Advanced Topics

Liquid-Liquid Phase Separation (LLPS) Biophysics

Summary

Liquid-liquid phase separation (LLPS) is the process by which proteins and nucleic acids demix from the cytoplasm to form concentrated, membrane-less condensates. Driven by multivalent weak interactions, LLPS underlies the formation of stress granules, nucleoli, and other biomolecular condensates.

Key Points

  • 1Demixing creates membrane-less compartments via thermodynamic phase separation
  • 2Driven by multivalent weak interactions between IDRs, modular domains, and RNA
  • 3Underlies nucleoli, stress granules, P-bodies, and other condensates
  • 4Dysregulation linked to neurodegeneration and cancer

Liquid-liquid phase separation has emerged as a fundamental organizing principle in cell biology, explaining how cells create distinct compartments without membranes.

Physical Principles

Phase Separation Basics

LLPS is governed by thermodynamics:

- Demixing: Homogeneous solution separates into two phases

- Dense phase: Concentrated in macromolecules (condensate)

- Dilute phase: Depleted of macromolecules (surrounding cytoplasm)

- Phase boundary: Sharp interface, no membrane required

Thermodynamic Framework

Phase separation occurs when:

ΔG_mix = ΔH_mix - TΔS_mix > 0

For demixing:

- Favorable interactions within each phase outweigh mixing entropy

- Temperature dependence: UCST (upper) or LCST (lower critical solution temperature)

- Concentration threshold: Critical concentration for phase separation

Flory-Huggins Theory

Polymer physics framework applied to biomolecules:

- χ parameter: Interaction strength between components

- Polymer length: Longer polymers phase separate more readily

- Binodal curve: Defines phase boundary

Molecular Drivers

Multivalent Interactions

LLPS requires multiple weak, transient interactions:

#### Protein-Protein

- Intrinsically disordered regions (IDRs): Flexible scaffolds

- Modular domains: SH3-PRM, SH2-pTyr pairs

- Electrostatic interactions: Oppositely charged patches

#### Protein-RNA

- RNA-binding domains: RRM, KH domains

- Low-complexity sequences: RGG motifs, RS domains

- RNA structure: Multivalent scaffold

Low-Complexity Domains (LCDs)

Regions enriched in specific amino acids:

- Glycine-rich: Flexibility

- Serine/Glutamine-rich: Hydrogen bonding

- Aromatic residues: Tyr, Phe for π-π stacking

- Arginine: Cation-π interactions with aromatics

Sticker-Spacer Model

Conceptual framework for LLPS-driving sequences:

- Stickers: Residues that mediate attractive interactions

- Spacers: Provide flexibility between stickers

- Valency: Number of stickers determines phase behavior

Biomolecular Condensates

Nucleolus

Site of ribosome biogenesis:

  • Multilayered organization (FC, DFC, GC)
  • Driven by nucleolar proteins (NPM1, fibrillarin)
  • rRNA processing compartmentalization

    • Stress Granules

    Form under cellular stress:

  • Contain stalled translation machinery
  • mRNAs, RNA-binding proteins (G3BP, TIA-1)
  • Transient, reversible assemblies

    • P-Bodies

    mRNA decay and storage:

  • Decapping enzymes, exonucleases
  • Translational repression
  • Dynamic exchange with cytoplasm

    • Cajal Bodies

    Nuclear bodies for snRNP assembly:

  • Coilin as scaffold protein
  • SMN complex
  • Telomerase maturation

    • Paraspeckles

    Nuclear bodies formed around lncRNA NEAT1:

  • RNA-dependent assembly
  • Sequester specific proteins and mRNAs

    • Material Properties

    Liquid-Like Behavior

    Hallmarks of liquid condensates:

    - Spherical shape: Minimizes surface tension

    - Fusion: Droplets coalesce upon contact

    - Internal mixing: Fluorescence recovery after photobleaching (FRAP)

    - Dripping/Wetting: Deformation under flow

    Viscoelasticity

    Condensates have complex rheology:

    - Viscosity: Resistance to flow

    - Elasticity: Tendency to return to shape

    - Maxwell model: Viscoelastic behavior

    Maturation and Aging

    Condensates can change over time:

    - Liquid → gel → solid: Progressive hardening

    - Fibril formation: Within condensates

    - Pathological aggregation: Disease-related transitions

    Regulation of LLPS

    Post-Translational Modifications

    PTMs tune phase behavior:

    - Phosphorylation: Often dissolves condensates

    - Methylation: Can promote or inhibit LLPS

    - Ubiquitination: Recruitment or exclusion signals

    RNA

    Nucleic acids modulate phase separation:

    - Scaffold role: RNA as multivalent platform

    - Buffering: RNA can dissolve protein condensates

    - Specificity: Sequence-dependent recruitment

    ATP

    Active processes influence condensates:

    - Chaperones: Dissolve aberrant assemblies

    - RNA helicases: Remodel RNA-protein interactions

    - Active dissolution: Energy-dependent fluidization

    Disease Connections

    Neurodegeneration

    LLPS linked to multiple diseases:

    #### ALS/FTD

  • FUS, TDP-43, hnRNPA1 mutations
  • Alter phase behavior
  • Promote pathological aggregation

    • #### Huntington's Disease

  • Polyglutamine expansion in huntingtin
  • Altered phase behavior
  • Toxic inclusions

    • Cancer

    Aberrant condensates in malignancy:

    - Oncogenic fusions: Create novel condensates

    - Transcriptional condensates: Super-enhancer regulation

    - Therapeutic targeting: Dissolving pathological condensates

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