Cell division, in which one living cell becomes two, is one of the most common occurrences in nature, but also one of the most complex and vital. The growth and reproduction of living beings depend on it, and diseases such as cancer are born from errors in this process, which uses sophisticated molecular machinery, made of proteins. Until now unknown details of the functioning of part of this equipment were revealed in research carried out by Edmarcia Elisa de Souza, from the National Biosciences Laboratory (LNBio), in her doctoral thesis, defended at the Institute of Biology (IB) at Unicamp.

The focus of Edmarcia's work was a kinase, a type of protein that “marks” other molecules, through a process called phosphorylation, activating them, deactivating them or altering their function, called Nek 7. “Nek 7 is a known regulator of cell division,” the researcher told Journal of Unicamp, but a little studied regulator. 

“One of the things that is done a lot in my group, when we study the function of a protein, is to identify its interaction partners, other proteins that interact with the target protein. Kinases are proteins that behave as a 'hub' or 'node', which are found as foci in networks of interactions and which generally interact with many other proteins”, added Jörg Kobarg, professor at Unicamp, researcher at LNBio and advisor of Edmarcia's thesis. “So her PhD approach was to identify Nek 7’s interaction partners.” 

With this, the researcher built the interactome – the map of interaction relationships – of Nek 7. “If a protein is physically interacting with another, it is an indication that it may be acting in the same function as that interactor. It's a first hypothesis. The second hypothesis is that they can act together, or independently associated, in a specific molecular mechanism or one that has not yet been discovered, for example”, explained Edmarcia. One of the discoveries of the study of the Nek 7 interactome was that its function is independent of that of another kinase, Nek 6, something that contradicted the literature available until then. This discovery led to Edmarcia publishing an article in Journal of Proteome Research, one of the main journals in the field.

“Our studies demonstrated a broad spectrum of proteins interacting with human Nek7, classified within multiple functional categories, especially cell division”, says the text of the thesis.

Segregating chromosomes

During mitosis, the process in which a cell divides into two cells identical to the original, the segregation of genetic material, which is present in the chromosomes housed in the nucleus, occurs during a phase called interphase. After replicating in interphase, the chromosomes need to be aligned correctly in the central plane of the cell and then separated, with each complete collection being pulled away from the other by a “crane” whose handles, microtubules, are molecular strings made of proteins. . 

In normal mitosis, structures called centrosomes migrate to opposite ends of the cell's interior, and produce the so-called mitotic spindle, a set of microtubule networks that grip the chromosomes, which have already been duplicated and aligned. From there, the chromosomes are pulled towards the poles occupied by the centrosomes.

Problems in these mechanisms can generate defects such as aneuploidy, when cells resulting from division have different numbers of chromosomes. Aneuploidy is a condition associated with malignant tumors, which can be caused by multipolar mitosis, in which chromosomes are pulled not in two, but three or more different directions, by the centrosomes. It is in this phase, of alignment and separation of chromosomes, that Nek 7 acts. “When the expression of Nek 7 is reduced, multipolar mitosis occurs”, explained the researcher. “The function of Nek 7 is, first, to correctly separate the centrosomes, and the error in separation causes this type of condition characteristic of cancer cells.”  

“This suggests that Nek 7, as a kinase, goes there to phosphorylate proteins and, in doing so, promotes an orderly process. And if it is missing, there are problems”, said the advisor, Jörg Kobarg.

In his work, Edmarcia discovered a close link between Nek 7 and the protein called RGS2. 

“We discovered, unexpectedly, that one of Nek7's interactors, the RGS2 protein, was located in key structures, essential for the cell division process, including the centrosome and the mitotic spindle,” said the researcher. A dysregulation of these structures can lead to cancer.

Technology

“And how do you reach these conclusions? Having the correct tools”, said Edmarcia. 

Edmarcia's discoveries were made with the help of cutting-edge technologies for producing microscopic images, which allow monitoring and recording the functioning, step-by-step, of a living cell. 

The researcher worked for five months in the laboratory of American researcher Stephen Doxsey, at the Medical University of Massachusetts, where she had access to equipment that allows “time-lapse” videos to be made – which concentrate a long period of time in a few seconds, like films of blooming flowers common in nature documentaries – of cellular processes.  

“We went to the USA to try to unravel the function of RGS2 and Nek 7 in the mitotic spindle, the structure essential for cell division and whose dysregulation is directly related to cancer,” said the researcher. “Professor Steve Doxsey's laboratory has cutting-edge equipment and his group has expertise in 'time-lapse imaging', which allows us to watch cell division and molecular dynamics in living cells, in real time. So, you make a movie of the cell dividing.” 

Other studies carried out by her, using confocal microscopy technology, which allows the production of three-dimensional images of microscopic structures, revealed that the RGS2 protein is necessary for the organization and orientation of the “cable” system that segregates chromosomes, the mitotic spindle.

“So, if you ask me, what the bottom line is: reducing the expression of Nek 7 or RGS2 caused similar effects on the mitotic spindle. Furthermore, the localization of RGS2 in the mitotic spindle is Nek7 dependent. This indicates a functional link between the two”, explained Edmarcia. “And they interact physically, too,” Kobarg recalled. “The similar effect of reducing one or the other is a strong sign that, in addition to physically interacting, they act in the same function.”

 The presentation of the discovery about the role of RGS2 in the mitotic spindle earned Edmarcia an award from the Brazilian Society of Cell Biology, and his use of confocal microscopy was recognized with an award at a workshop on the technology, held at LNBio.

“She applied all the resources that exist today to study molecular phenomena inside the living cell, and found new things,” said Kobarg.

Going deeper

“The fascinating thing is to see the molecular function: this translates into one thing for the cell, which in turn translates into another in the tissue, in the organ, in the formation of the tumor. All of these functions affect different levels of the organization, spilling over to the next level, so to speak,” said the advisor.

Edmarcia and Kobarg recognize that there is still much to discover about the functional relationship between Nek 7, RGS2 and the mechanisms of interaction between these molecules. “We don’t have the molecular mechanism, we don’t have the signaling cascade. And that's what we'd like to know: what Nek 7's position would be in these roles. It is our main interest”, said the researcher.

“Now we try to find out: does Nek 7 directly interact with and phosphorylate RGS2, and then does RGS2 play a role in the mitotic spindle and cell division? And know how this happens, what other molecules are involved”, added the researcher.  

“We know that Nek7 phosphorylates vitro to RGS2. Now, if we could, for example, identify the residues of phosphorylated RGS2 in vivo, or altering these residues, would be interesting”, said the advisor. “Knowing whether phosphorylation in one position of the protein makes a difference compared to another, does this mutation cause the same thing? If we had that kind of mechanistic detail, if we could molecularly connect something like the beginning of a step in cell division to phosphorylation. We are still, at the moment, at the protein level, but we have an atomic level, so to speak, a very fine level of regulation.”

Publication

Thesis: “Nek7 is a multifunctional kinase that acts on different biological processes and in concert with cell division signaling”
Author: Edmarcia Elisa de Souza
Advisor: Jörg Kobarg
Unity: Institute of Biology (IB)