Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here we show in contrast that condensed biomolecular systems-often termed condensates-sustain pH gradients without any external energy input. By studying individual condensates on the micrometre scale using a microdroplet platform, we reveal dense-phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that protein condensates can drive substantial alkaline and acidic gradients, which are compositionally tunable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterization of human proteomic condensate networks, we further highlight potential wide-ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a regulatory mechanism to modulate biochemical activity in living and artificial systems.

Cells deploy robust mechanisms to repair DNA damage, safeguarding genomic stability and cellular health, but the physical principles underlying these processes remain incompletely understood. Experiments show in vitro that upon a DNA double-strand break, a DNA–protein condensate can tether the broken DNA ends before they disperse away, a critical step for subsequent repair biochemistry. However, it remains puzzling how such condensation reliably achieves spatiotemporal localization at the break site and captures both broken ends despite intrinsic stochasticity. Here, we propose that broken DNA ends can trigger a conversion of proteins from a soluble state to a condensate-competent state. Combining this idea with Brownian dynamics simulations and theory, we propose a physical mechanism for reliable DNA-end tethering. Simulations show that such break-induced conversion can drive local DNA–protein condensation with two possible outcomes: successful or failed tethering. To rationalize this, we construct an effective free energy landscape, identify the corresponding stationary states, and demonstrate that tethering is governed by a kinetic competition between polymer relaxation and condensation dynamics. Together, our study shows that DNA end-dependent conversion, coupled with DNA–protein condensation, can reliably tether broken DNA ends.

Ribonucleoprotein (RNP) granules have been linked to translation regulation and disease, but their assembly and regulatory mechanisms are not well understood. Here, we show that the RNA-binding protein G3BP1 preferentially interacts with unfolded RNA, driving the assembly of RNP granule-like condensates that establish RNA-RNA interactions. These RNA-RNA interactions limit the mobility and translatability of sequestered mRNAs and stabilize the condensates. The DEAD-box RNA helicase DDX3X attenuates RNA-RNA interactions inside RNP granule-like condensates, rendering the condensates dynamic and enabling mRNA translation. Importantly, disease-associated and catalytically inactive DDX3X variants fail to resolve such RNA-RNA interactions. Inhibiting DDX3X in cultured cells accelerates RNP granule assembly and delays their disassembly, indicating that RNA-RNA interactions contribute to RNP granule stability in cells. Our findings reveal how RNP granules generate inhibitory RNA-RNA interactions that are modulated by DEAD-box RNA helicases to ensure RNA availability and translatability.

Cells use membraneless compartments to organize their interiors, and recent research has begun to uncover the molecular principles underlying their assembly. Here, we explore how site-specific and chemically specific interactions shape the properties and functions of condensates. Site-specific recruitment involves precise interactions at specific sites driven by partially or fully structured interfaces. In contrast, chemically specific recruitment is driven by complementary chemical interactions without the requirement for a persistent bound-state structure. We propose that site-specific and chemically specific interactions work together to determine the composition of condensates, facilitate biochemical reactions, and regulate enzymatic activities linked to metabolism, signaling, and gene expression. Characterizing the composition of condensates requires novel experimental and computational tools to identify and manipulate the molecular determinants guiding condensate recruitment. Advancing this research will deepen our understanding of how condensates regulate cellular functions, providing valuable insights into cellular physiology and organization.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.

Cytosolic aggregation of the RNA binding protein TDP-43 (transactive response DNA-binding protein 43) is a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that during oxidative stress, TDP-43 becomes SUMO2/3-ylated by the SUMO E3 ligase protein PIAS4 (protein inhibitor of activated STAT 4) and enriches in cytoplasmic stress granules (SGs). Upon pharmacological inhibition of TDP-43 SUMO2/3-ylation or PIAS4 depletion, TDP-43 enrichment in SGs is accompanied by irreversible aggregation. In cells that are unable to assemble SGs, SUMO2/3-ylation of TDP-43 is strongly impaired, supporting the notion that SGs are compartments that promote TDP-43 SUMO2/3-ylation during oxidative stress. Binding of TDP-43 to UG-rich RNA antagonizes PIAS4-mediated SUMO2/3-ylation, while RNA dissociation promotes TDP-43 SUMO2/3-ylation. We conclude that SUMO2/3 protein conjugation is a cellular mechanism to stabilize cytosolic RNA-free TDP-43 against aggregation.

The realization that the cell is abundantly compartmentalized into biomolecular condensates has opened new opportunities for understanding the physics and chemistry underlying many cellular processes1, fundamentally changing the study of biology2. The term biomolecular condensate refers to non-stoichiometric assemblies that are composed of multiple types of macromolecules in cells, occur through phase transitions, and can be investigated by using concepts from soft matter physics3. As such, they are intimately related to aqueous two-phase systems4 and water-in-water emulsions5. Condensates possess tunable emergent properties such as interfaces, interfacial tension, viscoelasticity, network structure, dielectric permittivity, and sometimes interphase pH gradients and electric potentials6–14. They can form spontaneously in response to specific cellular conditions or to active processes, and cells appear to have mechanisms to control their size and location15–17. Importantly, in contrast to membrane-enclosed organelles such as mitochondria or peroxisomes, condensates do not require the presence of a surrounding membrane.

Increasing insights into how sequence motifs in intrinsically disordered regions (IDRs) provide functions underscore the need for systematic motif detection. Contrary to structured regions where motifs can be readily identified from sequence alignments, the rapid evolution of IDRs limits the usage of alignment-based tools in reliably detecting motifs within. Here, we developed SHARK-capture, an alignment-free motif detection tool designed for difficult-to-align regions. SHARK-capture innovates on word-based methods by flexibly incorporating amino acid physicochemistry to assess motif similarity without requiring rigid definitions of equivalency groups. SHARK-capture offers consistently strong performance in a systematic benchmark, with superior residue-level performance. SHARK-capture identified known functional motifs across orthologs of the microtubule-associated zinc finger protein BuGZ. We also identified a short motif in the IDR of S. cerevisiae RNA helicase Ded1p, which we experimentally verified to be capable of promoting ATPase activity. Our improved performance allows us to systematically calculate 10,889 motifs for 2695 yeast IDRs and provide it as a resource. SHARK-capture offers the most precise tool yet for the systematic identification of conserved regions in IDRs and is freely available as a Python package (https://pypi.org/project/bio-shark/) and on https://git.mpi-cbg.de/tothpetroczylab/shark.

Biomolecular phase separation is an emerging theme for protein assembly and cellular organisation. The collective forces driving such condensation, however, remain challenging to characterise. Here we show that tracking the dilute phase concentration of only one component suffices to quantify composition and energetics of multicomponent condensates. Applying this assay to several disease- and stress-related proteins, we find that monovalent ions can either deplete from or enrich within the dense phase in a context-dependent manner. By analysing the effect of the widely used modulator 1,6-hexanediol, we find that the compound inhibits phase separation by acting as a solvation agent that expands polypeptide chains. Extending the strategy to in cellulo data, we even quantify the relative energetic contributions of individual proteins within complex condensates. Together, our approach provides a generic and broadly applicable tool for dissecting the forces governing biomolecular condensation and guiding the rational modulation of condensate behaviour.

Cytosolic aggregation of the nuclear protein TAR DNA-binding protein 43 (TDP-43) is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43-enriched phase within stress granules, which subsequently transition into pathological aggregates. Intra-condensate demixing of TDP-43 is observed in iPS-motor neurons, a disease mouse model, and patient samples. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We suggest that up-concentration inside condensates followed by intra-condensate demixing could be a general pathway for protein aggregation.

Upregulation of insulin mRNA translation upon hyperglycemia in pancreatic islet β-cells involves several RNA-binding proteins. Here, we found that G3BP1, a stress granule marker downregulated in islets of subjects with type 2 diabetes, binds to insulin mRNA in glucose concentration-dependent manner. We show in mouse insulinoma MIN6-K8 cells exposed to fasting glucose levels that G3BP1 and its paralog G3BP2 colocalize to cytosolic condensates with eIF3b, phospho-AMPKα Thr172 and Ins1/2 mRNA. Glucose stimulation dissolves G3BP1 + /2 + condensates with cytosolic redistribution of their components. The aldolase inhibitor aldometanib prevents the glucose- and pyruvate-induced dissolution of G3BP1 + /2 + condensates, increases phospho-AMPKα Thr172 levels and reduces those of phospho-mTOR Ser2448 . G3BP1 or G3BP2 depletion precludes condensate assembly. KO of G3BP1 decreases Ins1/2 mRNA abundance and translation as well as proinsulin levels, and impaires glucose-stimulated insulin secretion. Further, other insulin secretagogues such as exendin-4 and palmitate, but not high KCl, prompts the dissolution of G3BP1 + /2 + condensates. G3BP1 + /2 + /Ins mRNA + condensates are also found in primary mouse and human β-cells. Hence, G3BP1 + /2 + condensates represent a conserved glycolysis/aldolase-regulated compartment for the physiological storage and protection of insulin mRNA in resting β-cells.

During heat shock (HS), cells orchestrate a gene expression program that promotes the synthesis of HS proteins (HSPs) while simultaneously repressing the synthesis of other proteins, including growth-promoting housekeeping proteins. Recent studies show that mRNAs encoding housekeeping proteins, along with associated processing factors, form macromolecular assemblies during HS. These assemblies inhibit transcription, nuclear export, and translation of housekeeping mRNAs, and coincide with structural rearrangements in proteins. These findings reveal a mechanism linking temperature sensitivity through structural rearrangements and macromolecular assembly to the ‘shut down’ of housekeeping protein synthesis. This review delves into recent findings in yeast, with a focus on macromolecular assembly, offering perspectives into mechanisms that regulate gene expression during HS and how these processes may be conserved.

Inhibitors of the integrated stress response (ISR) have been used to explore the potential beneficial effects of reducing the activation of this pathway in diseases. As the ISR is in essence a protective response, there is, however, a risk that inhibition may compromise the cell's ability to restore protein homeostasis. Here, we show that the experimental compound ISRIB impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Accumulation of a UPS reporter substrate that is intercepted by ribosome quality control was comparable to the level observed after blocking the UPS with a proteasome inhibitor. Consistent with impairment of the cytosolic UPS, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. Our data suggest that the persistent protein translation during proteotoxic stress in the absence of a functional ISR increases the pool of DRiPs, thereby hindering the efficient clearance of cytosolic substrates by the UPS. Activation of the integrated stress response is important for preserving functionality of the ubiquitin-proteasome system.

Hyperglycemia upregulates insulin translation in pancreatic beta cells. Several RNA- binding proteins involved in this process have been identified, including G3BP1, a stress granule marker downregulated in islets of subjects with type 2 diabetes. We show that in mouse insulinoma MIN6-K8 cells exposed to fasting glucose levels G3BP1 and its paralog G3BP2 colocalize to cytosolic condensates with eIF3b and Ins1/2 mRNA. Upon glucose stimulation, the condensates dissolve and G3BP1/2, eIF3b, and insulin mRNAs redistribute throughout the cytosol. Intriguingly, G3BP1+ condensates in MIN6-K8 cells differ from sodium arsenate-induced stress granules in regards to eIF2α and AMPKα phosphorylation. Knockout of G3BP1 or G3BP2 prevented condensate assembly, but only G3BP1 deletion decreased the levels of Ins1/2 mRNA and proinsulin and impaired polysome formation. Like glucose, other insulin secretagogues such as Exendin-4 and palmitate, but not high KCl, prompted the dissolution of G3BP1+ condensates. G3BP1+/ Ins mRNA+ condensates were also present in mouse and human beta cells from normoglycemic donors. Hence, G3BP1+ condensates represent a glucose-regulated compartment for the physiological storage and protection of insulin mRNA in resting beta cells.### Competing Interest StatementThe authors have declared no competing interest.

SUMMARY Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

The Integrated Stress Response Inhibitor (ISRIB) is an experimental compound that has been used to explore the potential beneficial effects of reducing the activation of the integrated stress response (ISR). As the ISR is a protective response, there is, however, a risk that its inhibition may compromise the cell’s ability to restore protein homeostasis. Here, we show that ISRIB treatment impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Degradation of proteins intercepted by ribosome quality control (RQC) was particularly affected as accumulation of a UPS reporter substrate for ribosome quality control (RQC) was comparable to the level observed after proteasome inhibition. Consistent with impaired RQC, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. As depletion of the RQC ubiquitin ligase listerin partially restored ubiquitin-dependent proteasomal degradation, these data suggest that the persistent protein translation during proteotoxic stress in ISRIB-treated cells increases the pool of newly synthesized proteins targeted by RQC, which aggravates UPS dysfunction by overloading the cytosolic UPS. ### Competing Interest Statement The authors have declared no competing interest. This study does not contain deposited data. Original data can be provided upon request.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology. Physical characterisation of proteins is challenging. Here the authors report single-molecule microfluidic diffusional sizing (smMDS) to enable calibration-free single-molecule diffusional-sizing based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures.

Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells.For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1