1.4 From aggregation to division - phase separation
What is phase separation?
Phase separation refers to the spontaneous partitioning of a system into subsystems with distinct macroscopic properties (Weber et al,. 2018).
Phase separation in daily life:
There is one kind of cold sauce or dressing made from a mixture of vinegar, oil, pepper, and salt, to which various flavorings may be added called vinaigrette. People are sometimes find annoying when make vinaigrette and leave it, but only come back to find that the oil and vinegar have de-mixed into two different phases: an oil phase and a vinegar phase (you could only mix them add some other egg whites, which serves as an emulgator ).
This kind of liquid de-mixing is called emulsion, which usually distinguish from suspension (liquid-solid) mixture. One intriguing question is that: What drives demixing and how to sustain mixture state?
Before we answer this question, we first review some of the basic physics of different state (A. Hyman et al,. 2014).
A liquid is a state of matter in which components can easily rearrange. Roughly speaking, we can distinguish liquids from solids, in which components do not easily rearrange and exhibit a different degree of order. More precisely, in solids, particles are caged; in other words, particles keep their specific neighborhood for a long time. In liquids, particles change their neighborhood quickly.
A solid is a material that can be cast in an arbitrary shape, and the system keeps a memory of this reference shape for very long times. If it is deformed, it will tend to return to the initial shape, unless it breaks. The property with which it resists shape deformation is called shear elasticity.
A gel usually means a cross-linked network of polymeric structures. In biology, we usually think of biological gels as typical examples of physical gels, because they are held together by forces that are weaker than covalent bonds. One important type of gel in biology is a hydrogel. A hydrogel is a gel that has a high water content and cross-linked components that are water soluble. This means that water enters and swells the gel, and squeezing out water requires an external force (nuclear pore complex and formation of structures by RNA-binding proteins.
What drives mixing or demixing?
Mixing: Multicomponent systems often tend to mix spontaneously and are then found in a homogeneous mixed state.This is a consequence of a system’s tendency to increase entropy. Entropy characterizes the amount of dis-order in a system. The second law of thermodynamics states that entropy increases when processes happen spontaneously. Therefore, the mixing entropy generally drives the mixing of initially unmixed components.To mix, particles must be transported, which typically occurs via diffusion. How is this diffusive transport related to the mixing entropy? In general, particle flux is driven by differences in chemical potential.
Demixing: If mixing will increase the entropy, in the case of vinaigrette, why is entropy not driving the system to a mixed state? The separation into two phases is driven by the physical interactions between the oil molecules and “vinegar molecules.” Specifically, if oil molecules neighbor other oil molecules, the system has lower energy than if oil molecules neighbor “vinegar molecules.” It is this energy reduction by demixing that opposes entropy-driven mixing.
How to sustain mixing state?
Basically, there are two kinds of mechanism for a system to remain phase separation state (C. Lee at el,. 2018):
1) Equilibrium (passive) phase separation
Interactions between molecules can cause a homogeneous system to undergo a phase separation, i.e. the spontaneous partitioning of a system into multiple phases of distinct properties such as concentration. The transition from the homogeneous state to the phase-separated state is controlled by parameters such as temperature, pressure or concentrations.
If we look at those phase boundaries, diffusive fluxes are not generated by concentration differences across the phase boundary. This is because there is no chemical potential difference across the interface. It is possible to have two phases with different composition in which the chemical potentials are equal because the chemical potential changes nonmonotonically with concentrations. This means that there can exist two concentrations with the same chemical potential (A. Hyman et al,. 2014).
Figure3 shows a equilibrium phase diagram of a ternary mixture composed of molecules P (red disks), S (blue disks) and C (not shown). Outside the phase boundary (green curve) the system is homogeneous (‘ ’ symbol). Inside the phase boundary (‘♦’ symbol) the system phase separates into two phases ‘in’ and ‘out’ of distinct concentrations. The coexistence concentrations Pˆin,out, Sˆin,out are given by the intersections between the tie-lines (straight lines) and the phase boundary.
2) Non-equilibrium (active) phase separation
The presence of non-equilibrium chemical reactions (for instance, consuming ATP) have been proposed recently to explain multi-drop stability in the cytoplasm, as well as being a mechanism to control the formation, dissolution and size of membrane-less organelles. For example: centrioles (Zwicker et al,. 2014) , stress granules (JD Wurtz et al,. 2018). We will discuss more about non-equilibrium (active) phase separation in cell next.
Figure4 Model of non-equilibrium phase separation in the cell cytoplasm. We consider the ternary mixture from section 2.2 with the addition of chemical reactions. Molecules P can convert into S with the rate constant k and vice versa with the rate constant h (equation (6)). S does not participate to phase separation, i.e. its concentration remains continuous at the drop interfaces (Sˆin = Sˆout). © J. Phys. D: Appl. Phys. C. Lee at el,. 2018.
Phase separation in cell
Cell functions are the results from chains of reactions happen in the interior cell. A cell is a combo of many modules and elements. Be capable of precise functions, cells have evolved more than meticulous to be organized. On top of the functions, one serious problem is that: How do they organize complex biochemical reactions in space? It turns out that cells have at least two strategies nevertheless one is very familiar to most of us, the other type of strategy has mindblowed the community in recent years.
The first strategy is compartmentalization. Many compartments in cells are separated by membranes, such as mitochondria or lysosomes. Compartmentalization mechanism benefit the cell from at least two aspects:
Increase the reaction rates inside the compartment.
Sequester or store the molecules within the compartment.
However, the fact that many compartments do not have membranes confused people to wonder why they not simply mix with their surroundings and what's their functions?
Figure 5. Membrane-less organelles in HeLa cells. Protein markers are coilin (Cajal bodies), DCP1A (P bodies), MKI67 (nucleoli), G3BP1 (stress granules), and PML (PML bodies). © Alberti S. Phase separation in biology[J]. Current Biology, 2017, 27(20): R1097-R1102.
Cajal bodies are regions within the nucleus that are enriched in proteins and RNAs involved in mRNA processing. They are the main sites for the assembly of small nuclear ribonucleoproteins (snRNPs). (nature subjects - cajal body)
Nuclear speckles are localized adjacent to nucleoplasmic interchromatin regions, a distinct class of dynamic organelles. The composition of nuclear speckles, enriched in pre-mRNA splicing factors, such as small nuclear ribonucleoproteins (snRNPs) and serine/arginine-rich (SR) proteins, and poly(A)+ RNA , as well as their spatial proximity to sites of active transcription, suggest they may play a role in regulating gene expression by supplying or storing factors associated with the splicing of pre-mRNAs .
PML (promyelocytic leukaemia) bodies are spherical structures in the nucleus that can measure up to 1 µM in diameter. The principal organizing component of PML bodies is the PML protein. PML bodies vary in composition and have been implicated in cellular processes such as telomere lengthening and the DNA damage response. (nature subject - PML body)
The nucleolus (plural: nucleoli) is a non-membrane bound organelle inside the nucleus and it is responsible for the synthesis, processing, and assembly of ribosomes. It is also involved in several other cellular processes, such as mitosis, stress response, and cell cycle regulation. Structurally, the nucleoli consists of three subregions: the fibrillar centers, dense fibrillar components, and granular components (FC, DFC and GC). (protein atlas )
mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP (ribonucleoprotein) granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. It contain markers like repressor/decapping activator Rck/p54/dhh1 or the decapping enzyme Dcp2. (Since direct topics)
Stress granules are aggregates composed of proteins and RNA molecules, mostly stalled translation initiation complexes that usually form in a reversible manner upon cellular stress. Stress granules can also precipitate the formation of toxic protein aggregates such as those seen during the progression of certain types of neurological disease.(nature subject - stress granules)
A comprehensive introduction on different Nuclear membrane-less bodies function are reviewed by Mitrea et al,. 2016.
P granules in C.elegans embryos
In C. elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior (showed in the right side in the video) of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. In 2009, Brangwynne et al,. first proposed the liquid feature of P granule, which then have drawn much more interest into the filed of non-membrane cell organs and phase separation.
These granules are described as liquids for the following observations (Brangwynne et al,. 2009, A. Hyman et al,. 2014):
Two P granules can fuse after touching, and the two P granules together revert back to a
spherical shape.
P granules can also be seen to drip off nuclei. In other words, P granules deform in shear
flows in a manner similar to that of liquid droplets.
Although they exchange material with the cytoplasm, as measured by fluorescence recovery
after photobleaching, they are spherical. As mentioned above, the spherical shape is driven
by surface tension.
If you photobleach half a P granule, it will recover through internal rearrangement.
Many other non-membrane-bound compartments likely have the properties of liquids. Then we might wonder why the cell form these liquid-like organelles?
Why liquid-like phase separation?
As we talked about the advantages for cells to compartmentalization which is served to spatially confine and organize chemical reactions. liquid droplet could attract more of the same kind of molecules that resemble the ones inside the drop and grow. In the droplet, reactions may have happened that were not possible outside because there the concentrations were too low. On the other hand, the membrane less structure will allow fast dynamics of molecular rearrangement and exchange between the reactants and products which is more efficient. To summarize in three main aspects (Shin and Brangwynne 2017): ① Reaction concentration ② Sequestration ③ Organizational hub
How to form phase separation in cell?
The structure is largely dependent on the inside molecule residents inside those membrane-less organelles (mainly Proteins and RNAs). A mount of research has shown the composition of these organelles and reviewed in (Mitrea et al,. 2016 Table 1).
Protein elements: low complexity sequence and fold domains.
Proteins associated with membrane-less organelles often exhibit multivalent features (shown in Figure 6) which are manifested structurally in different ways. Folded domains are proteins segments which adopt discrete and stable secondary and tertiary structures. However, intrinsically disordered regions (IDRs) of protein are often dynamic and heterogenous. Low complexity sequences and disorder are overrepresented in proteins shown to phase separatein vitro. (Kato et al,. 2012, Elbaum-Garfinkle et al,. 2015, Nott et al,. 2015).
RNA elements:
RNA can promote phase separation events via RNA-protein and RNA-RNA interactions.
RNA-protein interactions:
Yuan et al,. 2015 demonstrate that IDRs on a number of RNA-binding proteins can phase separate alone (by the multivalent connections discussed above, IDR-IDRinteractions.) or in concert with RNA-binding domains and RNA. They generated full length RNP granules which has IDRs and observed that the full-length granule protein hnRNPA1 can phase separate in vitro, producing dynamic liquid droplets.
Maharana et al,. 2018. show that in vitro high amounts of short, nonspecific RNAs keep prion-like (have low complexity protein domain which resembles prion) RBPs soluble. Conversely, longer RNAs that can form higher-order secondary structures and which specifically bind RBPs can nucleate phase separation. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro.
RNA-RNA interactions:
In other cases, RNA alone can promote phase separate without the help of proteins, forming liquid or gel like droplets. This is because the propensity of an RNAs to form multivalent Watson–Crick base pairing mechanism themselves.
In 2017, Jain and Vale studied repeat-containing RNAs in vitro and found that interactions among CAG /CUG/ GGGGCC repeat-containing regions of RNA are sufficient to generate micrometer-scale structures in vitro that are reminiscent of disease-associated nuclear foci, this phenomenon is termed as repeat expansion disorders.A testable hypothesis that emerges from this study is that toxicity ensues when phase-separated RNA gels sequester key enzymes in vivo and thus interfere with their normal function.
Many diseases have been associated with repeat expansion. For instance, healthy individuals contain fewer than 20 repeats of the trinucleotide CAG in exon 1 of the huntingtin (HTT) gene. However, in patients with Huntington’s disease, the number increases to more than 35 repeats. Similarly, the number of GGG GCC repeats in intron 1 of the C9orf72 gene increases from 2–23 in healthy individuals to 700–1,600 in patients with amyotrophic lateral sclerosis (ALS) or fronto temporal dementia (FTD). (Saha and Hyman 2018)
Tools to study Phase Separation:
Corelets (Bracha et al,. 2018):
Corelets (Core scaffolds to promote droplets): a two-module optogenetic system that mimics the native oligomerization of IDR-rich proteins, using a light-activatable high valency core. The core is comprised of 24 human ferritin heavy chain(FTH1) protein subunits, which self-assemble to form a spherical particle of 12 nm diameter (herein referred to as a “Core”). When functionalized by self-interacting modules, these particles can give rise to supramolecular clusters. The whole system is FTH1-SspB-IDR module.
CasDrop (Shin et al,. 2018):
Taking advantage of well-characterized CRISPR-Cas9 technology for programmable targeting to specific genomic loci, Shin et al build dCas9-ST-GFP-iLID system allowing for visualization of seeded sites, at the same time providing light-inducible binding scaffolds for recruiting various protein components, including proteins that contain IDRs.
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