The work environment in the cell is divided into "offices", that is, well-defined organelles bounded within membranes, and "work corners" in the open space - organelles without membranes, which are a kind of droplets without defined boundaries that are created from encounters between proteins. In recent years, these protein droplets have aroused great research interest due to their many roles in the cell and their involvement in disease states, but the basic molecular forces acting to create them are not fully known. Weizmann Institute of Science scientists found recently A way to "walk between the drops" and study the process of their formation: the researchers from the laboratory of Dr. Emmanuel Levy In the Department of Structural Biology, yeast cells have been turned into tiny test tubes where synthetic proteins are produced that produce simple versions of these droplets.
Similar to oil droplets in water, the encounter between proteins may produce droplets in the cell space in a process called phase separation. Just as a mixture of oil and water can, under certain conditions, remain well mixed without separating into its components, so the phase separation process also depends on the conditions of the mixture. A thin line, known as the "phase boundary", determines when phase separation will occur and when it will not. "Chemists usually 'play' with the temperature or pressure in the mixture to identify the phase boundary," explains Meta Heidenreich, the research student who led the project in Dr. Levy's group. "Such phase boundaries also exist in living cells, but it is much more difficult to detect them."
When you pour an "instant pudding" mixture into a bowl of milk and mix - the sticky texture of the pudding is created as a result of bonds between the sugar and protein molecules in the bowl. Similarly, the researchers in Dr. Levy's laboratory designed two types of synthetic proteins, the chemical attraction between them may cause them to organize spontaneously in a viscous network-like configuration. If the synthetic proteins in the experiment are the pudding powder and the milk, the bowl is the yeast cells that the researchers have engineered so that each cell produces both types of proteins.
But when making pudding, too much sugar or protein, or too little of both, will not allow you to reach the desired texture. Similarly, too much of one of the proteins or too little of both will not result in the formation of the desired droplet. Therefore, in the first part of the experiment, the researchers checked what are the protein concentrations in which droplets are formed. The researchers tracked thousands of cells, each engineered to produce a different amount of each of the proteins. As expected, some cells formed droplets and some did not, and this is how the scientists were able to discover the natural phase boundaries.
Later, the research team added another variable: "affinity" - that is, the degree of strength of the bond between the proteins. Through tiny changes in the synthetic proteins, the researchers controlled the degree of affinity between the two types of proteins. And again, by tracking thousands of cells, they were able to show how the affinity changes affect the phase boundary. The findings showed that increasing the affinity between the proteins increased the probability of droplet formation, even when the protein concentrations in the cell were low. Similar findings were previously observed when working with proteins in test tubes, but not in living cells.
In collaboration with Prof. Shmuel (Sam) Shaffern From the Department of Chemical and Biological Physics and researchers from the University of Oxford, the researchers created a theoretical connection between the properties of the individual proteins and the properties of the droplet as a whole and intuitively and quantitatively explained the source of attraction between the proteins leading to phase separation. "You can compare the individual proteins to a large flock of birds," says Dr. Levy. "The structure of the flock seems uniform and cohesive to us, but in fact it is the product of the sum of the individual actions of hundreds of birds, each of which follows a few very simple rules. In our case, the simple rules are, for example, the degree of chemical attraction between one protein and another."
After showing that the production of synthetic proteins in yeast cells is an effective tool for revealing the dynamics of the formation of membrane-less organelles, the researchers used this method to understand how the proteins in the cell find each other in the first place. The scientists hypothesized that the proteins are already attracted to each other during their production, so that the entire production system - which includes messenger RNA molecules (which provide the recipe for protein production) and ribosomes (the protein production factories) - is located in the cell at the site of the formation of the drop. The findings in the yeast cells confirmed this hypothesis.
"Many research groups are looking for mechanisms responsible for the locations of messenger RNA molecules in the cell. Our findings show that it is possible that an attraction between proteins during the production process may be such a mechanism", says Dr. Levy.
Since disruptions in the cellular droplets have been observed in various disease states, including neurodegenerative diseases such as ALS, various types of dementia and Huntington's disease, Dr. Levy and Heidenreich hope that the method they developed will help researchers reach new insights into how disruptions occur in these diseases.
