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An illustration of a part of a synthetic organelle without membrane. We see here two layers that separate as oil and water, but both layers are water. There is no oil. Each layer contains a different solute that gives it its own chemical thermodynamics, separating it from the other. Chemical reactions pass from one layer to another in a chain reaction. The molecules shown on the outside are sugars called dextran, a solute. The gray intermediate layer contains an enzyme, represented by small yellow spheres that would perform a step in the reaction cascade. Credit: Georgia Tech
A couple of sugars, a mesh of enzymes, a pinch of salt, a drop of polyethylene glycol, carefully arranged in water baths. And the researchers made a synthetic organelle, which they used in a new study to explore a strange cellular biochemistry.
Researchers at the Georgia Institute of Technology have engineered the chemical mixture in the laboratory to mimic membrane-free organelles, mini-organs in cells that are not contained in a membrane but are pools of aqueous solutions. And their model showed how, with just a few ingredients, organelles could perform refined biological processes.
The researchers published the results of their study in the journal Applied materials and ACS interfaces for the September 26, 2018 issue. The research was funded by the National Institutes of Health's National Institute of General Medical Sciences and the National Science Foundation.
A quick look at membrane-free organelles should help to understand the meaning of research.
What are membrane-free organelles?
The discovery of organelles that are pools of aqueous solutions and not objects with membranes is fairly recent. A perfect example is the nucleolus. It lies inside the nucleus of the cell, which is an organelle that has a membrane.
In the past, researchers thought that the nucleolus had disappeared during cell division and reappeared later. Meanwhile, researchers have realized that the nucleolus has no membrane and that during cell division, it diffuses as water bubbles do in the vinaigrette that has been shaken.
In a city, three aqueous solutions separate into three layers. In membraneless organelles, chemical reactions occur at the interfaces of these layers, treating a reagent step by step and moving the reaction product from one layer to the other. Credit: Georgia Tech / Rob Felt
"After cell division, the nucleolus comes back into a single compartment," said Shuichi Takayama, principal investigator of the study and professor at the department of biomedical engineering Wallace E. Coulter of Georgia Tech and Emory University. .
Membrane-free organelles may consist of a few different aqueous solutions, each with different solutes such as proteins or sugar or RNA or salt. The thermodynamic differences of the solutions, ie the way their molecules bounce, prevent them from fusing into a single solution.
Instead, they separate the phases as do oil and water, even after they get mixed up. But there is no oil in this case.
"These are all waters," Takayama said. "They do not mix with each other because they have different solutes."
What realistic processes has the synthetic experience demonstrated?
During mixing, important things happen. The nucleolus, for example, is essential for the transcription of DNA. But the synthetic structure, a collection of aqueous solutions developed by the first author of the study, Taisuke Kojima, produced a simpler series of reactions demonstrating how organelles lacking membranes could lead to sugar processing.
Inside the nucleus, seen here as a violet sphere, is a smaller violet sphere, the nucleolus, which is the most important membranous organelle of our cells. Credit: CNX OpenStax / Free download at cnx.org/contents/[email protected] / creative commons license
"We had three phases of solutions that each contained different reagents," said Kojima. "It was like a three-layered bullet: an external solution, an intermediate solution and a basic solution.Gloss was in the outer layer, one enzyme, glucose oxidase, was in the second layer and peroxidase of horseradish with a colorimetric substrate which gave us a visible signal during the last reaction sought. "
Glucose in the outer layer interfaced with glucose oxidase in the second layer, which catalyzed glucose into hydrogen peroxide. It landed in the second layer and interfaced with the horseradish peroxidase in the middle layer, which catalyzed it in the middle layer with this compound that transforms the colors.
"This type of cascading reaction is what one would expect to see organelles without membrane functioning," Takayama said.
The cascade transported even each reaction product from one compartment to another, which was very common in biological processes, such as food digestion or transformation of organelle molecules.
What can we learn from a surprise discovery?
Part of the reaction caught the researchers by surprise, and this resulted in a new discovery.
The nucleolus, in the center of the nucleus of the cell, is the most important organelle without membrane. It was once thought that it disappeared during cell division and then reappeared. As it exists in solution, in reality, it turns into pieces that regroup again. Credit: CNX OpenStax / Download for free at cnx.org/contents/[email protected] / commons license
"When researchers think of membrane-free organelles, we often think that the reactions inside are more efficient when their enzymes and substrates are in the same compartment," said Takayama. "But in our experiments it slowed down the reaction, we said," Whoa, what's going on here? "
"When the substrate is in the same place where the reaction product accumulates, the enzyme is sometimes confused, which can hinder the reaction," said Kojima, a postdoctoral researcher at Takayama's lab. "I was pretty surprised to see it."
Kojima put the enzymes and substrate in separate solutions, which interfaced but did not fuse into a single solution, and the reaction in its synthetic organelle worked effectively. This has shown how unexpected the subtleties can be the development of organelle chemistry.
"It was a Goldilocks diet, not too much contact between the substrate and the enzyme, not too little," Takayama said.
"Sometimes in a cell, a substrate is not abundant and it may be necessary to concentrate it in its own small compartment and then bring it into contact with the enzyme," Takayama said. "On the other hand, some substrates can be very abundant in the nucleus, and it can be important to separate them from the enzymes to have just enough contact for the right type of reaction."
Explore more:
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More information:
Taisuke Kojima et al, membraneless compartmentalisation facilitates enzymatic cascade reactions and reduces substrate inhibition, Applied materials and ACS interfaces (2018). DOI: 10.1021 / acsami.8b07573
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