In the beginning was the phase separation

The question of the origin of life remains one of the oldest unanswered scientific questions. A team at the Technical University of Munich (TUM) has now shown for the first time that phase separation is an extremely efficient way of controlling the selection of chemical building blocks and providing advantages to certain molecules.

A team at the Technical University of Munich (TUM) has shown for the first time, that phase separation is an extremely
efficient way of controlling the selection of chemical building blocks, providing advantages to certain molecules.
This simple mechanism could have been decisive for the development of life. Left: clear solution; right: inside
 the tiny oil droplets instable molecules survive longer [Credit: Andreas Battenberg/TUM]

Life needs energy. Without energy, cells cannot move or divide, not even basic functions such as the production of simple proteins could be maintained. If energy is lacking, more complex connections disintegrate quickly, early life would die immediately.

Chemist Job Boekhoven and his team at TUM have now succeeded in using phase separation to find a mechanism in simple molecules that enables extremely unstable molecules such as those found in the primordial soup to have a higher degree of stability. They could survive longer, even if they had to survive a period without external energy supply.

The principle of simplicity

Job Boekhoven and his team were looking for a simple mechanism with primitive molecules that could produce life-like properties. "Most likely, molecules were simple in the primordial soup," says Boekhoven. The researchers investigated what happens when they "fed" various carboxylic acid molecules with high-energy carbodiimide condensing agents, thus bringing them out of equilibrium.

The reaction produces unstable anhydrides. In principle, these non-equilibrium products quickly disintegrate into carboxylic acids again. The scientists showed that the anhydrides that survived the longest were those that could form a kind of oil droplet in the aqueous environment.

Single droplets under a fluorescence microscope [Credit: Marta Tena-Solsona/TUM]

Molecules in the garage

The effect can also be seen externally: the initially clear solution becomes milky. The lack of water in the oil droplets is like a protection because anhydrides need water to disintegrate back into carboxylic acids.

Boekhoven explains the principle of phase separation with an analogy: "Imagine an old and rusty car: Leave it outside in the rain, and it continues to rust and decomposes because rusting is accelerated by water. Put it in the garage, and it stops rusting because you separate it from the rain."

In a way, a similar process occurs in the primordial soup experiment: Inside the oil droplet (garage) with the long-chain anhydride molecules there is no water, so its molecules survive longer. If the molecules compete with each other for energy, again those that can protect themselves by forming oil droplets are likelier to survive, while their competitors get hydrolyzed.

Next goal: viable information carriers

Since the mechanism of phase separation is so simple, it can possibly be extended to other types of molecular aggregations with life-like properties such as DNA, RNA or self-dividing vesicles. Studies have shown that these bubbles can divide spontaneously. "Soon we hope to turn primitive chemistry into a self-replicating information carrier that is protected from decay to a certain extent," says Boekhoven.

The study is published in Nature Communications.

Source: Technical University of Munich [May 23, 2018]

Missing ingredient to spark the fireworks of life discovered

Most people can name at least a few bones of the human body, but not many know about the cytoskeleton within our cells, let alone the "microtubules" that give it its shape. Now, a group of Princeton researchers has resolved a long-standing controversy by identifying exactly how the body creates these micron-sized filaments.

Credit: Princeton University

Using a novel imaging technique, Sabine Petry and the researchers in her lab were able to show that a protein called XMAP215, previously known only to help microtubules grow faster and longer, is necessary to creating the nucleus of each microtubule. Their work appears in the May issue of the journal Nature Cell Biology.

"Our study shows that microtubules in the cell are generated through cooperation between two molecules," said Akanksha Thawani, a fourth-year graduate student in Petry's lab who is the first author of the new paper. "XMAP215 functions together with a larger protein complex that forms ring shaped structures, gamma-tubulin ring complex (g-TuRC)."

"Microtubules are like the skeleton of the cell -- they give the cell its architecture," said Petry, an assistant professor of molecular biology and the senior author on the paper. "Beyond that, by positioning organelles, they can also serve as a highway for other components. Motor proteins can actually 'walk' along these microtubules. They really are fundamental to cell biology."

For 30 years, researchers in the field have known that the pillar-like microtubules are built of bricks called "tubulin" that grow from a tiny nucleus, and most agreed that g-tubulin was the only compound that could create that nucleus.

But there was a problem, said Petry. The few researchers who had succeeded in isolating g-TuRC found that when they put it in a test tube, it spectacularly underperformed at creating microtubule nuclei.

"Gamma-TuRC barely does anything," she said. "It nucleates a handful of microtubules, but it should make thousands."

Researchers have been puzzling over this for years, looking for some other factor that could activate or enhance g-TuRC. That search may now be over.

"The microtubule field has known [that g-tubulin] is not sufficient, and that other factors, that were not known, were also needed for full activity," said Eva Nogales, a professor of molecular and cell biology at the University of California-Berkeley who was not involved in this research. "The work by Petry and co-workers now shows that XMAP215, previously considered a microtubule 'polymerase' involved in microtubule elongation, acts synergistically with g-tubulin to promote efficient microtubule nucleation. The work provides an answer to this critical puzzle in our understanding of microtubule regulation in the cell."

These time-lapse videos show microtubules growing after the addition of XMAP215, the protein identified by researchers

 in Sabine Petry’s lab as a necessary component of microtubule nucleation. The different squares show the microtubules 

growing after adding different amounts of XMAP215. The observations are made in the “haystack” of the cell,

 revealing the “fireworks” of the rapidly growing microtubules. Elapsed time is shown in seconds 

and the white scale bar shows 10 μm [Credit: Princeton University]

Complicating their research was the difficulty in seeing the tiny structures at all, explained Thawani, a graduate student in in chemical and biological engineering. "The cell has tens of thousands of these polymers at any time, and our standard ways of observing these under a microscope do not allow for a good resolution," she said.

Petry used the classic metaphor of looking for a needle in a haystack, and she compared looking through a microscope with looking at the whole pile of hay. To solve the problem, she adapted a technique called total internal reflection fluorescence (TIRF) microscopy. Instead of illuminating the whole microscopic sample -- the whole haystack -- she instead imaged only a 100 nanometer-thick sliver. (For reference, a human hair is about 50,000 nanometers wide.)

"With TIRF microscopy, we don't illuminate anything outside that layer," she explained. "We don't see it. That's why we get a much higher signal-to-noise ratio: We don't see the other stuff that otherwise overlaps with the observation. So instead of seeing the haystack, which is still on top of it, we can see the needles against the glass. ... We can actually see the microtubules being born, we can see them grow, we can see what happens to them -- at the resolution of the needle."

A serendipitous find

Thawani hadn't set out to solve the mystery of why g-TuRC underperformed in test tubes. She had wanted to use TIRF microscopy to observe the growth of microtubules whose size she hoped to control with XMAP215, a protein known to encourage microtubule growth.

But instead of just growing longer microtubules, she saw that she was growing many more of them. "We added this protein, and it totally created a blast of microtubules," Thawani said. "That was one of the most mind-blowing bits in the whole process."

"The intention was just to study how these 'fireworks' form," said Petry. "So it was serendipity that Akanksha wanted to make them bigger, but then she saw -- 'Oh my gosh, there are more microtubules!' And the reason why she could see that more microtubules formed was that we have developed this imaging and extract capability. That's why no one else has seen it before."

Their team showed that the same phenomenon was seen in a test tube where g-TuRC generated microtubules together with XMAP215. This was chiefly the contribution of Rachel Kadzik, a postdoctoral researcher in Petry's lab and a co-first-author on the paper who purified the g-TuRC and, with Thawani, purified the XMAP215. G-TuRC is a 44-protein complex that has proven very difficult to purify and study in the 30 years since it was discovered.

Using the pure extractions of each protein, Petry's team was able to see that in a test tube, neither protein can nucleate microtubules without the other. "If we take XMAP215 out, not a single microtubule forms. If you take g-tubulin out, not a single microtubule forms," Petry said.

Their discovery required the combination of Thawani and Kadzik's experiments, viewed with Petry's TIRF microscopy imaging technique. "It was interesting," Petry said. "Rachel came from developmental biology, down to chemistry, and then Akanksha came from engineering up to biochemistry, so together, they were the super-team."

Source: Princeton University [May 16, 2018]

Scientists crack how primordial life on Earth might have replicated itself

Scientists have created a new type of genetic replication system which demonstrates how the first life on Earth - in the form of RNA - could have replicated itself. The scientists from the Medical Research Council (MRC) Laboratory of Molecular Biology say the new RNA utilises a system of genetic replication unlike any known to naturally occur on Earth today.

Liquid brine containing replicating RNA molecules is concentrated in the cracks between ice crystals,
as seen with an electron microscope [Credit: Philipp Holliger, MRC LMB]

A popular theory for the earliest stages of life on Earth is that it was founded on strands of RNA, a chemical cousin of DNA. Like DNA, RNA strands can carry genetic information using a code of four molecular letters (bases), but RNA can be more than a simple 'string' of information. Some RNA strands can also fold up into three-dimensional shapes that can form enzymes, called ribozymes, and carry out chemical reactions.

If a ribozyme could replicate folded RNA, it might be able to copy itself and support a simple living system.

Previously, scientists had developed ribozymes that could replicate straight strands of RNA, but if the RNA was folded it blocked the ribozyme from copying it. Since ribozymes themselves are folded RNAs, their own replication is blocked.

Now, in a paper published in the journal eLife, the scientists have resolved this paradox by engineering the first ribozyme that is able to replicate folded RNAs, including itself.

Normally when copying RNA, an enzyme would add single bases (C, G, A or U) one at a time, but the new ribozyme uses three bases joined together, as a 'triplet' (e.g. GAU). These triplet building blocks enable the ribozyme to copy folded RNA, because the triplets bind to the RNA much more strongly and cause it to unravel - so the new ribozyme can copy its own folded RNA strands.

The scientists say that the 'primordial soup' could have contained a mixture of bases in many lengths - one, two, three, four or more bases joined together - but they found that using strings of bases longer than a triplet made copying the RNA less accurate.

Dr Philipp Holliger, from the MRC Laboratory of Molecular Biology and senior author on the paper, said: "We found a solution to the RNA replication paradox by re-thinking how to approach the problem - we stopped trying to mimic existing biology and designed a completely new synthetic strategy. It is exciting that our RNA can now synthesise itself.

"These triplets of bases seem to represent a sweet spot, where we get a nice opening up of the folded RNA structures, but accuracy is still high. Notably, although triplets are not used in present-day biology for replication, protein synthesis by the ribosome - an ancient RNA machine thought to be a relic of early RNA-based life - proceeds using a triplet code.

"However, this is only a first step because our ribozyme still needs a lot of help from us to do replication. We provided a pure system, so the next step is to integrate this into the more complex substrate mixtures mimicking the primordial soup - this likely was a diverse chemical environment also containing a range of simple peptides and lipids that could have interacted with the RNA."

The experiments were conducted in ice at -7°C, because the researchers had previously discovered that freezing concentrates the RNA molecules in a liquid brine in tiny gaps between the ice crystals. This also is beneficial for the RNA enzymes, which are more stable and function better at cold temperatures.

Dr Holliger added: "This is completely new synthetic biology and there are many aspects of the system that we have not yet explored. We hope in future, it will also have some biotechnology applications, such as adding chemical modifications at specific positions to RNA polymers to study RNA epigenetics or augment the function of RNA."

Dr Nathan Richardson, Head of Molecular and Cellular Medicine at the MRC, said: "This is a really exciting example of blue skies research that has revealed important insights into how the very beginnings of life may have emerged from the 'primordial soup' some 3.7 billion years ago. Not only is this fascinating science, but understanding the minimal requirements for RNA replication and how these systems can be manipulated could offer exciting new strategies for treating human disease."

Source: Medical Research Council [May 15, 2018]