Phase Separation in Biological Systems Flashcards
Compartmentalisation of Cells
- in eukaryotic cells have many compartments / organelles with specfic functions and provide spatiotemparal control over cell materials / metabolic pathways etc.
- most organelles have a membrane boundary but there are also many membraneless organelles
- these are supramolecular assemblies of proteins and RNA molecules and typically form liquid droplets or hydrogels
Biophysical Assays of Membraneless Compartments
-established liquid nature of some membraneless compartments
Protein Granule Model of Membraneless Compartments
- protein granules are a good model of liquid membraneless compartments
- molecules diffuse freely within protein granule
- two protein granules can fuse into one
- one protein granule can bud of from another
- spherical shape from surface tension
Liquid-Liquid Phase Separation
Description
- entropy of mixing drives spontaneous mixing of components
- there is a predicted entropy increase with mixing and this drive to spontaneous mixing is always there
Liquid-Liquid Phase Separation
Volume Fractions
φ1 = volume fraction of component 1 φ2 = volume fraction of component 2
φ1 + φ2 = 1
Liquid-Liquid Phase Separation
Concentrations
φ = volume fraction υ = molecular volume c = concentration
c = φ/υ
Liquid-Liquid Phase Separation
De-Mixing
Description
-repulsion between components can lead to de-mixing
-two co-existing phases with volume fractions φ1=φs & φ1=φd
-there is no net-flux of molecules across the interface since:
μ1(φs) = μ1(φd)
Liquid-Liquid Phase Separation
De-Mixing
Free Energy of Mixing
F = E - T Smix
Liquid-Liquid Phase Separation
De-Mixing
Interaction Energy
E = χ12 V φ1 (1 - φ1)
-where χ12 is the FLory interaction parameter
Liquid-Liquid Phase Separation
De-Mixing
Chemical Potential
μ1 = υ1 / V dF/dφ1
Liquid-Liquid Phase Separation Surface Tension
Overview
- entails coarsening of the disperse phase (droplets)
- owing to the Laplace pressure one large droplet is favourable over many small droplets
- droplets may coarsen by fusion or Ostwald ripening
Liquid-Liquid Phase Separation Surface Tension
Laplace Pressure
ΔP = Pin - Pout = 2γ/R
-where γ is the surface tension
Liquid-Liquid Phase Separation Surface Tension
Ostwald Ripening
-droplets leave smaller droplet in preference of larger one
2D Compartments
Overview
- compartmentalisation also occurs in 2D
- e.g. in lipid bilayer membranes and polymer brushes
2D Compartments
Lipid Bilayer Membranes
- microdomains within lipid membranes
- same driving mechanisms as 3D
2D Compartments
Polymer Brushes
- glycocalyces may phase separate in 2D
- potentially driven by cross-linking of polysaccaride scaffold
2D Compartments
Perineural Nets
- HA brushes as invitro model of perineural nets
- demonstrate cross-linking by proteins can recapitulate granular and reticular phases
2D Compartments
Phases
- basic priniples are the same as for 3D
- granular and reticular phases represent polymer-rich phase being the dispose and continuous phase respectively
- a change to substrate provides extra effects (e.g. microphase separation if anchors are immobile and cannot move in plane)
Functions of Membraneless Compartments
List
- concentration of biochemical reactions
- sequestering harmful components
- storage of biomolecules
- sieving
- signal amplitude and noise reduction
Functions of Membraneless Compartments
Concentration of Biochemical Reactions
- benefits from concentrated liquid phase where reactants can easily meet
- composition may be dsitinct from surrounding continuous phase leading to different reactions
Functions of Membraneless Compartments
Sequestering Harmful Components
-protein aggregates in disease are harmful but could be the initial rescue mechanism of cells to sequester more toxic protein oligomers
Functions of Membraneless Compartments
Sieving
-nuclear pore permeability barrier controls biomolecule transport between the nucleus and cytoplasm
Functions of Membraneless Compartments
Signal Amplitude and Noise Reduction
- signal pathways amplified by conecntrating receptors in membrane micro domains
- liquid-liquid phase separation decrease protein concentration fluctuations thereby reducing noise in signalling pathways
Phase Separation as a Mechanism to Reduce Noise in Cells
- proteins are produced in the cell cytoplasm by ribosomes, there are many ribosomes but only a small number will be producing any particular protein at any given time
- this can lead to large variations in protein concentration across the cell but for biochemical reactions it is useful to reduce theis noise and acheive a more uniform concentration
- liquid-liquid phase separtion provides a mechanism to reduce this noise by the creation of two phases, the dilute phase of low protein concentration and the dispere phase of high protein concetration
- when total protein concentration changes, the size and number of the droplets changes but the concentrations in both the continuous and disperse phases remains constant
Does phase separtion theory hold in non-equilbirium conditions in real cells?
- the important factors are protein diffusion time and protien turnover time
- if diffusion time is < < turn over time the cell is essentially at equilibrium all the time
- good agreement with experiment
Dynamics of Phase Separation
Using Phase Separation in Cells
-to harness phase separation, cells need to control kinetics of phase separation and droplet size
Dynamics of Phase Separation
Initiation of New Droplet
-may require a nucleation event (as in crystlisation)
Dynamics of Phase Separation
Nucleation
- homogeneous nucleation, spontaneous nucleatoin via random fluctuatoin
- heterogeneous nucleation, at dedicated sites have regions which promote nucleation to help control where and how many components form
Dynamics of Phase Separation
Fusion
- spontaneous fusion of droplets may be avoided by:
- -physical separation e.g. entrapment in the cytoskeletal network
- -surface active molecules (e.g. polymer prush on chromosomes), molecules are essentially designed to repel each other
What are the requirements on proteins to phase separate?
- proteins known to drive phase separation have distinct features:
- -large number of interaction motifs (polyvalency)
- -individual interactions are weak (low affinity, few kbT)
- -intrinsically disordered linkers that separate interaction motifs (conformational flexibility)
Importance of Linker Properties for Phase Transition
- simulations highlight the importance of linker properties for phase transition:
- -flexible linkers promote phase transition and gelation
- -bulky linkers promote gelling without phase transition
Physics Challenges of Phase Separation
- important aspects of phase separation are still poorlt understood
- phase separation in multi component, multi compartment systems is complex
- same for phase separation in active cells
Experimental Challenges of Phase Separation
- compositional complexity of cytoplasm makes system control and quantitative analysis challenging
- combination of well-defined reconsitiuted in-vitro models and complex real cell models will be required to generate quantitative models of underpinning mechanisms and validate biological relevance
Phase Separation and Superselectivity
- polyvalency, low affinity and conformational flexibility are key ingredients for protein phase separation
- these are the same criteria as for superselectivity
- could there be a connection?
