeed, the strongest evidence for bacterial condensates to date comes from single-molecule tracking of proteins in polar microdomains in Caulobacter (113).…”

Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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“…The observation that RNPs do not appear to fuse with other RNPs even when closely associated within the germ plasm aggregate underscores the potential for substructure in phase-separated organelles (Anderson and Kedersha, 2007;Jain et al, 2016;Mitrea and Kriwacki, 2016), possibly depending on differential affinities of aggregate components in relation to each other and the cytoplasm. Indeed, many known cases of homotypic interactions within RNPs involve protein-protein interactions involving low complexity domains or IDRs (Gilks et al, 2004;Kato et al, 2012;Toretsky and Wright, 2014;Lin et al, 2015;Niewidok et al, 2018), but RNA-RNA interactions may provide additional levels of complexity for the development of such substructure (Van Treeck et al, 2018).…”

Section: Germ Plasm Is Composed Of Homotypic Rnps That Aggregate Segmentioning

confidence: 99%

Aggregation, segregation, and dispersal of homotypic germ plasm RNPs in the early zebrafish embryo

Eno

Hansen

Pelegri

2019

Developmental Dynamics

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Background In zebrafish and many other organisms, specification of primordial germ cells (PGCs) requires the transmission of maternally‐derived germ plasm. Zebrafish germ plasm ribonucleoparticles (RNPs) aggregate along the cleavage furrows during the first several cell cycles, segregate asymmetrically during the cleavage stages, and undergo cytoplasmic dispersal in the late blastula. Results For all tested germ plasm RNAs [carbonic anhydrase 15b (ca15b), deleted in azoospermia‐like (dazl), dead end (dnd), nanos 3 (nos3), regulator of G‐protein signaling14a (rgs14a), and vasa/DEAD box polypeptide 4 (vasa/ddx4)], RNPs are homotypic (containing a single RNA type), with RNPs packing tightly yet remaining distinct within germ plasm aggregates. Homotypic clustering of RNAs within RNPs is observed before aggregation in the cortex and is maintained through germ plasm recruitment, asymmetric segregation and RNP dispersal. We also identify a step of germ plasm fragmentation during the cleavage stages that precedes RNP dispersal. Conclusions Our findings suggest that germ plasm aggregates act as subcellular compartments that temporarily collect and carry single RNA‐type RNPs from fertilization until their cytoplasmic dispersal in PGCs at the end of the blastula period, and describe a previously unknown fragmentation step that allows for an increase in the pool of germ plasm‐carrying cells, presumably PGCs. Developmental Dynamics 248:306–318, 2019. © 2019 Wiley Periodicals, Inc.

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“…It has been reported that SGs are not homogenous and contain substructures. In particular, several studies support a model where SGs contain stable cores surrounded by a more dynamic shell (Cirillo et al, 2020;Jain et al, 2016;Niewidok et al, 2018). Previously, it was also shown in Drosophila embryos that gurken transcripts are translated on the outside edge of P-bodies, another type of membrane-less organelle, and not in their core (Davidson et al, 2016).…”

Section: Position Of Mrnas Within Sgs Is Similar For Translating and mentioning

confidence: 88%

Single-molecule imaging reveals translation of mRNAs localized to stress granules

Matějů

Eichenberger

Eglinger

et al. 2020

Preprint

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Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation. observation of biochemical reactions inside and outside of biomolecular condensates in order to understand their biological function. RESULTS Single-molecule imaging reveals SG-associated translationIn order to characterize the relationship between mRNA localization and translation during stress, we combined live-cell imaging of SGs with SunTag-based single-molecule imaging of translation (Pichon et al., 2016;Wang et al., 2016;Wu et al., 2016;Yan et al., 2016). This approach allows visualization of individual reporter mRNAs via MS2 stem-loops and simultaneous visualization of translation via the SunTag, which consists of an array of antibody epitopes that are recognized by genetically-encoded single-chain antibodies (scFv). To accomplish this, we engineered a HeLa cell line stably expressing GFP-tagged scFv and Halo-tagged MS2 coat protein (MCP) . The reporter mRNA encodes the SunTag array in frame with Renilla luciferase, enabling the measurement of protein synthesis by two complementary assays. The reporter mRNA further contains a destabilized FKBP domain to enhance the degradation of the mature protein, thereby facilitating imaging of nascent peptides during translation. To enable visualization of translation during stress, we further placed the 5'UTR of ATF4 in front of the SunTag array ( Figure 1A). This enables the mRNA to be efficiently translated during stress conditions when many other mRNAs are inhibited. The reporter mRNA was integrated into the genome at a site-specific locus and was under the control of a doxycycline-inducible promoter, which enables precise mRNA tracking and minimizes potential artifacts of overexpression. Using luciferase assays, we confirmed that the translation of this ATF4-SunTag reporter is specifically upregulated during arsenite stress and repressed under normal growth conditions ( Figure 1B), as previously reported for endogenous ATF4 (Andreev et al., 2015;Roybal et al., 2005). Importantly, ATF4-SunTag was efficiently translated at an oxidative stress condition (100 µM arsenite) that also triggered robust SG formation in all cells ( Figure 1...

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Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

Abstract: Niewidok et al. analyze the distribution and dynamics of RNA-binding proteins (RBPs) with single-molecule resolution in living neuronal cells, providing direct support for liquid droplet behavior of stress granules in living cells and revealing transient binding of RBPs in nanocores.

Cited by 120 publications

References 46 publications

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Aggregation, segregation, and dispersal of homotypic germ plasm RNPs in the early zebrafish embryo

Single-molecule imaging reveals translation of mRNAs localized to stress granules

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