New discovery on how cells build their internal skeleton

Inside every cell, a network of tiny filaments, called 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, termed microtubules, are hollow tubes that act as scaffold structures and transport tracks. 

Scientists have long been curious about how cells control the formation of these microtubules, a process essential for healthy cell function and division. This is an important question, since microtubules are also a prime target used for chemotherapy to kill cancer cells.

Two research teams, one at the Institute for Research in Biomedicine (IRB Barcelona), led by Dr. Jens Lüders, and one at the Centro Nacional de Investigaciones Oncológicas (CNIO), led by Dr. Óscar Llorca, have now made an important breakthrough in understanding how cells generate the microtubules that form their internal skeleton. 

Their findings, published in Developmental Cell, shed light on how a protein called CDK5RAP2 activates the microtubule nucleator γ-tubulin ring complex (γTuRC), a key component of this skeleton-building process, helping cells organize their interior and divide properly.

“Key to the success of this project was that we were able to reconstitute the activation of the microtubule nucleator gTuRC in vitro, providing us with sufficient amounts of high quality material for the cryo-EM analysis”, comments Dr. Jens Lüders, head of the Microtubule Organization in Cell Proliferation and Differentiation lab at IRB Barcelona.

“This work is a beautiful example of how visualizing individual molecules at high-resolution using cryo-EM and the subsequent processing of this information using neural network-based algorithms can reveal large molecules in action and how they work.”, says Dr. Óscar Llorca, from the Macromolecular Complexes in DNA Damage Response Group at CNIO.

 

Building the cell’s framework 

Microtubules are like scaffold 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 job is handled by γTuRC, which acts like a template for assembling the first pieces of the microtubule.

However, in its ground state gTuRC is not perfectly shaped for functioning as a template, and for years scientists have been puzzled about how γTuRC may take on the correct shape to start the building process. Researchers have now shown that CDK5RAP2 plays a central role in this process by binding to γTuRC and stimulating its activity. 

The protein attaches to five key sites on the γTuRC, helping it take on a more symmetrical, microtubule-like structure, which enables efficient microtubule nucleation. Without this activation, the γTuRC would remain in its asymmetric form, which is not well-suited to template microtubule formation.

"CDK5RAP2 is like a construction manager, ensuring that the cell’s skeleton gets built properly. This process is fundamental for cells to grow and divide", explain Marina Serna and Fabian Zimmermann, first authors of the study, researchers a 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 gTuRC. Through cryo-EM, they were able to observe how CDK5RAP2 binds to γTuRC, triggering structural changes in the complex. These detailed images provided unprecedented insights into how the complex adopts microtubule-like symmetry.

With cryo-EM, they were able to see how multiple copies of CDK5RAP2 bind around the outside of the cone-shaped γTuRC, allowing it to adopt a form that can efficiently start microtubule growth.

The study also discovered that during activation, γTuRC frequently releases a protein called actin, which is usually present inside the non-activated γTuRC structure. This release of actin may be important for allowing the complex to adopt its more functional, microtubule-like shape.

While this study reveals critical steps in how cells build their internal scaffolding, the researchers are now interested in whether defects in the activation of gTuRC may underlie certain rare neurodevelopmental disorders caused by mutations in the CDK5RAP2 gene and in genes encoding gTuRC subunits. 

Another important question is whether other, alternative gTuRC activation mechanisms exist. Such insights 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 intervention.

The work was funded by the Spanish Ministry of Science and Innovation, with additional support from the European Union's Horizon 2020 research and innovation program. The "la Caixa" Foundation also contributed through its fellowship program, alongside the European Union’s Marie Skłodowska-Curie Actions.



Related article:
CDK5RAP2 activates microtubule nucleator gTuRC by facilitating template formation and actin release.
Marina Serna, Fabian Zimmermann, Chithran Vineethakumari, Nayim Gonzalez-Rodriguez, Óscar Llorca, Jens Lüders.
Developmental Cell (2024). DOI: 10.1016/j.devcel.2024.09.001