Germ cells depend on specialized post-transcriptional regulation for proper development and function, much of which is mediated by dynamic RNA granules. These membrane-less organelles form through the condensation of RNA and proteins, governed by multivalent biomolecular interactions. RNA granules compartmentalize cellular components, selectively enriching specific factors and modulating biochemical reactions. Over recent decades, various types of RNA granules have been identified in germ cells across species, with extensive studies uncovering their molecular roles and developmental significance. This review explores the mRNA regulatory mechanisms mediated by RNA granules in germ cells. We discuss the distinct spatial organization of specific granule components and the variations in material states of germ granules, which contribute to the regulation of mRNA storage and translation. Additionally, we highlight emerging research on how changes in these material states, during developmental stages, reflect the dynamic nature of germ granules and their critical role in development.

The observation of ultrathin sections of cells and other specimens using transmission electron microscopy (TEM) is a standard technique in many biological research laboratories. Cells are typically collected, dehydrated through an ascending series of alcohols, and embedded in resin blocks before being sectioned with an ultramicrotome. This multi-step process can be time consuming and error prone. To address these challenges, we introduced modifications to improve sample visualization while avoiding hazardous substances like osmium tetroxide and epoxy resins (e.g., Araldite), which are increasingly regulated internationally. Specifically, we stained cells with Trypan Blue and used low melting point agar, facilitating visual identification of target areas and enabling precise embedding. As a result, visual tracking of samples prior to embedding for TEM was improved, preventing the cutting of empty blocks and ensuring efficient preparation of ultrathin sections.

Phase separation, a fundamental phenomenon in both natural and industrial settings, involves the coarsening of domains over time t to reduce interfacial energy. While well-understood for simple viscous liquid mixtures, the physical laws governing coarsening dynamics in complex fluids, such as colloidal suspensions, remain unclear. Here, we investigate colloidal phase separation through particle-based simulations with and without hydrodynamic interactions (HIs). The former incorporates many-body HIs through momentum conservation, while the latter simplifies their effects into a constant friction coefficient on a particle. In cluster-forming phase separation with HIs, the domain size ℓ grows as ℓ∝t1/3, aligning with the Brownian-coagulation mechanism. Without HIs, ℓ∝t1/5, attributed to an improper calculation of cluster thermal diffusion. For network-forming phase separation, ℓ∝t1/2 with HIs, while ℓ∝t1/3 without HIs. In both cases, network coarsening is governed by the mechanical stress relaxation of the colloid-rich phase, yet with distinct mechanisms: slow solvent permeation through densely packed colloids for the former and free draining for the latter. Our results provide a clear and concise physical picture of colloid-solvent dynamic coupling via momentum conservation, offering valuable insights into the self-organization dynamics of particles like colloids, emulsions, and globular proteins suspended in a fluid.

Intracellular compartmentalization plays a critical role in maintaining cellular homeostasis and regulating metabolic processes. A growing body of evidence suggests that various metabolic enzymes, including CTP synthase (CTPS), can dynamically assemble into membraneless filamentous structures. The formation of these membraneless organelles is precisely regulated by the cellular metabolic state. CTPS, a rate-limiting enzyme in the de novo biosynthesis of CTP, has been shown to assemble into filamentous structures known as cytoophidium. First identified in 2010 by three independent research groups, cytoophidia are evolutionarily conserved across diverse organisms, including bacteria, archaea, yeast, mammals, and plants, suggesting a fundamental biological function. Given the well-established advantages of Drosophila melanogaster as a genetic model, this organism provides a powerful system for investigating the physiological roles of cytoophidia. This review synthesizes current findings on CTPS cytoophidia in Drosophila, with a particular focus on their spatiotemporal distribution in tissues and their regulatory roles in three key biological processes: intestinal homeostasis, lipid metabolism, and reproductive physiology. Furthermore, we discuss the challenges and future directions in cytoophidia research, offering insights into their broader implications in cellular metabolism and physiology.

Nucleic acids have the potential to form not only duplexes, but also various non-canonical secondary structures in living cells. Non-canonical structures play regulatory functions mainly in the central dogma. Therefore, nucleic acid targeting molecules are potential novel therapeutic drugs that can target 'undruggable' proteins in various diseases. One of the concerns of small molecules targeting nucleic acids is selectivity, because nucleic acids have only four different building blocks. Three- and four-stranded non-canonical structures, triplexes and quadruplexes, respectively, are promising targets of small molecules because their three-dimensional structures are significantly different from the canonical duplexes, which are the most abundant in cells. Here, we describe some basic properties of the triplexes and quadruplexes and small molecules targeting the triplexes and tetraplexes.

Ion-protein interactions regulate biological processes and are the basis of key strategies of modulating protein phase diagrams and stability in drug development. Here, we report the mechanisms by which H-bonds and electrostatic interactions in ion-protein systems determine phase separation and amyloid formation. Using microscopy, small-angle X-ray scattering, circular dichroism and atomistic molecular dynamics (MD) simulations, we found that anions specifically interacting with insulin induced phase separation by neutralising the protein charge and forming H-bond bridges between insulin molecules. The same interaction was responsible for an enhanced insulin conformational stability and resistance to oligomerisation. Under aggregation conditions, the anion-protein interaction translated into the activation of a coalescence process, leading to amyloid-like microparticles. This reaction is alternative to conformationally-driven pathways, giving rise to elongated amyloid-like fibrils and occurs in the absence of preferential ion-protein binding. Our findings depict a unifying scenario in which common interactions dictated both phase separation at low temperatures and the occurrence of pronounced heterogeneity in the amyloid morphology at high temperatures, similar to what has previously been reported for protein crystal growth.

The disruption of transactive response DNA binding protein 43 kDa (TDP-43) shuttling leads to the depletion of nuclear localization and the cytoplasmic accumulation of TDP-43. We aimed to evaluate the mechanism underlying the behavior of TDP-43 in ischemic stroke. Adult male C57BL/6 J mice were subjected to 30 or 60 min of transient middle cerebral artery occlusion (tMCAO), and examined at 1, 6, and 24 h post reperfusion. Immunostaining was used to evaluate the expression of TDP-43, G3BP1, HDAC6, and RAD23B. The total and cytoplasmic number of TDP-43-positive cells increased compared with sham operation group and peaked at 6 h post reperfusion after tMCAO. The elevated expression of G3BP1 protein peaked at 6 h after reperfusion and decreased at 24 h after reperfusion in ischemic mice brains. We also observed an increase of expression level of HDAC6 and the number of RAD23B-positive cells increased after tMCAO. RAD23B was colocalized with TDP-43 24 h after tMCAO. We proposed that the formation of stress granules might be involved in the mislocalization of TDP-43, based on an evaluation of G3BP1 and HDAC6. Subsequently, RAD23B, may also contribute to the downstream degradation of mislocalized TDP-43 in mice tMCAO model.

Cells maintain spatiotemporal control over biochemical processes through the formation and dissolution of biomolecular condensates, dynamic membraneless organelles formed via liquid-liquid phase separation. Composed primarily of proteins and nucleic acids, these condensates regulate key cellular functions, and their properties are influenced by the concentration and type of molecules involved. The structural versatility challenges the de novo design and assembly of condensates with predefined properties. Through feedback between computational and experimental approaches, we introduce a modular system for assembling condensates using nucleic acid nanotechnology. By utilizing programmable oligonucleotides and orthogonal synthesis methods, we control the structural parameters, responsive behavior, and immunorecognition of the products. Dissipative particle dynamics simulations predict some conditions to produce larger, well-defined condensates with compact, globular cores, while others result in smaller, more diffuse analogs. Fluorescence microscopy confirms these findings and microrheology demonstrates the viscoelastic adaptability of tested condensates. Nucleases trigger disruption of structures, and ethidium bromide intercalation protects condensates from digestion. Immunostimulatory assays suggest condensate-specific activation of the IRF pathway via cGAS-STING signaling. This study provides a framework for developing biomolecular condensates with customizable properties and immunorecognition for various biological applications.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) play a crucial role in organizing biochemical processes within living cells. The phase transition of these condensates from a functional liquid-like state to a pathological gel-like or solid-like state is believed to be linked to cellular dysfunction and various diseases. Here, we present a biomimetic model to demonstrate that endogenous enzyme-catalyzed crosslinking within condensate-mimicked coacervate microdroplets can promote a liquid-to-gel phase transition. We identify the transformation in physical characteristics of the densely packed microdroplets including reduced internal mobility, increased storage modulus, selective blocking of large nanoparticles, and enhanced salt resistance. The reversible dynamics of gel-like microdroplets mediated by ionic strength exhibited a limited release and recapture of sequestered positively charged guest molecules. Furthermore, we validate that the phase transition contributes to a restricted biochemical process through an enzymatic cascade. Overall, this work represents an adaptive in vitro platform for exploring the phase transitions associated with the physiological functions of biomolecular condensates and offers chemical insights and perspectives for investigating potential mechanisms involved in phase transitions.

Biomolecular condensates are membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically disordered proteins (IDPs) often function as condensate scaffolds, fueled by liquid-liquid phase separation (LLPS) dynamics. Intracellular probing of condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins. Conformational heterogeneity in IDPs, however, renders them uniquely susceptible to artifacts from tagging. Probing epidermal condensates in skin, we recently introduced genetically-encoded LLPS-sensors that circumvent the need for molecular-level tagging of skin IDPs. Departing from subcellular tracking of IDP-scaffolds, LLPS-sensors report on the assembly and liquid-like dynamics of their condensates. Here, we demonstrate biomolecular approaches for the evolution and tunability of epidermal LLPS-sensors and assess their impact in the early and late stages of intracellular phase separation. Benchmarking against scaffold-bound fluorescent reporters, we discovered that tunable ultraweak scaffold-sensor interactions uniquely enable the sensitive and innocuous probing of nascent and established biomolecular condensates. Our LLPS-sensitive tools pave the way for the high-fidelity intracellular probing of IDP-governed biomolecular condensates across biological systems.

Cytoplasmic aggregation and nuclear depletion of TAR DNA-binding protein 43 (TDP-43) are hallmarks of several neurodegenerative disorders. Yet, recapitulating both features in cellular systems has been challenging. Here, we produced amyloid-like fibrils from recombinant TDP-43 low-complexity domain and demonstrate that sonicated fibrils trigger TDP-43 pathology in human cells, including induced pluripotent stem cell (iPSC)-derived neurons. Fibril-induced cytoplasmic TDP-43 inclusions acquire distinct biophysical properties, recapitulate pathological hallmarks such as phosphorylation, ubiquitin, and p62 accumulation, and recruit nuclear endogenous TDP-43, leading to its loss of function. A transcriptomic signature linked to both aggregation and nuclear loss of TDP-43, including disease-specific cryptic splicing, is identified. Cytoplasmic TDP-43 aggregates exhibit time-dependent heterogeneous morphologies as observed in patients-including compacted, filamentous, or fragmented-which involve upregulation/recruitment of protein clearance pathways. Ultimately, cell-specific progressive toxicity is provoked by seeded TDP-43 pathology in human neurons. These findings identify TDP-43-templated aggregation as a key mechanism driving both cytoplasmic gain of function and nuclear loss of function, offering a valuable approach to identify modifiers of sporadic TDP-43 proteinopathies.

The phase separation of intrinsically disordered proteins is emerging as an important mechanism for cellular organization. However, efforts to connect protein sequences to the physical properties of condensates, that is, the molecular grammar, are hampered by a lack of effective approaches for probing high-resolution structural details. Using a combination of multiscale simulations and fluorescence lifetime imaging microscopy experiments, we systematically explored a series of systems consisting of diblock elastin-like polypeptides (ELPs). The simulations succeeded in reproducing the variation of condensate stability upon amino acid substitution and revealed different microenvironments within a single condensate, which we verified with environmentally sensitive fluorophores. The interspersion of hydrophilic and hydrophobic residues and a lack of secondary structure formation result in an interfacial environment, which explains both the strong correlation between ELP condensate stability and interfacial hydrophobicity scales, as well as the prevalence of protein-water hydrogen bonds. Our study uncovers new mechanisms for condensate stability and organization that may be broadly applicable.

Biomolecular condensates formed through liquid-liquid phase separation play key roles in intracellular organization and signaling, yet their function in extracellular environments remains largely unexplored. Here, we establish a model using heparan sulfate, a key component of the extracellular matrix, to study extracellular condensate-cell interactions. We demonstrate that heparan sulfate can form condensates with a positively charged counterpart in serum-containing solutions, mimicking the complexity of extracellular fluid, and supporting cell viability. We observe that these condensates adhere to cell membranes and remain stable, enabling a versatile platform for examining extracellular condensate dynamics and quantifying their rheological properties as well as their adhesion forces with cellular surfaces. Our findings and methodology open new avenues for understanding the organizational roles of condensates beyond cellular boundaries.

The heterochromatin protein HP1α consists of an N-terminal disordered tail (N-tail), chromodomain (CD), hinge region (HR), and C-terminal chromo shadow domain (CSD). While CD binds to the lysine9-trimethylated histone H3 (H3K9me3) tail in nucleosomes, CSD forms a dimer bridging two nucleosomes with H3K9me3. Phosphorylation of serine residues in the N-tail enhances both H3K9me3 binding and liquid-liquid phase separation (LLPS) by HP1α. We have used integrative structural methods, including nuclear magnetic resonance, small-angle X-ray scattering (SAXS), and multi-angle-light scattering combined with size-exclusion chromatography, and coarse-grained molecular dynamics simulation with SAXS, to probe the HP1α dimer and its CSD deletion monomer. We show that dynamic intra- and intermolecular interactions between the N-tails and basic segments in CD and HR depend on N-tail phosphorylation. While the phosphorylated HP1α dimer undergoes LLPS via the formation of aggregated multimers, the N-tail phosphorylated mutant without CSD still undergoes LLPS, but its structural unit is a dynamic intermolecular dimer formed via the phosphorylated N-tail and a basic segment at the CD end. Furthermore, we reveal that mutation of this basic segment in HP1α affects the size of heterochromatin foci in cultured mammalian cells, suggesting that this interaction plays an important role in heterochromatin formation in vivo.

Age-dependent transition of metastable, liquid-like protein condensates to amyloid fibrils is an emergent phenomenon of numerous neurodegeneration-linked protein systems. A key question is whether the thermodynamic forces underlying reversible phase separation and maturation to irreversible amyloids are distinct and separable. Here, we address this question using an engineered version of the microtubule-associated protein Tau, which forms biochemically active condensates. Liquid-like Tau condensates exhibit rapid aging to amyloid fibrils under quiescent, cofactor-free conditions. Tau condensate interface promotes fibril nucleation, impairing their activity to recruit tubulin and catalyze microtubule assembly. Remarkably, a small molecule metabolite, L-arginine, selectively impedes condensate-to-fibril transition without perturbing phase separation in a valence and chemistry-specific manner. By heightening the fibril nucleation barrier, L-arginine counteracts age-dependent decline in the biochemical activity of Tau condensates. These results provide a proof-of-principle demonstration that small molecule metabolites can enhance the metastability of protein condensates against a liquid-to-amyloid transition, thereby preserving condensate function.

TIP60 is a tumor suppressor with histone acetyltransferase (HAT) activity, playing a crucial role in regulating chromatin architecture by acetylating histones to enhance accessibility for other regulatory factors. Its function is vital for several key cellular processes, including DNA damage repair, apoptosis, and autophagy. While the downregulation of TIP60 has been associated with various cancers, the effects of naturally occurring mutations in TIP60 on its function in malignancies remain poorly understood. In this study, we explored how cancer-related mutations in TIP60 impact its structure and function. Several deleterious and destabilizing missense mutations were identified and analyzed for structural changes. Molecular dynamics simulations revealed alterations in protein conformational stability and radius of gyration due to these mutations, supported by significant changes in TIP60's solvent accessibility and intramolecular hydrogen bonding. Biochemical assays with recombinant proteins showed a loss of catalytic activity in the G400W mutant. Live cell imaging indicated abnormal localization of the G400W mutant within the nucleus. Additionally, we observed aberrant phase separation of TIP60 caused by the G400W mutation. Notably, the G400W mutation impairs TIP60's catalytic function, preventing effective DNA repair and leaving the genome vulnerable to further mutations. Our findings highlight cancer-associated mutations in TIP60 that may contribute to the molecular mechanisms underlying cancer initiation and progression.

Engineering of nuclear condensates with chemically inducible gene switches is highly desired but challenging for precise and on-demand regulation of mammalian gene expression. Here, we harness the phase-separation capability of biomolecular condensates and describe a versatile strategy to chemically program ligand-dependent gene expression at various stages of interest. By engineering synthetic anchor proteins capable of tethering various genetically encoded condensate structures toward different cellular compartments or gene products of interest, inducible regulation of transcriptional and translational activities was achieved at different endogenous and episomal loci using the same sets of anchor proteins and synthetic solid-state condensates. Using such a holistic condensate-based strategy, we not only achieved regulation performances comparing favorably to state-of-the-art strategies described for CRISPR-Cas9 activity and transcriptional silencing but further showed that chemically inducible retention of mRNA molecules into engineered condensate structures within the nucleus can become a remarkably efficient alternative for translational regulation.

Biomolecular condensates, such as stress and germ granules, often contain subcompartments. For instance, the Caenorhabditis elegans germ granule, which localizes near the outer nuclear membrane of germ cell nuclei, is composed of at least four ordered compartments, each housing distinct sets of proteins and RNAs. How these compartments form and why they are spatially ordered remains poorly understood. Here, we show that the conserved DEAD-box RNA helicase DDX-19 defines another compartment of the larger C. elegans germ granule, which we term the D compartment. The D compartment exhibits properties of a liquid condensate and forms between the outer nuclear pore filament and other compartments of the germ granule. Two nuclear pore proteins, NPP-14 and GLEL-1, are required for its formation, suggesting that the D compartment localizes adjacent to the outer nuclear membrane through interactions with the nuclear pore. The loss of DDX-19, NPP-14 or GLEL-1 leads to functional defects, including aberrant formation of the other four germ granule compartments, a loss of germline immortality and dysregulation of small RNA-based transgenerational epigenetic inheritance programs. Hence, we propose that a function of the D compartment is to anchor larger germ granules to nuclear pores, enabling germ granule compartmentalization and promoting transgenerational RNA surveillance.

Keeping condensates in liquid-like states throughout the biosynthesis process in microbial cell factories remains an ongoing challenge. Here, we present a light-controlled phase regulator, which maintains the liquid-like features of synthetic condensates on demand throughout the biosynthesis process upon light induction, as demonstrated by various live cell-imaging techniques. Specifically, the tobacco etch virus (TEV) protease controlled by light cleaves intrinsically disordered proteins (IDPs) to alter their valency and concentration for controlled phase transition and programmable fluidity of cellular condensates. As a proof of concept, we harness this capability to significantly improve the production of squalene and ursolic acid (UA) in engineered Saccharomyces cerevisiae. Our work provides a powerful approach to program the solid-liquid phase transition of biomolecular condensates for improved biosynthesis.

Ferroptosis is an iron-dependent regulated form of cell death implicated in various diseases, including cancers, with its progression influenced by iron-dependent peroxidation of phospholipids and dysregulation of the redox system. Whereas the extracellular matrix of tumors provides mechanical cues influencing tumor initiation and progression, its impact on ferroptosis and its mechanisms remains largely unexplored. In this study, we reveal that heightened mechanical tension sensitizes cells to ferroptosis, whereas decreased mechanics confers resistance. Mechanistically, reduced mechanical tension reduces intracellular free iron levels by enhancing FTH1 protein expression. Additionally, low mechanics significantly diminishes NCOA4, pivotal in mediating FTH1 phase separation-induced ferritinophagy. Targeting NCOA4 effectively rescues ferroptosis susceptibility under low mechanical tension through modulation of FTH1 phase separation-driven autophagy. In conclusion, our findings demonstrate that mechanics regulates iron metabolism via NCOA4-FTH1 phase separation-mediated autophagy, thereby influencing ferroptosis sensitivity and offering promising therapeutic avenues for future exploration.Abbreviations: ACO1: aconitase 1; ATG5: autophagy related 5; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FER-1: ferrostatin-1; FTH1: ferritin heavy chain 1; FTL: ferritin light chain; GPX4: glutathione peroxidase 4; IR: ionizing radiation; IREB2: iron responsive element binding protein 2; NCOA4: nuclear receptor coactivator 4; NFE2L2: NFE2 like bZIP transcription factor 2; NOPP: norepinephrine; PBS: phosphate-buffered saline; PI: propidium iodide; RSL3: (1S,3 R)-RSL3; TCGA: The Cancer Genome Atlas; WWTR1: WW domain containing transcription regulator 1; YAP1: Yes1 associated transcriptional regulator.

Protein responses to environmental stress, particularly temperature fluctuations, have long been a subject of investigation, with a focus on how proteins maintain homeostasis and exhibit thermoresponsive properties. While UCST-type (upper critical solution temperature) phase behavior has been studied extensively and can now be predicted reliably using computational models, LCST-type (lower critical solution temperature) phase transitions remain less explored, with a lack of computational models capable of accurate prediction. This gap limits our ability to probe fully how proteins undergo phase transitions in response to temperature changes. Here, we introduce Mpipi-T, a residue-level coarse-grained model designed to predict LCST-type phase behavior of proteins. Parametrized using both atomistic simulations and experimental data, Mpipi-T accounts for entropically driven protein phase separation that occurs upon heating. Accordingly, Mpipi-T predicts temperature-driven protein behavior quantitatively in both single- and multi-chain systems. Beyond its predictive capabilities, we demonstrate that Mpipi-T provides a framework for uncovering the molecular mechanisms underlying heat stress responses, offering new insights into how proteins sense and adapt to thermal changes in biological systems.

Liquid-liquid phase separation (LLPS) is a cornerstone of cellular organization, driving the formation of biomolecular condensates that regulate diverse biological processes and inspire innovative applications. This review explores the molecular mechanisms underlying peptide-mediated LLPS, emphasizing the roles of intermolecular interactions such as hydrophobic effects, electrostatic interactions, and π-π stacking in phase separation. The influence of environmental factors, such as pH, temperature, ionic strength, and molecular crowding on the stability and dynamics of peptide coacervates is examined, highlighting their tunable properties. Additionally, the unique physicochemical properties of peptide coacervates, including their viscoelastic behavior, interfacial dynamics, and stimuli-responsiveness, are discussed in the context of their biological relevance and engineering potential. Peptide coacervates are emerging as versatile platforms in biotechnology and medicine, particularly in drug delivery, tissue engineering, and synthetic biology. By integrating fundamental insights with practical applications, this review underscores the potential of peptide-mediated LLPS as a transformative tool for advancing science and healthcare.

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GC's spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GC's spatial heterogeneity. SURF6's association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates-suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that arrests the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

Parkinson's disease (PD), a neurodegenerative disorder without cure, is characterized by the pathological aggregation of α-synuclein (α-Syn) in Lewy bodies. Classic deposition pathway and condensation pathway contribute to α-Syn aggregation, and liquid-liquid phase separation is the driving force for condensate formation, which subsequently undergo liquid-solid phase separation to form toxic fibrils. Traditional Chinese Medicine (TCM) has a long history in treating neurodegenerative disease; herein, we identified chemicals from herbs that inhibit α-Syn aggregation. We screened commonly prescribed TCMs for PD from the CNKI database and registered patents, 13 chemicals were identified in the TCMSP databases as candidate inhibitors, among which three phenols, forsythoside B (FTSB), echinacoside (ECH), and 4-hydroxyindole (C4-OH) efficiently inhibit α-Syn aggregation. Moreover, FTSB and ECH increase α-Syn fluidity within condensates, inhibit α-Syn transition into amyloid fibrils and reduce fibril-induced toxicity in SH-SY5Y cells. Importantly, they disaggregated preformed α-Syn amyloid fibrils. Notably, in an α-Syn overexpressing NL5901 C. elegans PD model, either FTSB or ECH treatment significantly extended the lifespan and improved the PD-like movement disorders, both in the preventive and therapeutic treatment approaches, by reducing toxic α-Syn inclusion formation and improving the fluidity of α-Syn. Together, we offer new therapeutic candidates targeting phase separation-associated aggregation for PD.

In this review, we aim to explore the multifaceted roles of galectins in host defense from a broader perspective, particularly regarding their functions when host integrity is compromised. Numerous comprehensive reviews on galectin functions in immunity have already been published. For researchers new to the field, this wealth of information may create an impression of galectins as proteins involved in a wide array of biological processes. Furthermore, due to the heterogeneity of galectin ligands, glycans, there is a risk of perceiving galectin-specific functions as ambiguous, potentially obscuring their core biological significance. To address this, we revisit foundational aspects, focusing on the significance of the recognition of galactose, a "late-comer" monosaccharide in evolutionary terms, provide an overview of galectin glycan binding specificity, with emphasis on the potential biological importance of each carbohydrate-recognition domain. We also discuss the biological implications of the galectin location paradox wherein these cytosolic lectins function in host defense despite their glycan ligands being synthesized in the secretory pathway. Additionally, we examine the role of galectins in liquid-liquid phase separation on membranes, which may facilitate their diverse functions in cellular responses. Through this approach, we aim to re-evaluate the complex and diverse biological roles of galectins in host defense.

Molecular imprinting technology (MIT), a synthetic strategy to create tailor-made molecular specificity, has recently achieved significant advancements. Epitope imprinting strategy, an improved MIT by imprinting the epitopes of biomolecules (e.g., proteins and nucleic acids), enables to target the entire molecule through recognizing partial epitopes exposed on it, greatly expanding the applicability and simplifying synthesis process of molecularly imprinted polymers (MIPs). Thus, epitope imprinting strategy offers promising solutions for the fabrication of smart biomaterials with molecular targeting and exhibits wide applications in various biomedical scenarios. This review explores the latest advances in epitope imprinting techniques, emphasizing selection of epitopes and functional monomers. We highlight the significant improvements in specificity, sensitivity, and stability of these materials, which have facilitated their use in bioanalysis, clinical therapy, and pharmaceutical development. Additionally, we discuss the application of epitope-imprinted materials in the recognition and detection of peptides, proteins, and cells. Despite these advancements, challenges such as template complexity, imprinting efficiency, and scalability remain. This review addresses these issues and proposes potential directions for future research to overcome these barriers, thereby enhancing the efficacy and practicality of epitope molecularly imprinting technology in biomedical fields.

Protein cis-regulatory elements (CREs) are regions that modulate the activity of a protein through intramolecular interactions. Kinases, pivotal enzymes in numerous biological processes, often undergo regulatory control via inhibitory interactions in cis. This study delves into the mechanisms of cis regulation in kinases mediated by CREs, employing a combined structural and sequence analysis. To accomplish this, we curated an extensive dataset of kinases featuring annotated CREs, organized into homolog families through multiple sequence alignments. Key molecular attributes, including disorder and secondary structure content, active and ATP-binding sites, post-translational modifications, and disease-associated mutations, were systematically mapped onto all sequences. Additionally, we explored the potential for conformational changes between active and inactive states. Finally, we explored the presence of these kinases within membraneless organelles and elucidated their functional roles therein. CREs display a continuum of structures, ranging from short disordered stretches to fully folded domains. The adaptability demonstrated by CREs in achieving the common goal of kinase inhibition spans from direct autoinhibitory interaction with the active site within the kinase domain, to CREs binding to an alternative site, inducing allosteric regulation revealing distinct types of inhibitory mechanisms, which we exemplify by archetypical representative systems. While this study provides a systematic approach to comprehend kinase CREs, further experimental investigations are imperative to unravel the complexity within distinct kinase families. The insights gleaned from this research lay the foundation for future studies aiming to decipher the molecular basis of kinase dysregulation, and explore potential therapeutic interventions.

PurposeTo detect the prognostic importance of liquid-liquid phase separation (LLPS) in lung adenocarcinoma.MethodsThe gene expression files, copy number variation data, and clinical data were downloaded from The Cancer Genome Atlas cohort. LLPS-related genes were acquired from the DrLLPS website. The prognostic model based on LLPS was constructed by the Cox regression and LASSO regression analyses after the identification of LLPS-related differentially expressed genes (DEGs). Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed. The LLPS-related prognostic risk score was validated by GSE31210 and GSE72094. The overall survival of lung adenocarcinoma patients was predicted by plotting a nomogram. The biological features of the high-risk lung adenocarcinoma were evaluated by the CIBERSORT, ESTIMATE, Gene Set Variation Analysis, and Genomics of Drug Sensitivity in Cancer. Reverse transcription-quantitative polymerase chain reaction detected hub gene expression.ResultsA total of 91 DEGs were screened out in LLPS, among which 9 genes were discovered as prognostic biomarkers of lung adenocarcinoma. GRIA1, CRTAC1, MAGEA4, and MAPK4 were identified as hub genes by the LASSO Cox regression analysis. High-risk and low-risk groups were divided according to the risk index, with the high-risk group displaying a markedly worse outcome. CRTAC1 expression was significantly decreased, MAGEA4 and MAPK4 expressions were increased, while GRIA1 expression was altered in lung adenocarcinoma cells. Tumor microenvironment, signaling pathway enrichment, and drug sensitivity significantly differed between different risk groups.ConclusionsThis work proposed a prognostic tool based on the LLPS-related gene signature to offer prospective and effective biomarkers for lung adenocarcinoma prognosis.

Macroautophagy is a catabolic process that maintains cellular homeostasis by recycling intracellular material through the use of double-membrane vesicles called autophagosomes. In turn, autophagosomes fuse with vacuoles (in yeast and plants) or lysosomes (in metazoans), where resident hydrolases degrade the cargo. Given the conservation of autophagy, Saccharomyces cerevisiae is a valuable model organism for deciphering molecular details that define macroautophagy pathways. In yeast, macroautophagic pathways fall into two subclasses: selective and nonselective (bulk) autophagy. Bulk autophagy is predominantly upregulated following TORC1 inhibition, triggered by nutrient stress, and degrades superfluous random cytosolic proteins and organelles. In contrast, selective autophagy pathways maintain cellular homeostasis when TORC1 is active by degrading damaged organelles and dysfunctional proteins. Here, selective autophagy receptors mediate cargo delivery to the vacuole. Now, two groups have discovered a new hybrid autophagy mechanism, coined cargo hitchhiking autophagy (CHA), that uses autophagic receptor proteins to deliver selected cargo to phagophores built in response to nutrient stress for the random destruction of cytosolic contents. In CHA, various autophagic receptors link their cargos to lipidated Atg8, located on growing phagophores. In addition, the sorting nexin heterodimer Snx4-Atg20 assists in the degradation of cargo during CHA, possibly by aiding the delivery of cytoplasmic cargos to phagophores and/or by delaying the closure of expanding phagophores. This review will outline this new mechanism, also known as Snx4-assisted autophagy, that degrades an assortment of cargos in yeast, including transcription factors, glycogen, and a subset of ribosomal proteins.

Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 h post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.

Biomolecular condensates (BMCs) are crucial for cellular organization and function, and their dysregulation is linked to neurological, oncologic and inflammatory diseases. This highlights the need for advanced investigative tools leading to targeted BMC therapeutics. To address this need, Nereid Therapeutics uses Corelet™ technology and an automated high-throughput screening (HTS) platform to precisely quantify phase separation events and identify BMC modulators for previously undruggable targets. Hundreds of thousands of small molecules have been screened utilizing Corelet technology, yielding small molecule BMC-modulating compounds which serve as the basis for the development of targeted therapies for diseases with high unmet need.

Spatiotemporally controlled laser-induced phase separation (LIPS) offers unique research avenues and has potential for biological and biomedical applications. However, LIPS conditions often have drawbacks for practical use, which limit their applications. For instance, LIPS droplets are unstable and diminish after the laser is terminated. Here, we developed a novel LIPS method using laser-induced Soret effect with a simple setup to solve these problems. We generate liquid-liquid phase-separated (LLPS) droplets using LIPS in an aqueous two-phase system (ATPS) of dextran (DEX) and polyethylene glycol (PEG). When DEX-rich droplets were generated in the DEX/PEG mix on the phase boundary, the droplets showed unprecedently high longevity; the DEX droplets were retained over 48 h. This counterintuitive behaviour suggests that the droplet is in an unknown metastable state. By exploiting the capability of DEX-rich droplets to enrich nucleic acid polymers, we achieved stable DNA enrichment in LIPS DEX droplets with a high enrichment factor of 1400 ± 400. Further, we patterned DNA-carrying DEX-rich droplets into a designed structure to demonstrate the stability and spatiotemporal controllability of DEX-rich droplet formation. This is the first report for LIPS droplet generation in a DEX/PEG system, opening new avenues for biological and medical applications of LIPS.

Biomolecular condensate formation driven by intrinsically disordered proteins (IDPs) is regulated by interactions between various domains of the proteins. Such condensates are implicated in various neurodegenerative diseases. The presynaptic intrinsically disordered protein, α-Syn is involved in the pathogenesis of Parkinson's disease. The central non-amyloid β-component (NAC) domain in the protein is considered to be a major driver of pathogenic aggregation, although recent studies have suggested that the P1 domain from the flanking N-terminal region can act as a 'master controller' for α-Syn function and aggregation. To gain molecular insight into the phase behavior of the P1 domain itself, we investigate how assemblies of P1 (residues 36-42) chains phase separate with varying temperatures using all-atom molecular dynamics simulations. The simulations reveal that P1 is able to phase separate above a lower critical solution temperature. Formation of a condensed phase is driven by exclusion of water molecules by the hydrophobic chains. P1 chain density in the condensate is determined by weak multi-chain interactions between the residues. Moreover, tyrosine (Y39) is involved in the formation of strongest contacts between residue pairs in the dense phase. These results provide a detailed picture of condensate formation by a key segment of the α-Syn molecule.

Liquid-liquid phase separation (LLPS) enables the formation of membraneless organelles, essential for cellular organization and implicated in diseases. We introduce catGRANULE 2.0 ROBOT, an algorithm integrating physicochemical properties and AlphaFold-derived structural features to predict LLPS at single-amino-acid resolution. The method achieves high performance and reliably evaluates mutation effects on LLPS propensity, providing detailed predictions of how specific mutations enhance or inhibit phase separation. Supported by experimental validations, including microscopy data, it predicts LLPS across diverse organisms and cellular compartments, offering valuable insights into LLPS mechanisms and mutational impacts. The tool is freely available at https://tools.tartaglialab.com/catgranule2 and https://doi.org/10.5281/zenodo.14205831 .

The Balbiani body (Bb), which was discovered about 170 years ago, is a membraneless organelle in the oocyte in most species. In organisms like Xenopus and Zebrafish, Bb accumulates mitochondria, endoplasmic reticulum (ER), and germline determinants and regulates the proper localization of germline determinants. The Bb forms around the centrosome in the oocyte during early oogenesis. The mechanism behind its assembly has gained attention only very recently. Here, we report that overexpression of the germ plasm matrix protein Xvelo leads to the formation of a 'Bb-like' structure in somatic cells. The 'Bb-like' structure assembles around the centrosome and selectively recruits mitochondria, ER, and germline determinants. Taking advantage of this system, we investigated the roles of centrosome components on the assembly of Xvelo. Our results reveal that multiple components of the centrosome, including Sas6, Cenexin, and DZIP1, interact with Xvelo and promote its assembly, with Sas6 exhibiting the most prominent activity. Importantly, knocking down Sas6, Cenexin, and DZIP1 individually or in combination resulted in reduced Xvelo aggregates. Taken together, our work suggests that the centrosome may function as a nucleation center to promote the initiation of Xvelo assembly, resulting in the formation of the Bb around the centrosome.

Understanding the interaction between nanomaterials and cellular structures is crucial for nanoparticle applications in biomedicine. We have identified a subtype of stress granules, called nanomaterial-provoked stress granules (NSGs), induced by gold nanorods (AuNRs). These NSGs differ from traditional SGs in their physical properties and biological functions. Uptake of AuNRs causes reactive oxygen species accumulation and protein misfolding in the cell, leading to NSG formation. Physically, NSGs have a gel-like core and a liquid-like shell, influenced positively by HSP70 and negatively by HSP90 and the ubiquitin-proteasome system. AuNRs promote NSG assembly by interacting with G3BP1, reducing the energy needed for liquid-liquid phase separation (LLPS). NSGs impact cellular functions by affecting mRNA surveillance and activating Adenosine 5'-monophosphate (AMP)-activated protein kinase signaling, crucial for a cellular stress response. Our study highlights the role of LLPS in nanomaterial metabolism and suggests NSGs as potential targets for drug delivery strategies, advancing the field of nanomedicine.

The formation of self-perpetuating protein aggregates such as amyloids is associated with various diseases and provides a basis for transmissible (infectious or heritable) protein isoforms (prions). Many human proteins involved in the regulation of transcription contain potentially amyloidogenic regions. Here, it is shown that short N-terminal isoforms of the human protein PHC3, a component of the chromatin-modifying complex PRC1 (Polycomb repressive complex 1), can form prion-like aggregates in yeast assays, exhibit amyloid properties in the E. coli-based C-DAG assay, and produce detergent-resistant aggregates when ectopically expressed in cultured human cells. Moreover, aggregates of short isoforms can sequester the full-length PHC3 protein, causing its accumulation in the cytosol instead of the nucleus. The introduction of an aggregating short PHC3 isoform alters the transcriptional profile of cultured human cells. It is proposed that the aggregation of short isoforms is involved in the feedback downregulation of PRC1 activity, leading to more open chromatin configuration.

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart-failure syndromes remains mechanistically unexamined. We observed mislocalization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post-myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by the R120G mutation in the cognate chaperone protein CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine 59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phosphomimetic mutation of serine 59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates, and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knockin was sufficient to induce desmin mislocalization and myocardial protein aggregates, while S59A CRYAB knockin rescued left ventricular systolic dysfunction after myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine 59 phosphorylation and rescued post-myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

PIWI-interacting (pi) RNAs play an essential role in protecting the genomic integrity of germ cells from the disruptive transpositions of selfish genetic elements. One of the most important model systems for studying piRNA biogenesis is the ovary derived BmN4 cell line of the silkworm Bombyx mori. In recent years, many steps and components of the pathways involved in this process have been unraveled. However, a holistic description of piRNA biogenesis in BmN4 cells is still unavailable. In this paper, we review the state of the art and propose a novel model for piRNA biogenesis in BmN4 cells. This model was built considering the latest published data and will empower researchers to plan future experiments and interpret experimental results.

Uncovering what drives select biomolecules to form phase-separated condensates in vivo and identifying their physiological significance are topics of fundamental importance. Here we show that nitrogen-starved Escherichia coli produce long-chain polyphosphates, which scaffold the RNA chaperone Hfq into phase-separating high molecular weight complexes together with components of the RNA translation and processing machinery. The presence of polyphosphate within these condensates, which we termed HP-bodies, controls Hfq function by selectively stabilizing polyadenylated RNAs involved in transcription and protein translation, and promoting interactions with translation- and RNA-metabolism-associated proteins involved in de novo protein synthesis. Lack of polyphosphate prevents HP-body formation, which increases cell death and significantly hinders recovery from N-starvation. In functional analogy, we demonstrate that polyP contributes specifically to the formation of Processing (P)-bodies in human cell lines, revealing that a single, highly conserved and ancestral polyanion serves as the universal scaffold for functional phase-separated condensate formation across the tree of life.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein containing low-complexity domains (LCDs), which are known to regulate protein behavior under stress conditions. This study demonstrates the ability to control hnRNPK's transitions into four distinct material states-monomer, soluble aggregate, liquid droplet, and fibrillar hydrogel-by modulating environmental factors such as temperature and protein concentration. Importantly, the phase-separated and hydrogel states are newly identified for eGFP-hnRNPK, marking a significant advancement in understanding its material properties. A combination of biophysical techniques, including DLS and SEC-LS, were used to further characterize hnRNPK in monomeric and soluble aggregate states. Structural methods, such as SANS, SAXS, and TEM, revealed the elongated morphology of the hnRNPK monomer. Environmental perturbations, such as decreased temperature or crowding agents, drove hnRNPK into phase-separated or gel-like states, each with distinct biophysical characteristics. These novel states were further analyzed using SEM, X-ray diffraction, and fluorescence microscopy. Collectively, these results demonstrate the complex behaviors of hnRNPK under different conditions and illustrate the properties of the protein in each material state. Transitions of hnRNPK upon condition changes could potentially affect functions of hnRNPK, playing a significant role in regulation of hnRNPK-involved processes in the cell.

Biomolecular condensates perform diverse physiological functions. Previous work showed that VASP, a processive actin polymerase, forms condensates that assemble and bundle actin. Here, we show that this behavior does not require proteins with specific polymerase activity. Specifically, condensates composed of Lamellipodin, a protein that binds actin but is not an actin polymerase, were also capable of assembling actin filaments. To probe the minimum requirements for condensate-mediated actin bundling, we developed an agent-based computational model. Guided by its predictions, we hypothesized that any condensate-forming protein that binds filamentous actin could bundle filaments through multivalent crosslinking. To test this, we added a filamentous-actin-binding motif to Eps15, a condensate-forming protein that does not normally bind actin. The resulting chimera formed condensates that facilitated efficient assembly and bundling of actin filaments. Collectively, these findings broaden the family of proteins that could organize cytoskeletal filaments to include any filamentous-actin-binding protein that participates in protein condensation.

Translation initiation factor 2 (IF2) plays an essential role in bacterial cells by delivering the fMet-tRNAfMet to the ribosome pre-initiation complex. IF2 is known to have an N-terminal disordered region which is present across bacterial species, yet its function is not fully understood. Deletion of the IDR in E. coli showed no phenotypes at normal growth temperature (37°C); however, this IDR was found to be required for growth at cold temperatures (15°C). Since large IDRs can drive phase separation of various RNA binding proteins into biomolecular condensates, we investigated whether E. coli IF2 could phase separate. We discovered that IF2's N-terminal IDR drives phase separation in E. coli and C. crescentus, suggesting that IF2 condensation is a conserved property. Finally, using E. coli, we found that the IDR strongly drives phase separation in the cold, suggesting IF2 condensates promote fitness during cold stress.