Biomolecular condensates and disease pathogenesis

Biomolecular condensates or membraneless organelles(MLOs)formed by liquid-liquid phase separation(LLPS)divide intracellular spaces into discrete compartments for specific functions.Dysregulation of LLPS or aberrant phase transition that disturbs the formation or material states of MLOs is closely correlated with neurodegeneration,tumorigenesis,and many other pathological processes.Herein,we summarize the recent progress in development of methods to monitor phase separation and we discuss the biogenesis and function of MLOs formed through phase separation.We then present emerging proof-of-concept examples regarding the disruption of phase separation homeostasis in a diverse array of clinical conditions including neurodegenerative disorders,hearing loss,cancers,and immunological diseases.Finally,we describe the emerging discovery of chemical modulators of phase separation.

High-throughput experimental methods for investigating biomolecular condensates

Background:The concept of biomolecular condensate was put forward recently to emphasize the ability of certain cellular compartments to concentrate molecules and comprise proteins and nucleic acids with specific biological functions,from ribosome genesis to RNA splicing.Due to their unique role in biological processes,it is crucial to investigate their compositions,which is a primary determinant of condensate properties.Results:Since a wide range of macromolecules comprise biomolecular condensates,it is necessary for researchers to investigate them using high-throughput methodologies while low-throughput experiments are not efficient enough.These high-throughput methods usually purify interacting protein libraries from condensates before being scanned in mass spectrometry.It is possible to extract organelles as a whole for specific condensates for further analysis,however,most condensates do not have a distinguishable marker or are sensitive to shear force to be extracted as a whole.Affinity tagging allows a comprehensive view of interacting proteins of target molecule yet only proteins with strong bonds may be pulled down.Proximity labeling serves as a complementary method to label more dynamic proteins with weaker interactions,increasing sensitivity while decreasing specificity.Image-based fluorescent screening takes another path by scanning images automatically to illustrate the condensing state of biomolecules within membraneless organelles,which is a unique feature unlike the previous mass spectrometry-based methods.Conclusion:This review presents a rough glimpse into high-throughput methodologies for biomolecular condensate investigation to encourage usage of bioinformatic tools by researchers in relevant fields.

From molecule to cell:the expanding frontiers of plant immunity

In recent years,the field of plant immunity has witnessed remarkable breakthroughs.During the co-evolution between plants and pathogens,plants have developed a wealth of intricate defense mechanisms to safeguard their survival.Newly identified immune receptors have added unexpected complexity to the surface and intracellular sensor networks,enriching our understanding of the ongoing plant–pathogen interplay.Deciphering the molecular mechanisms of resistosome shapes our understanding of these mysterious molecules in plant immunity.Moreover,technological innovations are expanding the horizon of the plant–pathogen battlefield into spatial and temporal scales.While the development provides new opportunities for untangling the complex realm of plant immunity,challenges remain in uncovering plant immunity across spatiotemporal dimensions from both molecular and cellular levels.

Plant RNA-binding proteins:Phase separation dynamics and functional mechanisms underlying plant development and stress responses

RNA-binding proteins(RBPs)accompany RNA from synthesis to decay,mediating every aspect of RNA metabolism and impacting diverse cellular and developmental processes in eukaryotes.Many RBPs undergo phase separation along with their bound RNA to form and function in dynamic membraneless biomolecular condensates for spatiotemporal coordination or regulation of RNA metabolism.Increasing evidence suggests that phase-separating RBPs with RNA-binding domains and intrinsically disordered regions play important roles in plant development and stress adaptation.Here,we summarize the current knowledge about how dynamic partitioning of RBPs into condensates controls plant development and enables sensing of experimental changes to confer growth plasticity under stress conditions,with a focus on the dynamics and functional mechanisms of RBP-rich nuclear condensates and cytoplasmic granules in mediating RNA metabolism.We also discuss roles of multiple factors,such as environmental signals,protein modifications,and N6-methyladenosine RNA methylation,in modulating the phase separation behaviors of RBPs,and highlight the prospects and challenges for future research on phase-separating RBPs in crops.

PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment

Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.

Recent progress on the activation of the cGAS–STING pathway and its regulation by biomolecular condensation

The cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthetase (cGAS)–stimulator of interferon genes(STING) pathway, comprising the DNA sensor cGAS, the second messenger cyclic GMP–AMP (cGAMP), and the endoplasmicreticulum (ER) adaptor protein STING, detects cytoplasmic double-stranded DNA (dsDNA) to trigger type I-interferon responses forhost defense against pathogens. Previous studies defined a model for the allosteric activation of cGAS by DNA-binding, but recentwork reveals other layers of mechanisms to regulate cGAS activation such as the phase condensation and metal ions, especially thediscovery of Mn^(2+) as a cGAS activator. Activation of the 23-cGAMP sensor STING requires translocating from the ER to the Golgiapparatus. The sulfated glycosaminoglycans at the Golgi are found to be the second STING ligand promoting STING oligomerizationand activation in addition to 23-cGAMP, while surpassed levels of 23-cGAMP induce ER-located STING to form a highly organizedER membranous condensate named STING phase-separator to restrain STING activation. Here, we summarize recent advances in theregulation of cGAS–STING activation and their implications in physiological or pathological conditions, particularly focusing on theemerging complexity of the regulation.

Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis

The shoot meristem generates the entire shoot system and is precisely maintained throughout the life cycle under various environmental challenges.In this study,we identified a prion-like domain(PrD)in the key shoot meristem regulator SHOOT MERISTEMLESS(STM),which distinguishes STM from other related KNOX1 proteins.We demonstrated that PrD stimulates STM to form nuclear condensates,which are required for maintaining the shoot meristem.STM nuclear condensate formation is stabilized by selected PrD-containing STM-interacting BELL proteins in vitro and in vivo.Moreover,condensation of STM promotes its interaction with the Mediator complex subunit MED8 and thereby enhances its transcriptional activity.Thus,condensate formation emerges as a novel regulatory mechanism of shoot meristem functions.Furthermore,we found that the formation of STM condensates is enhanced upon salt stress,which allows enhanced salt tolerance and increased shoot branching.Our findings highlight that the transcription factor partitioning plays an important role in cell fate determination and might also act as a tunable environmental acclimation mechanism.

Phase separation in cGAS-STING signaling

Biomolecular condensates formed by phase separation are widespread and play critical roles in many physiological and pathological processes.cGAS-STING signaling functions to detect aberrant DNA signals to initiate anti-infection defense and antitumor immunity.At the same time,cGAS-STING signaling must be carefully regulated to maintain immune homeostasis.Interestingly,exciting recent studies have reported that biomolecular phase separation exists and plays important roles in different steps of cGAS-STING signaling,including cGAS condensates,STING condensates,and IRF3 condensates.In addition,several intracellular and extracellular factors have been proposed to modulate the condensates in cGAS-STING signaling.These studies reveal novel activation and regulation mechanisms of cGAS-STING signaling and provide new opportunities for drug discovery.Here,we summarize recent advances in the phase separation of cGAS-STING signaling and the development of potential drugs targeting these innate immune condensates.

Tuning the rheostat of immune gene translation

Biomolecular condensates assembled through phase transitions regulate diverse aspects of plant growth,develop-ment,and stress responses.How biomolecular condensates control plant immunity is poorly understood.In Nature Plants,a new study(Zhou et al.,Nat Plants 9:289-301,2023)reveals how plants assemble translational condensates to balance tissue health and disease resistance.

Liquid-liquid phase separation as a major mechanism of plant abiotic stress sensing and responses

Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research.Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes.Therefore,the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced.Recently,accumulating evidence has emerged to suggest that liquid-liquid phase separation(LLPS)is a major mechanism for environmental stress sensing and response in plants.In this review,we briefly introduce LLPS regarding its concept,compositions,and dynamics,and then summarize recent progress of LLPS research in plants,emphasizing the contribution of LLPS to the sensing of various environmental stresses,such as dehydration,osmotic stress,and low and high temperatures.Finally,we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS,and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops.

Regulating FUS Liquid-Liquid Phase Separation via Specific Metal Recognition

Liquid-liquid phase separation(LLPS)or biomolecular condensation that leads to formation of membraneless organelles plays a critical role in many biochemical processes.Mechanism study of regulating LLPS is therefore central to the understanding of protein aggregation and disease-relevant process.We report a fused in sarcoma protein(FUS)-derived low complexity(LC)sequence that undergoes LLPS in the presence of metal ions.The LC protein was constructed by fusing a hexhistidine-tag to the N-terminal low complexity domain(the residues 1–165 in QGSY-rich segment)of FUS.Spontaneous condensation of the intrinsic disordered protein into coacervate droplets was observed in the presence of metal ions that chelate oligohistidine moieties to form protein matrix.We demonstrate the key role of metal ion-histidine coordination in governing LLPS behaviours and the fluidity of biomolecular condensates.By taking advantage of competitive binding using chelators,we show the possibility of regulating dynamic behaviors of disease-relevant protein droplets,and developing a potential approach towards controllable biological encapsulation/release.

Mechanisms and regulation underlying membraneless organelle plasticity control

Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments,such as membrane-bound and membraneless organelles(MLOs),for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control.Emerging evidence and view support the notion that MLOs are built by multivalent interactions of biomolecules via phase separation and transition mechanisms.In healthy cells,dynamic chemical modifications regulate MLO plasticity,and reversible phase separation is essential for cell homeostasis.Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders,cancer,and other diseases.In this review,we provide molecular underpinnings on(i)mechanistic understanding of phase separation,(ii)unifying structural and mechanistic principles that underlie this phenomenon,(iii)various mechanisms that are used by cells for the regulation of phase separation,and(iv)emerging therapeutic and other applications.

Liquid–liquid phase separation in plants:Advances and perspectives from model species to crops

Membraneless biomolecular condensates play important roles in both normal biological activities and re-sponses to environmental stimuli in living organisms.Liquid‒liquid phase separation(LLPS)is an organi-zational mechanism that has emerged in recent years to explain the formation of biomolecular conden-sates.In the past decade,advances in LLPS research have contributed to breakthroughs in diseasefields.By contrast,although LLPS research in plants has progressed over the past 5 years,it has been concentrated on the model plant Arabidopsis,which has limited relevance to agricultural production.In this review,we provide an overview of recently reported advances in LLPS in plants,with a particular focus on photomorphogenesis,flowering,and abiotic and biotic stress responses.We propose that many potential LLPS proteins also exist in crops and may affect crop growth,development,and stress resistance.This possibility presents a great challenge as well as an opportunity for rigorous scientific research on the biological functions and applications of LLPS in crops.