Chapter 2 – Orientation for the Bio-Curious  21

form of which results in the well-​known MRSA superbug found in hospitals, need to with­

stand an internal osmotic pressure equivalent to ~25 atmospheres.

2.2.6  LIQUID–​LIQUID PHASE-​SEPARATED (LLPS) BIOMOLECULAR CONDENSATES

A feature of life is information flow across multiple scales, yet the physical rules that govern

how this occurs in a coordinated way from molecules through to cells are unclear; there is not,

currently, a Grand Unified Information Theory of Physical Biology. However, observations

from recent studies implicate liquid–​liquid phase separation (LLPS) in cell information

processing (Banani, 2017). Phase transitions are everywhere across multiples scales, from

cosmological features in the early universe to water boiling in a kettle. In biomolecular LLPS,

a mixture of biomolecules (typically proteins and RNA, which you will find out about later

in this chapter) coalesce inside a cell to form liquid droplets inside the cytoplasm. The transi­

tion of forming this concentrated liquid state comprising several molecules from previously

isolated molecules that are surrounded by solvent molecules of water and ions involves an

increase in overall molecular order, so the reduction in entropy since the number of access­

ible free energy microstates is lower.

In essence, the biomolecules are transitioning from being well-​mixed to demixed. Such a

process would normally be thermodynamically unfavorable, however, in this case it is driven

by a net increase in the free energy due to attractive enthalpic interactions between the

molecules in the liquid droplet on bringing them closer together. When considering the net

enthalpic increase, we need to sum up all the possible attractive interactions (often interactions

between different types of molecules) and subtract all of the total repulsive interactions (often

interactions between the same type of molecule)—​see Worked Case Example 2.1.

These liquid droplets are broadly spherical but have a relatively unstable structure; their

shape can fluctuate due to thermal fluctuations of the surrounding molecules in the cyto­

plasm, they can also grow further by accumulating of “nucleating” more biomolecules, and

also shrink reversibly, depending upon factors such as the local bimolecular concentrations

and the mixture of biomolecules and the physicochemical environment inside the cell. They

comprise components held by weak noncovalent interactions, imparting partial organiza­

tion via emergent liquid crystallinity, microrheology, and viscoelasticity, qualities that enable

cooperative interaction over transient timescales. Weak forces permit dynamics of diffusion

and molecular turnover in response to triggered changes of fluidity to facilitate the release

of molecular components. A traditional paradigm asserts that compartmentalization, which

underpins efficient information processing, is confined to eukaryotic cells’ use of membrane-​

bound organelles to spatially segregate molecular reagents. However, an alternative picture

has recently emerged of membraneless LLPS droplets as a feature of all cells that enable

far more dynamic spatiotemporal compartmentalization. Their formation is often associated

with the cell being under stress, and the big mystery in this area of research is what regulates

their size (anything from tiny droplets of a few nanometers of diameter up to several hundred

nanometers), since classical nucleation physics theory would normally predict that under the

right conditions a liquid–​liquid phase transition goes to completion, that is, a droplet will

continue to grow in size until all the biomolecular reagents are used up, but this is not what

occurs (see Chapter 8 for more details on this).

If we consider the pressure difference between the inside and outside of a droplet or radius

r as ΔP, then the force due to this exerted parallel to any circular cross-​section is simply

the total area of that cross-​section multiplied by ΔP, or FP=​ΔP.πr². This is balanced by an

opposing force due the surface tension T per unit length (a material property relating to the

biomolecular droplet and the surrounding water solvent) that acts around the circumference

of this cross-​section, of FT=​T.2πr. In steady state, FP=​ FT so ΔP=​2T/​r. What this simple ana­

lysis shows is that smaller droplets have a higher pressure difference between the inside and

the outside, so more work must be done for droplet molecules to escape. However, the total

work for a finite volume of all droplets is small for larger droplets due to a lower overall sur­

face area to volume ratio, so overall surface tension favors droplet growth, and this growth

becomes more likely the larger droplets become.