Phase Separation in Biological Systems

Question 1

Compartmentalisation of Cells

Answer 1

• 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

Question 2

Biophysical Assays of Membraneless Compartments

Answer 2

-established liquid nature of some membraneless compartments

Question 3

Protein Granule Model of Membraneless Compartments

Answer 3

• 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

Question 4

Liquid-Liquid Phase Separation

Description

Answer 4

• 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

Question 5

Liquid-Liquid Phase Separation

Volume Fractions

Answer 5

φ1 = volume fraction of component 1
φ2 = volume fraction of component 2

φ1 + φ2 = 1

Question 6

Liquid-Liquid Phase Separation

Concentrations

Answer 6

φ = volume fraction
υ = molecular volume
c = concentration

c = φ/υ

Question 7

Liquid-Liquid Phase Separation
De-Mixing
Description

Answer 7

-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)

Question 8

Liquid-Liquid Phase Separation
De-Mixing
Free Energy of Mixing

Answer 8

F = E - T Smix

Question 9

Liquid-Liquid Phase Separation
De-Mixing
Interaction Energy

Answer 9

E = χ12 V φ1 (1 - φ1)

-where χ12 is the FLory interaction parameter

Question 10

Liquid-Liquid Phase Separation
De-Mixing
Chemical Potential

Answer 10

μ1 = υ1 / V dF/dφ1

Question 11

Liquid-Liquid Phase Separation Surface Tension

Overview

Answer 11

• 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

Question 12

Liquid-Liquid Phase Separation Surface Tension

Laplace Pressure

Answer 12

ΔP = Pin - Pout = 2γ/R

-where γ is the surface tension

Question 13

Liquid-Liquid Phase Separation Surface Tension

Ostwald Ripening

Answer 13

-droplets leave smaller droplet in preference of larger one

Question 14

2D Compartments

Overview

Answer 14

• compartmentalisation also occurs in 2D

- e.g. in lipid bilayer membranes and polymer brushes

Question 15

2D Compartments

Lipid Bilayer Membranes

Answer 15

• microdomains within lipid membranes

- same driving mechanisms as 3D

Question 16

2D Compartments

Polymer Brushes

Answer 16

• glycocalyces may phase separate in 2D

- potentially driven by cross-linking of polysaccaride scaffold

Question 17

2D Compartments

Perineural Nets

Answer 17

• HA brushes as invitro model of perineural nets

- demonstrate cross-linking by proteins can recapitulate granular and reticular phases

Question 18

2D Compartments

Phases

Answer 18

• 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)

Question 19

Functions of Membraneless Compartments

List

Answer 19

• concentration of biochemical reactions
• sequestering harmful components
• storage of biomolecules
• sieving
• signal amplitude and noise reduction

Question 20

Functions of Membraneless Compartments

Concentration of Biochemical Reactions

Answer 20

• benefits from concentrated liquid phase where reactants can easily meet
• composition may be dsitinct from surrounding continuous phase leading to different reactions

Question 21

Functions of Membraneless Compartments

Sequestering Harmful Components

Answer 21

-protein aggregates in disease are harmful but could be the initial rescue mechanism of cells to sequester more toxic protein oligomers

Question 22

Functions of Membraneless Compartments

Sieving

Answer 22

-nuclear pore permeability barrier controls biomolecule transport between the nucleus and cytoplasm

Question 23

Functions of Membraneless Compartments

Signal Amplitude and Noise Reduction

Answer 23

• signal pathways amplified by conecntrating receptors in membrane micro domains
• liquid-liquid phase separation decrease protein concentration fluctuations thereby reducing noise in signalling pathways

Question 24

Phase Separation as a Mechanism to Reduce Noise in Cells

Answer 24

• 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

Question 25

Does phase separtion theory hold in non-equilbirium conditions in real cells?

Answer 25

- 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

Question 26

Dynamics of Phase Separation | Using Phase Separation in Cells

Answer 26

-to harness phase separation, cells need to control kinetics of phase separation and droplet size

Question 27

Dynamics of Phase Separation | Initiation of New Droplet

Answer 27

-may require a nucleation event (as in crystlisation)

Question 28

Dynamics of Phase Separation | Nucleation

Answer 28

- 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

Question 29

Dynamics of Phase Separation | Fusion

Answer 29

- 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

Question 30

What are the requirements on proteins to phase separate?

Answer 30

- 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)

Question 31

Importance of Linker Properties for Phase Transition

Answer 31

- simulations highlight the importance of linker properties for phase transition: - -flexible linkers promote phase transition and gelation - -bulky linkers promote gelling without phase transition

Question 32

Physics Challenges of Phase Separation

Answer 32

- 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

Question 33

Experimental Challenges of Phase Separation

Answer 33

- 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

Question 34

Phase Separation and Superselectivity

Answer 34

- 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?