It is a cellular process that has been going on for a billion years, but we are not able to reproduce it, nor to fully understand it. Mitosis, the cell division mechanism so important to life, involves more than 100 proteins in its heart. Today, the group of Professor Andrea Musacchio from the Max Planck Institute for Molecular Physiology in Dortmund was able to fully reconstruct the engine of the mitosis machinery, called the kinetochore. Being able to model a functional kinetochore is the first step towards making artificial chromosomes, which could one day be used to restore missing functions in cells. The results appear in the newspaper this week Scientists progress.
A wonder of nature
When a human cell begins to divide, its 23 chromosomes duplicate into identical copies that stay joined in a region called a centromere. This is where the kinetochore is located, a complicated assembly of proteins that binds to thread-like structures called microtubules. As mitosis progresses, the kinetochore gives the green light for microtubules to tear up copies of DNA, to new cells being formed. âThe kinetochore is a beautiful, flawless machine: you hardly ever lose a chromosome in a normal cell! Â», Explains Musacchio. âWe already know the proteins that constitute it, but important questions about the functioning of the kinetochore remain open: how is it reconstructed during the replication of chromosomes? How does it bind to microtubules? And how does he control them? ”
The effort of a lifetime
Musacchio’s quest for answers began over 20 years ago and was guided by a simple motto: âBefore we understand how things go wrong, we better understand why and how things work. He therefore embarked on the mission of reconstructing the kinetochore in vitro. In 2016, he was able to synthesize a partial kinetochore composed of 21 proteins. In the new publication, Musacchio, graduate student Kai Walstein and their colleagues at MPI Dortmund were able to completely reconstruct the system: all subunits, from those that bind the centromere to those that bind microtubules, are now present in the correct numbers and stoichiometry. Scientists have proven that the new system works well, successfully replacing parts of the original kinetochore in the cell with artificial parts. âThis is a real milestone in the reconstruction of an object that has existed, unchanged, in all eukaryotic cells for over a billion years! Â», Explains Musacchio. This breakthrough paves the way for the production of synthetic chromosomes that carry functions that can be replicated in organisms. âThe potential for biotechnology applications could be huge,â he says.
In the protein factory
Scientists at MPI had to overcome a major hurdle to reconstruct the kinetochore, namely to fully reconstruct the highly flexible centromeric protein C (CENP-C). It is an essential protein that connects the centromeric region to the external proteins of the kinetochore. The researchers reconstructed CENP-C by “gluing” its two ends.
A highly organized, factory-like laboratory is fundamental for the reconstruction of complex protein assemblies. For each protein in the kinetochore, scientists at MPI built a production pipeline to isolate genes, express them in insect cells, and collect them. âWhen we put them together in vitro, these proteins snap together to form the kinetochore, just like the LEGO parts following the instructions,â he says. Besides the famous toys, each kinetochore protein has a different interface and interaction with closer proteins.
The group will now move to the next level of complexity: studying how the kinetochore works and interacts in the presence of microtubules and supplied energy (in the form of ATP). The project was recently awarded an ERC Synergy grant and will be carried out by an international team comprising Musacchio’s group and researchers from Cambridge, UK, and Barcelona, ââSpain.
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