Tag Archives: Pi pi contact

Neuroscience can no longer ignore phase separation

As a budding organic chemist, I always found physical chemistry rather dull, particularly phase diagrams. Organic reactions give you a very clear and intuitive picture of energy and entropy without the math.

In past few years cell biology has been finding phase changes everywhere. Now it comes to neuroscience as the synaptic active zone (where vesicles are released) is an example of a phase change (macromolecular condensation, liquid liquid phase separation, biomolecular condensates — it goes by a lot of names as the field is new). If you are new to the field, have a look at an excerpt from an earlier post before proceeding further — it is to be found after the ****

Although the work [ Nature vol. 588 pp. 454 – 458 ’20 ] was done in C. elegans with proteins SYD2 (aka liprinAlpha) and ELKS1, humans have similar proteins.

Phase separation accounts for a variety of cellular organelles not surrounded by membranes. The best known example is the nucleolus, but others include Cajal bodies, ProMyelocytic Leukemia Bodies (PML bodies), gemline P granules, processing bodies, stress granules.

These nonmembranous organelles have 3 properties in common

l. They arise as a phase separation from the surrounding milieu

2. They remain in a liquid state, but with properties distinct from those in the surrounding cellular material

3. They are dynamic. Proteins move in and out of them in seconds (rather than minutes, hours or longer as is typical for stable complexes.

They are usually made of proteins and RNA, and proteins in them usually have low complexity sequences (one example contains 60 amino acids of which 45 are one of alanine, serine, proline and arginine)

Back to the synaptic active zone. The ELKS1 and SYD2 both have phase separation regions (which aren’t of low complexity but they both have lots of amino acids capable of making pi pi contacts). They undergo phase separation during an early stage of synapse development. Later they solidify and bind other proteins found in the active presynaptic zone. You can make mutant ELKS1 and SYD2 lacking the low complexity regions, but the synapses they form are abnormal.

The liquid phase scaffold formed by SYD2 and ELK1 can be reconstituted in vitro. It binds and incorporates downstream synaptic components. Both proteins are large (SYD2 has 1,139 amino acids, ELKS1 has 836).

What is remarkable is that you can take a phase separation motif from human proteins (FUS which when mutated can cause ALS, or from hnRNPA2) put them into SYD2 and ELK1 mutants lacking their low complexity region and have the proteins form a normal presynaptic active zone.

This is remarkable and exciting stuff

*****

Advances in cellular biology have largely come from chemistry.  Think DNA and protein structure, enzyme analysis.  However, cell biology is now beginning to return the favor and instruct chemistry by giving it new objects to study. Think phase transitions in the cell, liquid liquid phase separation, liquid droplets, and many other names (the field is in flux) as chemists begin to explore them.  Unlike most chemical objects, they are big, or they wouldn’t have been visible microscopically, so they contain many, many more molecules than chemists are used to dealing with.

These objects do not have any sort of definite stiochiometry and are made of RNA and the proteins which bind them (and sometimes DNA).  They go by any number of names (processing bodies, stress granules, nuclear speckles, Cajal bodies, Promyelocytic leukemia bodies, germline P granules.  Recent work has shown that DNA may be compacted similarly using the linker histone [ PNAS vol.  115 pp.11964 – 11969 ’18 ]

The objects are defined essentially by looking at them.  By golly they look like liquid drops, and they fuse and separate just like drops of water.  Once this is done they are analyzed chemically to see what’s in them.  I don’t think theory can predict them now, and they were never predicted a priori as far as I know.

No chemist in their right mind would have made them to study.  For one thing they contain tens to hundreds of different molecules.  Imagine trying to get a grant to see what would happen if you threw that many different RNAs and proteins together in varying concentrations.  Physicists have worked for years on phase transitions (but usually with a single molecule — think water).  So have chemists — think crystallization.

Proteins move in and out of these bodies in seconds.  Proteins found in them do have low complexity of amino acids (mostly made of only a few of the 20), and unlike enzymes, their sequences are intrinsically disordered, so forget the key and lock and induced fit concepts for enzymes.

Are they a new form of matter?  Is there any limit to how big they can be?  Are the pathologic precipitates of neurologic disease (neurofibrillary tangles, senile plaques, Lewy bodies) similar.  There certainly are plenty of distinct proteins in the senile plaque, but they don’t look like liquid droplets.

It’s a fascinating field to study.  Although made of organic molecules, there seems to be little for the organic chemist to say, since the interactions aren’t covalent.  Time for physical chemists and polymer chemists to step up to the plate.