Microbial biomolecular condensates: from conserved principles to synthetic biology opportunities.

Targeting of biomolecular condensates to the autophagy pathway.

Membrane-less organelles or compartments are considered to be dynamic reaction centers for spatiotemporal control of diverse cellular processes in eukaryotic cells. Although their formation mechanisms have been steadily elucidated via the classical concept of liquid–liquid phase separation, biomolecular behaviors such as protein interactions inside these liquid compartments have been largely unexplored. Here we report quantitative measurements of changes in protein interactions for the proteins recruited into membrane-less compartments (termed client proteins) in living cells. Under a wide range of phase separation conditions, protein interaction signals were vastly increased only inside compartments, indicating greatly enhanced proximity between recruited client proteins. By employing an in vitro phase separation model, we discovered that the operational proximity of clients (measured from client–client interactions) could be over 16 times higher than the expected proximity from actual client concentrations inside compartments. We propose that two aspects should be considered when explaining client proximity enhancement by phase separation compartmentalization: (1) clients are selectively recruited into compartments, leading to concentration enrichment, and more importantly, (2) recruited clients are further localized around compartment-forming scaffold protein networks, which results in even higher client proximity.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organize different substrates in concentric subcompartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides expanding our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

Membrane-less compartments, such as biomolecular condensates, play a crucial role in cellular organization by enhancing enzymatic reaction efficiency through compartmentalization and crowding. This has considerable potential for improving in vitro diagnostics. However, the practical biomedical applications of intracellular microenvironments for in vitro diagnostics remain in their infancy. Herein, inspired by membrane-less compartments from biomolecular condensates, a crowded hydrogel-based approach to mimic the intracellular microenvironment through the biocompatible porous structure of hydrogels, thereby promoting the compartmentalization and aggregation of enzymatic reactions to achieve highly sensitive in vitro nucleic acid diagnosis, is proposed. This hydrogel-based system demonstrates significantly enhanced polymerase chain reaction amplification efficiency through enhanced primer-template binding and enzyme activity-mediated single-stranded DNA extension. This enhancement primarily arises from improved molecular interactions driven by excluded-volume effects in crowded environments as well as hydrogel compartmentalization. With high enzymatic efficiency, a two-orders-of-magnitude lower detection limit for pathogens is achieved. These findings suggest that crowded hydrogels have the potential to bridge the gap between the intracellular environment and in vitro applications, offering a novel strategy for advanced molecular diagnostics.

Biomacromolecular phase separation or phase transition is a cutting-edge area of biological research which has been rapidly developing in recent years. Cellular liquid-liquid phase separation, which is mediated by related biomacromolecules, drives the formation of many membrane-less compartments, such as nucleoli, centromeres, centrosomes, P granules, stress granules (SGs) and some signal transduction complexes. These biomolecular condensates are micron-scale membrane-less compartments in cells that function to concentrate biomacromolecules and they originate from liquid-liquid phase separation (LLPS), which is driven by multivalent interactions among biomacromolecules, including proteins and nucleic acids. Biomacromolecular phase separation has extensive biological functions. First, it regulates biochemical reactions in cells. For example, concentrating reactants inside biomolecular condensates can change reaction kinetics and substrate specificities. Second, it allows for fast changes in molecules upon sensing and responding to stress. Third, it can buffer cellular concentrations of biomacromolecules. Finally, it allows direct communication between membrane-less and membrane-bound organelles. Aberrant phase separation has been proven to be associated with many human diseases. Proteins involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) are components of biomolecular condensates. Dysregulation in the formation of these components leads to pathological aggregates. Aberrant phase separation is also involved in cancer. An example is the tumor suppressor speckle-type POZ protein (SPOP), which is involved in the ubiquitination and proteasomal degradation of related substrates. The substrates are proto-oncogenic proteins that trigger phase separation of SPOP and its co-localization in a membrane-less compartment. Mutations in SPOP that interfere with its substrate interactions cause prostate and breast cancer, as well as other solid tumors. Emerging evidence suggests that aberrant phase separation is also related to infectious diseases. For example, many viruses produce factors that inhibit SG formation or promote changes in SG composition that suppress cellular stress responses and promote viral replication. Therefore, targeting the regulators of phase separation could be a promising therapeutic approach for these human diseases. Although there have been rapid developments in biomacromolecular phase separation worldwide, this emerging field has attracted increased attention in China over the past 5 years. A series of outstanding achievements have established a prominent role for China in the international community of biomacromolecular phase separation research. Moreover, many excellent research teams have been established in Chinese universities and institutes. Currently, China is in a critical period of striving to be at the international frontier of biomacromolecular phase separation research. To further enhance China’s advantage in this emerging field, the following developmental proposals are discussed in this review: (1) Establishing academic research standards for biomacromolecular phase separation; (2) enhancing interdisciplinary research and developing a biomacromolecular phase separation theory; (3) developing related cutting-edge technologies and methods; (4) strengthening functional in vivo studies for biomacromolecular phase separation that elucidate the underlying molecular mechanisms and their relationships with human diseases; and (5) gathering outstanding scientists through the full utilization of academic organizations.

Eukaryotic cells contain many membrane-less compartments, such as P granules, stress granules and p62 bodies, which are composed of highly concentrated biomacromolecules. These compartments are involved in various cellular processes, including germ cell specification, stress response, and selective autophagy. They are called “biomolecular condensates” and function in organizing cellular biochemical reactions, to make them relatively independent, highly ordered, and highly efficient. According to recent studies, biomolecular condensates exhibit liquid-like properties, such as a spherical shape, fusion upon contact and high dynamics. They arise from “liquid-liquid phase separation” (LLPS) driven by multivalent interactions among macromolecules. There are many ways to achieve macromolecular multivalence: Linear modular domains with similar function, like PRM repeats in N-WASP; oligomerization domains, like the PB1 domain in the selective autophagy receptor p62; similar post-translational modifications at multiple sites; poly-ubiquitination at one site; degenerate binding of RNA to proteins, etc. Additionally, intrinsically disordered regions (IDRs), sometimes called low complexity domains (LCDs), which contain high frequencies of a few specific amino acid types, are a common way to mediate multivalent interactions and drive LLPS. Many IDR-containing proteins, such as the P granule component PGL-3, the nuclear pore protein Nup98, and the RNA-binding protein FUS, were shown to undergo phase separation in vitro and localize to specific biomolecular condensates in vivo . RNAs with disease-related nucleotide repeat expansions, such as CUG repeats, CAG repeats, and GGGGCC repeats, can also form phase-separated puncta in vitro and in patients’ cells. Mechanisms for regulating phase separation include modulating the relationship between the concentration and saturation concentration of biomacromolecules, and altering biomolecular interactions. Regulation of transcription, translation and degradation can change biomacromolecule concentrations. Temperature, crowding agents, and ATP (which functions as a hydrotrope), can impact protein solubility. Protein modifications and novel interactions with other biomacromolecules can impact inter-molecular or/and intra-molecular interactions, thus disrupting phase separation. A special example is the nuclear transport receptor Kapβ2, which suppresses normal and aberrant phase separation of some RNA-binding proteins with IDRs, including FUS, by interacting with their PY-NLS region. The components that are essential for establishing phase separation are called “scaffolds”, and they enrich their “clients” via interaction through their free binding sites. The composition of clients can be altered by changing the stoichiometries of scaffolds. Droplets formed by different biomacromolecules, such as FIB1 and NPM, can co-exist to form immiscible multi-phase condensates, which are analogous to multi-layered membrane-less organelles in vivo , such as nucleoli and stress granules. Many in vitro phase-separation systems have been established, which can induce receptor clustering, activate actin or tubulin polymerization, facilitate enzymatic reactions, etc. Some studies have suggested a link between phase separation and heterochromatin formation or gene control. Based on reconstituted phase separation systems in vitro , and their links to biomolecular condensates in vivo , we can gain deep understanding of the assembly, dissolution, composition, physical properties, biochemical activities and cellular functions of biomolecular condensates.

Networking and Dynamic Switches in Biological Condensates

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates.

Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

An increasing number of proteins involved in bacterial cell cycle events have been recently shown to undergo phase separation. The resulting biomolecular condensates play an important role in cell cycle protein function and may be involved in development of persister cells tolerant to antibiotics. Here we report that the E. coli chromosomal Ter macrodomain organizer MatP, a division site selection protein implicated in the coordination of chromosome segregation with cell division, forms biomolecular condensates in cytomimetic systems. These condensates are favored by crowding and preferentially localize at the membrane of microfluidics droplets, a behavior probably mediated by MatP-lipid binding. Condensates are negatively regulated and partially dislodged from the membrane by DNA sequences recognized by MatP ( matS ), which partition into them. Unexpectedly, MatP condensation is enhanced by FtsZ, a core component of the division machinery previously described to undergo phase separation. Our biophysical analyses uncover a direct interaction between the two proteins, disrupted by matS sequences. This binding might have implications for FtsZ ring positioning at mid-cell by the Ter linkage, which comprises MatP and two other proteins that bridge the canonical MatP/FtsZ interaction. FtsZ/MatP condensates interconvert with bundles in response to GTP addition, providing additional levels of regulation. Consistent with discrete foci reported in cells, MatP biomolecular condensates may facilitate MatP's role in chromosome organization and spatiotemporal regulation of cytokinesis and DNA segregation. Moreover, sequestration of MatP in these membraneless compartments, with or without FtsZ, could promote cell entry into dormant states that are able to survive antibiotic treatments.

Biomolecular condensates (BCs), formed through liquid–liquid phase separation (LLPS), are membraneless compartments that dynamically regulate key cellular processes. Beyond their canonical roles in energy metabolism and apoptosis, Mitochondria harbor distinct BCs, including mitochondrial RNA granules (MRGs), nucleoids, and degradasomes, that coordinate RNA processing, genome maintenance, and protein homeostasis. These structures rely heavily on proteins with intrinsically disordered regions (IDRs), which facilitate the transient and multivalent interactions necessary for LLPS. In this review, we explore the composition and function of mitochondrial BCs and their emerging involvement in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, and Huntington’s disease. We provide computational evidence identifying IDR-containing proteins within the mitochondrial proteome and demonstrate their enrichment in BC-related functions. Many of these proteins are also implicated in mitochondrial stress responses, apoptosis, and pathways associated with neurodegeneration. Moreover, the evolutionary conservation of phase-separating proteins from bacteria to mitochondria underscores the ancient origin of LLPS-mediated compartmentalization. Comparative analysis reveals functional parallels between mitochondrial and prokaryotic IDPs, supporting the use of bacterial models to study mitochondrial condensates. Overall, this review underscores the critical role of mitochondrial BCs in health and disease and highlights the potential of targeting LLPS mechanisms in the development of therapeutic strategies.

Intracellular macromolecules have the ability to form membraneless compartments, such as vacuoles and hollow condensates, through liquid-liquid phase separation (LLPS) in order to adapt to changes in their environment. The development of artificial non-homogeneous compartments, such as multiphase hollow or multicavity condensates, has gained significant attention due to their potential to uncover the mechanisms underlying the formation of artificial condensates and biomolecular condensates. However, the complexity of design and construction has hindered progress, particularly in creating dynamic non-homogeneous compartments. In this study, we present a dynamic membraneless compartment using peptide-oligonucleotide conjugates derived from short elastin-like polypeptides (sELP-ONs), which undergo pH-mediated phase transition. Below pH 8.8, the microcompartment exists as microdroplets that transform into non-homogeneous hollow condensates above pH 8.8. Notably, these hollow condensates retain liquid properties and high molecular ordering, and effectively sequester guest molecules with a hollow condensed layer. Furthermore, our sELP-ON microcompartments exhibit a feedback-induced phase transition in response to environmental pH fluctuations generated by complex enzymatic reactions mimicking cellular metabolism, providing a novel dynamic model for creating biomimetic membraneless compartments.

Distinct membraneless organelles within cells collaborate closely to organize crucial functions. However, biosynthetic communicating membraneless organelles have yet to be created. Here we report a binary population of membraneless compartments capable of coexistence, biological communication and controllable feedback under cellular environmental conditions. The compartment consortia emerge from two orthogonally phase-separating proteins in a cell-free expression system. Their appearance can be programmed in time and order for on-demand delivery of molecules. In particular, the consortia can sense, process and deliver functional protein cargo in response to a protease message or a DNA message that encodes the protease. Such DNA-based molecular programs can be further harnessed by installing a feedback loop that controls the information flow at the messenger RNA level. These results contribute to understanding crosstalk among membraneless organelles and provide a design principle that can guide construction of functional compartment consortia.

Dysregulation of Phase Separation in Cancer

Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.