Designing tiny platforms in living cells to observe proteins in 3D

<p>Oriol Gallego put together an international team with expertise in cell bioengineering, localization microscopy, and bioinformatics</p>
Oriol Gallego put together an international team with expertise in cell bioengineering, localization microscopy, and bioinformatics
  • <p>Oriol Gallego put together an international team with expertise in cell bioengineering, localization microscopy, and bioinformatics</p>
  • <p>PhD student Irene Pazos contributed the Cell paper verifying the new technique with a protein complex with known architecture, COG</p>

Ground-breaking research sometimes arises as a result of coincidence, multidisciplinarity and, of course, perseverance. Biochemist Oriol Gallego (Barcelona, 1978) is a Ramón y Cajal Research Associate in Raúl Méndez’s Translational Control of Cell Cycle and Differentiation Lab. Oriol led a promising study that has just been published in Cell. Combining genetic engineering, super-resolution microscopy, and biocomputation, he was able to see a 3D “protein nanomachine” in a living yeast cell. This breakthrough paves the way for inspiring future discoveries, such as the live observation of how viruses use protein complexes to infect cells.

 

“Six years ago, this was not a project,” confesses Gallego talking about the start of this research. “I was interested in cell bioengineering. I wanted to build nano-platforms within cells to be able to, for example, anchor enzymes to cellular membranes in order to control their lipid composition.”

At that time, a friend of his, an Italian physicist at EMBL in Germany, was working on the development of localization microscopy in living cells, which consists of determining the position of a fluorescent label down to a few nanometres. Together, they came up with the idea to study whether the two techniques could be combined to measure distances between the components of a protein complex by anchoring the complex to a nano-platform. Their goal was challenging because protein complexes in live cells move continuously, making it impossible to measure the space between its subunits. And even if it were possible, only the distance of the 2D projection of a 3D object would be obtained.

“This proof-of-concept for our new method worked as expected,” says Oriol. At that point, the researchers decided to invite scientists from a third field of expertise – bioinformatics – to join them. The bioinformaticians were able to reconstruct the 3D shapes from distance measurements obtained from 2D images. Oriol was just finishing up his postdoc at EMBL and moved to IRB Barcelona with a 5-year Ramón y Cajal contract. By 2012, they had already produced the first engineered cells and their idea was ready to be tested.

The next step was to concentrate on exocytosis, a mechanism used by the cell to transport cargo to the cell periphery and to communicate with the cell exterior. As explained here, the researchers were able to construct an artificial structure within the yeast cell, where they could anchor the active protein machinery of the exocytosis. The platform served as a reference point from which to measure the distance between the components of the protein complex. With these data and using a process analogous to the way GPS works, they reconstructed in detail how each of the eight proteins in the complex are organised in a living yeast cell.

The fact that this method is applicable to a living cell is as compelling as being able to observe a motor while functioning, instead of having to reconstruct how it works from its separate pieces.

“We were very confident that our method was solid,” says Oriol, “but one of the reviewers asked us to reconstruct the molecular architecture of the conserved oligomeric Golgi (COG), a protein complex of known architecture, to verify the reliability of our approach.” PhD student Irene Pazos carried out this part of the work.

The next step of the research will be to study cell compartmentalisation, that is, how the cell produces and maintains confined areas with a specific molecular composition in function of its necessities. As Oriol explains, “Some compartments are delimited by lipid barriers, like in some of cell organelles, while others don’t have this barrier,” he adds. “We are now focusing on describing these processes from a mechanistic perspective. We want to characterise the temporal and spatial coordination of protein complexes within the cell. Processes are required to maintain the asymmetric distribution of cellular components.”

This new stage of the research will also require an integrated approach involving fluorescence microscopy techniques and in vitro structural biology.

But the most ambitious part of the research is to transfer their discoveries to human cells. “Modifying yeast cells is much easier. First, they are rigid and it is easier to anchor a platform to them. Indeed, the platform has to be immobile, otherwise we cannot reliably measure its orientation. Second, because treating and modifying human cells is more expensive, our work will call for new technologies,” concludes the biochemist. A daunting challenge for anyone. But this young Catalan isn’t disheartened: “Challenging questions and problems always inspire me,” he says.

Oriol hopes that this paper will facilitate his career options. “My grant is about to finish, and after I go to Germany and Japan for short stays, my plan is to find a place to set up my group,” he confesses. He’s very aware, though, that things are never straightforward in science. “I have been lucky, my paper was published in Cell. But we all know that if you do good research, you won’t necessarily be able to publish in the best journals, nor is it true that having a good publication record will pave the way to a principal investigator position in Spain. You have to be realistic and keep your options open.”

“What has really been important in my career,” he concludes, “was to do a postdoc in a top level international centre such as EMBL, where there is very little hierarchy and networking is encouraged. This gave me the possibility to explore original ideas, and establish informal contacts that have proved essential to continue my work here at IRB Barcelona.”