Researchers at the Centre for Genomic Regulation (CRG), the Spanish National Cancer Research Center (CNIO), and the Spanish National Research Council (IBMB-CSIC) have captured the world’s first high-resolution images of the earliest moments of microtubule formation inside human cells. The findings lay the foundations for potential breakthroughs in treating many different types of diseases ranging from cancer to neurodevelopmental disorders.
“Microtubules are critical components of cells, but all the images we see in textbooks describing the first moments of their creation are models or cartoons based on structures in yeast,” said ICREA research professor Thomas Surrey, PhD, at the Centre for Genomic Regulation. “Here we capture the process in action inside human cells. Now that we know what it looks like, we can explore how it’s regulated. Given the fundamental role of microtubules in cell biology, this could eventually lead to new therapeutic approaches for a wide range of disorders.” Surry is lead author of the team’s published paper in Science, titled, “Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.”
One of the most important components are microtubules.These tiny tubular structures, made of proteins—and with a length of thousandths of a millimeter and a diameter of nanometers [millionths of a millimeter]—act like bridges or roads that help move things around cells and give the cell its shape. Cells in the human body are also constantly dividing, and with each division the genetic information contained in the chromosomes is duplicated, so that each daughter cell receives a complete copy of the genetic material. Achieving this involves a sophisticated mechanism that involves refined and fast changes within the cell. Microtubules are critical for cell division, ensuring that two new cells can be generated from a parent cell. And in neurons, microtubules form highways for transport over long distances.
Co-senior author Oscar Llorca, PhD, director of the structural biology program at CNIO, further described what happens inside the cell when cell division begins. “The chromosomes, once they have duplicated genetic information, move to the center of the cell and the cell, in a very remarkable way, quickly sprouts from its two ends large tubes that hook the chromosomes and pull each of the copies towards the two poles of the cell. Only then is it possible to encapsulate a copy of all our genetic material in each daughter cell.”
The structures that are launched “like long ropes that reach the chromosomes to divide them,” are microtubules, Llorca pointed out. “That’s why we say that microtubules play a key role in cell division. We need to understand very well the mechanisms that trigger the formation of these microtubules, at the right place and at the right time.”
Microtubules are built by a large assembly of proteins known as the gamma-tubulin ring complex (γ-TuRC). The proteins work like a blueprint, laying down tubulin building blocks in a specific order. This process is called microtubule nucleation, which is like laying the foundation stones of a bridge. Once the foundation is set, tubulins are added to make the bridge as long as necessary.
For the cell to work correctly, microtubules need to be made of thirteen different rows of tubulins. A few years ago, researchers were baffled to discover that human γ-TuRC exposes fourteen rows of tubulins. This was confusing because researchers expected it to be a perfect template for microtubules, which did not seem to be the case. But high-resolution structures had previously only been pictured of either γ-TuRC or microtubules in isolation and never together.
One of the challenges was how to deal with the high speed of the microtubule construction process. The CRG group succeeded in slowing it down in the laboratory, and also stopping the growth of microtubules in order to better analyze the initial stages of the process.
To observe γ-TuRC while it was actively forming microtubules, researchers prepared samples at the CRG in Barcelona and the Electronic Microscopy Center at ALBA (EMCA), where they were flash-frozen in a thin layer of ice—preserving the natural shape of the molecules involved and helping discern fine details of structures at near atomic level. “We had to find conditions that allowed us to image over a million microtubules in the process of nucleation before they grow too long and obscure the action of γ-TuRC,” explained Cláudia Brito, PhD, postdoctoral researcher at the CRG and co-first author of the study. “We were able to achieve this using the molecular toolbox of our lab and then freeze the microtubule stubs in place.”
The best frozen samples were then sent to BREM (Basque Resource for Electron Microscopy) for imaging, and the resulting images were transferred to Marina Serna, PhD, and Llorca at the CNIO for analysis and determination of the three-dimensional structures at atomic resolution.
In practice, having more than a million microtubules in different stages of growth is equivalent to having many frames of a movie in high resolution. You “just” have to arrange them in the right order to see the movie in progress. That task fell to the CNIO team, which used artificial intelligence techniques to complete the work.
“Determining the three-dimensional structure of growing microtubules from microscope images has been extremely complex. We needed multiple digital image-processing tools,” explained Serna. For Llorca, “the great challenge has been to analyze at high resolution the images of a dynamic process, where we were observing several stages at the same time. This has been possible thanks to the use of neural networks, which have allowed us to organize all this complexity.”
The results are three-dimensional structures at atomic resolution that represent the different stages of how the construction of a microtubule begins, and how the γ-TuRC ring becomes the mold that launches the formation of microtubules. The findings revealed that as γ-TuRC starts the nucleation process and as the microtubule begins to form, it cleverly changes its shape. Initially in an open state, it progressively closes as the microtubule grows. The change makes γ-TuRC stow away one of its 14 tubulins, effectively matching the design of the microtubule that needs only 13 rows. The whole process is facilitated by a newly discovered latch mechanism, revealing that it’s the growing microtubule itself which helps the template find its correct shape. “Our structure shows how the complex undergoes a major conformational change as the length of the nucleated microtubule increases to become a perfect template for the nucleation of microtubules with precisely 13-protofilaments as present in the cytoplasm of human cells,” the authors wrote.
“Together, the cryo-EM maps of microtubule nucleating γ-TuRC revealed the conformational transitions that transform the open conformation of γ-TuRC before nucleation into a fully closed conformation after it has nucleated a microtubule.”
Llorca noted, “We have visualized the process that initiates microtubule formation, and we see that human γ-TuRC is an open ring that closes to effectively become a perfect template to nucleate microtubules. But we also discovered that this ring, in order to close, needs the ‘first brick’ of a microtubule to be put in place; when this happens, a region of the human γ-TuRC acts as an anchor that engages this ‘first brick’ to then close the ring and launch the formation of the microtubules.” In their paper, the investigators added, “When the nucleated microtubule is sufficiently long and the γ-TuRC is in its fully closed conformation, the latch does not seem to be further required to maintain the closed state.”
The most well-known consequence of microtubule malfunction is cancer, a disease characterized by uncontrolled cell proliferation. Neurodevelopmental disorders such as microcephaly also occur when microtubule processes go wrong, as do other conditions ranging from respiratory problems to heart disease. This new, fundamental knowledge will be important to help scientists learn how to correct errors in the functioning of microtubules.
Some cancer drugs work by targeting microtubules, preventing them from disassembling or forming in the first place. However, these disrupt microtubules indiscriminately in both cancerous and healthy cells, leading to side effects. Tumors also develop resistance to these drugs. Understanding the precise mechanism of how microtubules are laid down could lead to the development of more targeted and effective cancer treatments, as well as new therapies for a broader range of conditions.
“Some of the drugs used today to treat cancer prevent the formation or dynamics of microtubules,” added Llorca. “However, these drugs affect microtubules indiscriminately, both in cancer cells and in healthy cells, leading to side effects. Knowing in detail how microtubules are formed may contribute to the development of more targeted treatments that affect microtubule formation and allow progress in the treatment of cancer and other diseases.”
In addition to being key to cell division, microtubules act as highways for moving cellular components between different areas of the cell. They are also structural elements that shape the cell itself, among other tasks. A good understanding of their formation has implications for multiple areas of biomedicine.
Surrey said that the next steps in understanding microtubules will involve deciphering how microtubule formation is regulated. “The process of nucleation decides where the microtubules are in a cell and how many you have in the first place. It is likely that the conformational changes we observe are controlled by yet-to-be-found regulators in cells. Several candidates have been described in other studies, but their mechanism of action is unclear … As further work clarifies how regulators bind to γ-TuRC and how they affect the conformational changes during nucleation, it may transform our understanding of how microtubules work, and eventually offer alternative sites that one might want to target to prevent cancer cells from going through the cell cycle.”
In conclusion in their discussion, the authors further stated, “Future structural work will reveal the mechanisms by which distinct regulators control the activity of the complex by modulating its conformational changes to ensure the correct levels of nucleation activity at the right place and time.”