Side roads

Dr. Shuldiner and Chalil Ast examined the pathways that proteins use to enter a cell organelle called the "endoplasmic reticulum": a series of folded membranes that form a labyrinthine structure where the proteins are folded, inspected, and sent to their destination. The proteins that pass through this organelle are eventually sent outside the cell

"Biologists hope to discover in their research 'textbook examples', that is, typical, general cases," says Dr. Maya Schuldiner from the Department of Molecular Genetics. To illustrate her point, she opens a biology textbook and points to a diagram depicting a molecular pathway - a series of interactions between molecules whose purpose is to bring a certain protein, or a certain process, from state A to state B. Since many proteins tend to use In one molecular pathway, such "textbook" examples can reveal important insights into how the cell works, and lay the foundations for further research and discoveries.

But behind textbook examples, claims Dr. Shuldiner, a much more complex reality is hidden: "In general, the pathways that are discovered in the early stages of research in any field are considered the most important. When we discover proteins that use other pathways, we tend to treat them as outliers. "Scientists often don't stop to ask how many proteins are really used in this or that pathway."

Dr. Shuldiner and research student Tsliil Est decided that it was time to stop and ask this question. The reason for this, among other things, is the existence of new technology that allows them to test a large amount of proteins at once: the advanced microscopic equipment and computational methods used by Dr. Shuldiner, allow her to discover the pathways used by hundreds of proteins in the cell, in a fraction of the time previously required to test a protein or two.

Dr. Shuldiner and Chalil Ast examined the pathways that proteins use to enter a cell organelle called the "endoplasmic reticulum": a series of folded membranes that form a labyrinthine structure where the proteins are folded, inspected, and sent to their destination. The proteins that pass through this organelle are eventually sent outside the cell : some of them are hormones and external signaling molecules, and some are proteins that stop at the outer surface of a membrane the cell and remain attached to it - like receptors. It is these proteins that allow the cell to sense the environment and to communicate with other cells. In fact, proteins are secreted in almost every disease. The pathway in which proteins enter the endoplasmic reticulum was discovered in the 70s -20, is called SRP, and it has been studied a lot. Other tracks that have been discovered since then are considered unimportant, and are simply called "non-tracks." depend on SRP".

Is the SRP pathway indeed the main entry pathway into the endoplasmic reticulum? The scientists tested all the proteins found in the endoplasmic reticulum of a baker's yeast cell - about 1,300 proteins. The answer was clear: only about half of them used the SRP route to get there. The rest used other routes: some were identified in the past and were known, but the findings also indicated additional routes, which are not known. The scientists discovered that, at least for yeast, there is a clear division: the proteins that use the SRP pathway are the ones that remain attached to the cell membrane afterwards, while proteins that do not use the SRP pathway are secreted out of the cell. Since these pathways have been well conserved during evolution, Dr. Schuldiner believes that a similar division also exists in human cells. This means that the proteins that use the other pathways include important proteins that are secreted outside the cell, such as hormones and signaling molecules.

These days the team is trying to create a more complete picture of the alternative routes. The final goal is to identify all the molecular pathways used by all the proteins that go outside the cell. The scientists expect that the result of this research will not be a simple and ideal model, but a complex and complicated picture, which will more accurately reflect the behavior of the proteins.

This may be bad news for textbook writers, but good news for our ability to understand living cells. "Ultimately, we want to get a completely new picture of how the cell works," says Dr. Schuldiner. "We want researchers to stop looking under the torch of conventional models, and expand the range to include all possibilities."

traffic laws

Where exactly do the proteins that circulate around the cell go? Scientists interested in this question will now be able to search for the answer. Dr. Maya Schuldiner and research student Michal Barker recently created a useful "atlas" that shows the changes in the location of proteins and their distribution in yeast cells under stress conditions. The new atlas - which will be accessible online - presents an enormous wealth of new information, and it will be an important research tool for Many scientists use yeast cells as a model of how the living cell works.
Dr. Schuldiner and Michal Barker began their work with a special type of library - strain libraries. This type of library contains strains of yeast in which certain genetic changes have been made. Baker's yeast cells contain 6,000 genes, each of which codes for the creation of a protein. In each of the "volumes A "bookmark" was placed in the strain library - a genetic change was made in one of the genes in the yeast cell, which connects the protein produced from it For a fluorescent marker, which shines with green light under a special microscope. In Dr. Schuldiner's laboratory, which is equipped with advanced automatic microscopic equipment, it is possible to examine all the yeast strains in the library at once. There are other types of libraries, including those where one of the genes is deleted in each "volume". Mixing and combining different libraries makes it possible to create new genetic compositions. The entire array is then automatically scanned by a robot to discover which proteins are produced in any given experimental condition and where they are located in the cell.

This unique setup allowed Dr. Shuldiner and Michal Barker to follow the movement of proteins - one of the biggest questions in cell biology. According to Dr. Shuldiner, accurate information about the movement and location of proteins in the cell can help answer several important research questions: How many of the cell's proteins are they mobile Under what conditions do they move? How does the cell use the movement of proteins to maintain its health, and continue to divide in a variety of situations and conditions?

By exposing the yeast cell libraries to different conditions and transferring them through the robotic system, the researchers were able to follow the movement of each of the proteins in the yeast cells. The result: a complete and detailed map describing the movement paths of the proteins, as well as documentation of the amounts of the different proteins formed in different situations.

An overview of the data reveals a picture of constant turmoil in the cells: At any given time hundreds of proteins are in motion. However, the data obtained in the experiment regarding the amounts of proteins were surprising: In many of the studies conducted today, protein levels are determined through experiments that actually check the amount of messenger RNA (the molecule that carries the final instructions to create a protein) - a move similar to using the construction plans, instead of the actual buildings, to map a city. However, programs may not be executed, or, alternatively, one program may be reused. Tracking the proteins themselves revealed to scientists that the amounts of messenger RNA and the amounts of proteins do not always correspond to each other. Dr. Shuldiner: "Many studies have already shown in the past that the production of proteins is controlled in many ways even after the stage in which the RNA molecule leaves the cell nucleus. Our new findings imply that the last control steps may be more important than previously thought in determining the level of proteins in the cell."

The online atlas is called Loqate LOcalization and Quantization Atlas of the yeast proteome. "The atlas can be used by scientists who wish to investigate specific questions, such as: Which proteins are involved in a certain cellular activity? When and where do they work?", says Dr. Schuldiner. "In addition, scientists who wish to get a clearer picture of life in a cell by combining different types of information They will find it an essential tool."

Power calculation

Contemporary research methods in the field of molecular biology advanced her beyond the basics of scientific research: raising a hypothesis and testing it through an experiment. The use of fast, powerful, and fully automatic equipment, most of which was built according to Dr. Shuldiner's special needs, allowed her and her group members to examine all the options at the same time, and extract significant information from them. "Thanks to these tools, the research is completely freed from bias," she says. "If we once started the work with an educated guess, for example, with the hypothesis that protein A affects protein B, and then tried to prove the hypothesis through an experiment, now we can ask 'which proteins are involved in the activity of B?'. Thanks to this we may discover that proteins C, D and E also affect protein A. If in the past a research student devoted his entire research work to testing a limited hypothesis, today he can get answers to questions. wide within a few weeks".