Developmental Cell. New discovery on how cells build their internal skeleton

Macromolecular Complexes in DNA Damage Response Group. Óscar Llorca is third from the left, and Marina Serna, first from the right. / Amparo Garrido. CNIO

Inside each cell, a network of tiny filaments, the microtubule cytoskeleton, helps maintain the cell’s shape, allows it to divide, and transports vital materials from one part of the cell to another. The filaments that form this network, called microtubules, are hollow tubes that act as scaffolding structures and transport pathways. One of the big questions in cell biology is how the cell controls the formation of these microtubules, a process essential for proper functioning and cell division. This is important because microtubules are also a key target used in chemotherapy to kill cancer cells.

Two research teams, one at the Institute for Research in Biomedicine (IRB Barcelona), led by Jens Lüders, and the other at the Spanish National Cancer Research Center (CNIO), led by Óscar Llorca, have achieved an important advance in the understanding of how cells generate the microtubules that form their internal skeleton. Their findings, published in Developmental Cell, explain how a protein called CDK5RAP2 activates the γ-tubulin ring complex (γTuRC), a key component in this skeleton-building process, helping cells to organize their interior and divide correctly.

“The key to the success of this project was that we were able to reconstitute the activation of the microtubule nucleator γTuRC in vitro, which provided us with sufficient high-quality material for analysis by electron cryomicroscopy,” says Jens Lüders, head of the Microtubule Organization in Cell Proliferation and Differentiation lab at IRB Barcelona.

“This work is a nice example of how visualizing single molecules at high resolution using electron cryomicroscopy, and then processing this information using algorithms based on neural networks, can reveal large molecules in action and how they function,” says Óscar Llorca, director of the Structural Biology program at the CNIO.

Building the cell skeleton

Microtubules are like scaffolding structures, and just like when constructing a building, the cell needs to assemble them in the right places, in the right orientation, and at the right times. This task is performed by the γTuRC, which acts as a template for assembling the first pieces of the microtubule.

However, in its basal state, γTuRC does not have the ideal shape to function as a template, and for years scientists have wondered how γTuRC adopts the correct shape to initiate the construction process. Researchers have now shown that CDK5RAP2 plays a central role in this process by binding to γTuRC and stimulating its activity. The protein binds to five key sites on γTuRC, helping it to adopt a more symmetrical, microtubule-like structure, allowing efficient microtubule nucleation. Without this activation, the γTuRC would remain in its asymmetric form, which is not suitable to serve as the basis for microtubule formation.

“CDK5RAP2 is like a construction manager, making sure that the skeleton of the cell is built correctly. This process is essential for cells to grow and divide,” explain Marina Serna and Fabian Zimmermann, first authors of the study and researchers at the CNIO and IRB Barcelona, respectively.

The power of advanced imaging

To uncover this mechanism, the team used cryo-electron microscopy (cryo-EM), a cutting-edge technique that allows scientists to capture high-resolution images of purified macromolecular complexes, such as γTuRC. Through cryo-EM, they were able to observe how CDK5RAP2 binds to γTuRC, triggering structural changes in the complex. These detailed images provided unprecedented insight into how the complex adopts a microtubule-like symmetry.

With cryo-EM they were able to see how multiple copies of CDK5RAP2 bind around γTuRC in a cone shape, allowing it to adopt a configuration that can efficiently initiate microtubule growth.

The study also found that during activation, γTuRC frequently releases a protein called actin, which is usually present within the unactivated structure of γTuRC. This actin release could be important for the complex to adopt its more functional, microtubule-like form.

Although this study reveals critical steps in how cells build their internal scaffolding, researchers are now interested in whether defects in γTuRC activation could underlie certain neurodevelopmental disorders caused by mutations in the CDK5RAP2 gene and in genes encoding γTuRC subunits. Another important question is whether there are other alternative mechanisms of γTuRC activation. This knowledge will lead to a deeper understanding of how cells assemble their microtubule cytoskeleton, which is a prerequisite for identifying disease mechanisms and ultimately opportunities for therapeutic interventions.

This work has been funded by the Spanish Ministry of Science and Innovation, with support from the European Union’s Horizon 2020 research and innovation program. The “la Caixa” Foundation has also contributed through its fellowship program, together with the Marie Skłodowska-Curie Actions of the European Union.

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