The first atomic-scale “film” of microtubules is built—a key process in cell division.

Microtubules under construction on a γTuRC “ring”, visualized using cryo-electron microscopy (cryo-EM). Side and top view (bottom left). Photo: Marina Serna/CNIO.

Publication in the journal Science of a solution to the most important biological problem: how human cells build their microtubules. The work of researchers from the Center for Genomic Regulation (CRG), the National Center for Cancer Research (CNIO) and IBMB-CSIC.

When cells divide, microtubules act like long, nanometer-thick “ropes” inside cells, pulling chromosomes so that each daughter cell retains a copy of the genetic material.

This work lays the foundation for future advances in the treatment of diseases ranging from cancer to neurodevelopmental disorders.

First steps in creating microtubules. Photo: Marina Serna/CNIO.

In our body, cells are constantly dividing. With each division, the genetic information contained in the chromosomes is duplicated, and each daughter cell receives a complete copy of the genetic material. It is a complex process, a clockwork mechanism that involves subtle and rapid changes within the cell. To make this possible, the cell contains microtubules – tiny tube-like structures. It has been a long time since we tried to understand how they are formed.

Now, for the first time, researchers from Center for Genomic Regulation (CRG)He National Cancer Research Center (CNIO) And Superior Council for Scientific Research (IBMB-CSIC) have achieved the equivalent of making a movie showing how human cells initiate the formation of their microtubules.

Results published today Online In the magazine The sciencesolve a problem raised many years ago and thus lay the foundation for future advances in the treatment of diseases ranging from cancer to neurodevelopmental disorders.

Long “ropes” that help divide chromosomes

Oscar Lorca, director of the structural biology program at the CNIO and the main co-author of the paper, describes what happens in a cell when cell division begins: “Chromosomes, once they duplicate the genetic information, are placed in the center of the cell and this, in an unusual way, quickly forms large tubes at its two ends , which hook chromosomes and pull each copy to the two poles of the cell. Only then will it be possible to encapsulate a copy of all our genetic material in each daughter cell.”

The structures that run, “like long ropes that reach the chromosomes and separate them,” Llorca explains, are microtubules. “That’s why we say microtubules play a key role in cell division. “We need to understand very well the mechanisms that trigger the formation of these microtubules in the right place and at the right time.”

They are also “cellular backbones”.

Microtubules are tubes thousandths of a millimeter long and nanometers (millionths of a millimeter) in diameter. In addition to playing a key role in cell division, they act as motorways to transport cellular components between different areas of the cell. They are also structural elements that, among other things, give shape to the cell itself. A good understanding of their preparation has implications for many areas of biomedicine.

“Microtubules are essential components of cells. Here we will show how the process of its formation occurs inside human cells. Given the fundamental role of microtubules in cell biology, this could lead in the future to new therapeutic approaches for a wide range of diseases,” explains the ICREA research professor. Thomas SurreyCRG researcher and co-author of the article in The science.

A molecular ring that triggers the formation of microtubules.

The now-obtained high-resolution images answer a question that has been floating around for many years: how microtubule formation begins in the early stages of cell division.

We now know that it all starts by closing itself to form a ring, a complex structure made up of several proteins called gTuRC (pronounced “gammaturk”)

Path gTuRC, its three-dimensional structure, was discovered several years ago and surprised researchers. It was expected that gTuRC This was a closed ring that served as the basic template on which the microtubule was built; But gTuRC This was shown as an open puck. Its size and shape were incompatible with the microtubule matrix.

Microtubules under construction on a γTuRC “ring”, visualized using cryo-electron microscopy (cryo-EM). Side and top view (bottom left). Photo: Marina Serna/CNIO.

New work by CRG and CNIO reveals the mechanism by which gTuRC locks into a ring and effectively becomes the perfect template to drive microtubule formation. Closing of gTuRC occurs upon attachment of the first molecular fragment of a microtubule.

“This is a trick the cell uses to close the gTuRC,” Llorca explains. “Once that first brick goes in, the gTuRC area can catch it and, like a lasso, act as hardware that pulls on the ring until it closes and starts the process.”

Visualizing this process required purifying gTuRC from human cells and reproducing the microtubule initiation process in vitro. The samples were observed using cryo-electron microscopes, and artificial intelligence was used to analyze the data.

A million film frames on an atomic scale

One of the problems was the high speed of the microtubule building process. The CRG group was able to slow it down in the laboratory and also stop the growth of microtubules so that they could analyze the initial stages of the process.

“We needed to find conditions that would allow us to image more than a million nascent microtubules before they became too large, obscuring the action of γ-TuRC. We achieved this by using molecular techniques in our laboratory and then freezing microtubule samples,” he explains. Claudia BritoCRG Research Fellow and first author of the study.

Microtubules under construction were observed on the IBMB-CSIC electron cryomicroscopy platform located at the Joint Electron Microscopy Center (JEMCA) at the ALBA synchrotron.

“They were frozen in a thin layer of ice, preserving the natural shape of the molecules involved,” he explains. Pablo Guerra, responsible for this Platform. In this way, the best experimental conditions for observing microtubule formation were determined. The best frozen samples were sent to BREM (Basque Resource for Electron Microscopy) for imaging and then transferred to Marina Serna and Oscar Lorca of CNIO for the analysis and determination of 3D structures at atomic resolution.

Artificial intelligence for assembly

In practice, having more than a million microtubules in different growth phases is equivalent to having many high-resolution movie frames. You “just” need to position them correctly to see the movie. This work fell to the CNIO team, which used artificial intelligence techniques to complete it.

“Determining the three-dimensional structure of growing microtubules from microscopic images has been extremely difficult. We needed several digital image processing tools,” explains Marina Serna, researcher at CNIO.

For Llorca, “the big challenge was analyzing high-resolution images of a dynamic process, where we observed several stages at the same time. This was made possible by using neural networks, which allowed us to organize all this complexity.”

The results are 3D, atomic-resolution structures that represent the different steps of how microtubule construction begins and how the γ-TuRC ring becomes the template that drives microtubule formation.

γ-TuRC initiating the nucleation process (left) and γ-TuRC closure (right).  Once closed, the base is fixed and tubulins can be added to make the microtubule the desired length.  Photo: Marina Serna/CNIO
γ-TuRC initiating the nucleation process (left) and γ-TuRC closure (right). Once closed, the base is fixed and tubulins can be added to make the microtubule the desired length. Photo: Marina Serna/CNIO

Health implications

As Llorca explains, “This discovery is relevant because we looked at a very simple mechanism of cell division that we didn’t know happened in humans.”

This is useful background knowledge for learning how to correct errors in microtubule function that have been linked to cancer, neurodevelopmental disorders, and other conditions ranging from respiratory problems to heart disease.

“Some drugs used today to treat cancer prevent the formation or dynamics of microtubules,” says Llorca. “However, these drugs indiscriminately target microtubules in both cancer and healthy cells, causing side effects. “Detailed knowledge of how microtubules form could help develop more targeted treatments that affect microtubule formation and lead to advances in the treatment of cancer and other diseases.”

Next step: understand the rules

Thomas Surrey, for his part, explains the next steps in understanding microtubules, which include understanding how their formation is regulated: “The process of nucleation decides where microtubules are in the cell and how many there are. The conformational changes we observed are likely controlled by as yet undiscovered regulators in cells. Several candidates have been described in other studies, but their mechanism of action is unclear.”

Future work “that elucidates how regulators bind to γ-TuRC and how they influence conformational changes during nucleation” could change our understanding of microtubule function and, over time, suggest alternative sites that could be targeted to prevent further development cancer cells. cell cycle,” concludes Surrey.

Financing

The work carried out in the Surrey laboratory has received support from the Spanish Ministry of Science and Innovation, EMBL, the Severo Ochoa Center of Excellence and the CERCA program of the Generalitat of Catalonia, as well as the Francis Crick Institute, which receives core funding from Cancer Research UK, the UK Medical Research Council and Wellcome Trust. TS also appreciates the support of the European Research Council and the Spanish Ministry of Science and Innovation. CB was supported by an EMBO and a Marie Curie fellowship.

Work in Lorca’s laboratory was funded by the Government Research Agency of the Ministry of Science and Innovation; The OL laboratory was also supported by the Carlos III National Institute of Health of the CNIO. The IBMB-CSIC CryoEM platform is supported by the project (IU16-014045 (CRYO-TEM)) of the Generalitat of Catalonia and the “ERDF: The Path to Creating Europe” of the European Union.

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