Phase Separation Science: Unveiling Cell Biology’s Hidden Order

Cells rely on intricate organization to function efficiently, yet not all structures are enclosed by membranes. Scientists have uncovered that biomolecules can self-organize through phase separation, forming dynamic compartments without physical barriers. This process regulates biological activities and maintains cellular order.

Understanding phase separation is transforming cell biology, with implications for gene regulation, signaling, and disease. Advances in imaging and molecular techniques have provided deeper insights, reshaping fundamental concepts in the field.

Biophysical Dynamics

Phase separation in cells is governed by thermodynamic and molecular interactions. Biomolecules, including proteins and nucleic acids, can demix from the surrounding cytoplasm or nucleoplasm, forming liquid-like compartments. This behavior is driven by weak multivalent interactions, where intrinsically disordered regions (IDRs) and low-complexity domains (LCDs) enable transient yet specific associations. Unlike membrane-bound organelles, these condensates assemble and disassemble in response to environmental cues. Their stability depends on enthalpic and entropic forces, influenced by temperature, ionic strength, and molecular crowding.

Studies using fluorescence recovery after photobleaching (FRAP) have shown that molecules within these condensates exchange rapidly with their surroundings, highlighting their liquid-like nature. However, some condensates can transition into gel-like or solid states, a process linked to pathological protein aggregation. Intermolecular interactions, including π-π stacking, electrostatic forces, and hydrogen bonding, dictate their internal organization and functionality.

Post-translational modifications, such as phosphorylation and methylation, alter molecular affinities, shifting phase boundaries and condensate composition. Molecular chaperones and RNA help regulate assembly, preventing aberrant phase transitions. ATP also acts as a hydrotrope, maintaining biomolecular solubility and preventing unwanted phase separation, indicating cells actively manage phase behavior through metabolic and signaling pathways.

Molecular Interactions In Cells

Phase separation and condensate formation depend on weak, transient molecular interactions, including hydrogen bonding, electrostatic attractions, and van der Waals forces. IDRs play a key role, enabling multivalent contacts that promote condensate assembly. Unlike structured domains with rigid binding sites, IDRs allow dynamic associations, adapting to cellular conditions. LCDs further stabilize condensates through π-π stacking and cation-π interactions.

RNA molecules act as scaffolds, recruiting proteins through sequence-specific and nonspecific binding. Their length and secondary structure influence condensate composition. RNA-binding proteins like FUS and TDP-43 exhibit phase separation behavior modulated by RNA concentration, balancing RNA availability and condensate stability. Post-translational modifications such as phosphorylation and ubiquitination modify charge distributions and steric properties, regulating phase behavior.

Molecular crowding amplifies weak interactions, promoting condensate formation under physiological conditions. Crowding also affects diffusion rates, shaping condensate assembly and disassembly kinetics. ATP, beyond its energy role, modulates protein solubility and prevents aberrant aggregation, suggesting cells regulate phase behavior by leveraging metabolic intermediates.

Membraneless Condensates

Cells organize biochemical reactions through membraneless condensates, which concentrate biomolecules without lipid enclosures. Unlike traditional organelles, these condensates emerge from protein and nucleic acid interactions, creating microenvironments for enzymatic activity, molecular storage, and signal transduction. Their rapid assembly and disassembly enable efficient cellular responses.

Condensates resemble liquid droplets, exhibiting surface tension, fusion, and internal fluidity. Super-resolution imaging and FRAP studies confirm their dynamic nature, with molecules exchanging between the condensate and surrounding cytoplasm. Some condensates display viscoelastic properties, behaving like gels, while others transition into solid-like assemblies over time. Their physical state depends on molecular composition, including intrinsically disordered proteins, RNA, and post-translational modifications.

Beyond molecular crowding, condensates actively regulate biochemical reactions by concentrating substrates and cofactors while excluding nonessential components. This enhances reaction rates and ensures spatial control over cellular processes. Stress granules, for example, sequester untranslated mRNAs and translation factors during stress, while nucleoli facilitate ribosomal RNA processing by localizing enzymatic machinery in a dense, phase-separated environment.

Gene Expression Roles

Phase separation plays a key role in gene regulation by creating specialized nuclear microenvironments that concentrate transcription factors, RNA polymerases, and coactivators. These condensates enhance transcription efficiency, assembling rapidly in response to signaling cues. Their formation at enhancer and promoter regions increases transcriptional output by recruiting key regulatory proteins while excluding inhibitors.

Super-enhancers, regions with high transcriptional activity, form condensates rich in mediator complexes and transcription factors, amplifying gene expression. Single-molecule imaging shows these clusters exhibit liquid-like properties, with molecules dynamically exchanging with the nucleoplasm. This fluidity enables cells to fine-tune gene expression in response to developmental and environmental changes. Disruptions in phase separation at enhancer sites are linked to diseases like cancer, where dysregulated gene expression drives uncontrolled cell proliferation.

Cell Signaling Influences

Phase separation organizes and regulates signaling pathways by concentrating signaling molecules, enhancing reaction rates and specificity. Many cascades rely on rapid protein complex assembly in response to stimuli, and phase-separated compartments achieve this organization without membranes. Scaffold proteins, often containing IDRs, facilitate the formation of dynamic condensates, ensuring efficient signal transmission.

MAPK signaling condensates regulate cell proliferation and differentiation. Components like MEK and ERK accumulate in liquid-like compartments, enhancing phosphorylation and signal amplification. Similarly, T-cell receptor signaling involves phase-separated microclusters that concentrate kinases and adaptor proteins at the plasma membrane, facilitating immune activation.

Condensates assemble and disassemble rapidly, allowing precise control over signaling responses. Dysregulation of phase-separated signaling complexes is implicated in diseases like cancer and neurodegeneration, where aberrant condensate formation leads to sustained oncogenic activation or disrupted cellular processes.

Protein Aggregation Links

While phase separation is essential for cellular function, its dysregulation can lead to pathological protein aggregation, a hallmark of neurodegenerative diseases. Normally, condensates remain dynamic, allowing proteins to diffuse in and out. However, prolonged persistence or aberrant transitions can result in solid-like aggregates resistant to cellular clearance.

Proteins linked to neurodegeneration, such as TDP-43, FUS, and tau, contain LCDs that promote phase separation. Under stress or mutation, these proteins transition into irreversible fibrillar aggregates. In ALS, for example, TDP-43 mutations enhance phase separation while reducing solubility, leading to persistent stress granules that form toxic aggregates. Similarly, tau undergoes phase separation under normal conditions, but hyperphosphorylation drives its transition into neurofibrillary tangles in Alzheimer’s disease.

Research suggests phase-separated states may act as precursors to pathological aggregation. Scientists are exploring therapeutic strategies targeting early-stage condensates to prevent irreversible aggregation. Small molecules that modulate phase separation, such as ATP-competitive inhibitors of aggregation-prone kinases, are being investigated to mitigate neurodegeneration.

Imaging And Biochemical Methods

Advances in imaging and biochemical techniques have enabled real-time visualization and manipulation of biomolecular condensates. Super-resolution microscopy, including stimulated emission depletion (STED) and structured illumination microscopy (SIM), has revealed their dynamic nature and spatial organization. FRAP has been instrumental in assessing material properties, providing insights into molecular exchange rates and phase transitions.

Biochemical methods such as turbidity assays and optogenetic tools have refined phase separation studies. Turbidity measurements quantify condensate formation by detecting light scattering, while optogenetics allows controlled induction of phase separation through light-activated protein clustering, enabling precise functional analysis.

Cryo-electron tomography has provided ultrastructural insights into condensates, revealing how molecular interactions govern phase-separated compartments. These methodological advancements continue to push phase separation research forward, shedding light on its roles in cellular regulation and disease.