Cecilia Lee, Illustrations Editor

A team of Yale researchers discovered how a protein called Ndc1 coordinates nuclear pore and envelope assembly after cell division, an important component of understanding how cell nuclei change in cancer.

The study’s principal investigator, Shirin Bahmanyar, associate professor of molecular, cellular and developmental biology, and first author of the paper Michael Mauro GRD ’22, a former Yale graduate student and current postdoctoral associate at Columbia University, used quantitative fluorescence microscopy to study the process of nuclear growth in C. elegans worms. Their research, published earlier this summer, explains one of the ways cells regulate nuclear size, which could help understand how cell nuclei change when a person has cancer.

“The idea here is: what is Ndc1 doing specifically during the early steps of building nuclear pores after cell division?” Mauro said. “What we wanted to understand is how Ndc1 does this. These complexes are built very rapidly after mitosis and then stably incorporated into the nuclear membrane.”

The nucleus of a cell serves as a compartment within the cell that houses the genome, or basic instructions of the cell. The nuclear envelope acts as a barrier that delineates the nucleus and protects the DNA within the nucleus from the harsh environment of the cytoplasm, according to Mauro.

However, within the nuclear envelope, there are nuclear pores, which are selectively permeable openings that allow the passage of certain molecules while restricting others. Importants are factors that recognize specific amino acid sequences in proteins that are designed to enter the nucleus and ferry those proteins into the nucleus through the nuclear pore. Exportants operate with the same mechanism but ferry proteins out of the nucleus into the cytoplasm, according to Bahmanyar.

The study uses C. elegans, a worm, as the model organism for the study of nucleus growth. Mauro explained that there were several reasons for the selection of this particular model organism.

First, the early embryo of C. elegans lends itself nicely to fluorescence microscopy, the main method utilized by the study.

Second, the C. elegans embryo used in the experiments exhibited transcriptional quiescence, a state of the cell in which it is easier to induce changes through RNA insertions. Finally, the mechanism for building nuclear pores is highly conserved between C. elegans and humans.

“Worms are closer to humans than yeast, making them a better organism to study this process since the organization of the nucleus and nuclear envelope is conserved from worms to humans,” Bahmanyar said. “The other thing is, our study really requires watching the dynamics of the process — the growth of the nucleus — which happens in a rapid manner in the embryo of the organism. This allows us to monitor, quantitatively, the process using high-resolution microscopy.”

The need to monitor nuclear growth as a dynamic process also explains why the researchers chose to use fluorescence microscopy. In comparison to traditional light microscopy, fluorescent microscopy allows researchers to localize specific molecules with high resolution and observe their dynamics, according to Mauro.

The researchers also employed a tactic called fluorescence recovery after photobleaching. In this technique, the scientists bleach a certain region of the nuclear envelope that they had previously tagged using a fluorescent marker. Then, they measure how long it takes for the unbleached, fluorescent molecules to move into the bleached area by tracking the change in color of the bleached region. This allows them to measure the mobility of the nuclear pore structure.

“The nuclear pore complex is a very stable complex; it is made up of a scaffold,” Bahmanyar said. “If you bleach the scaffold, they essentially do not recover. They do not get exchanged.”

However, when studying these dynamics in the absence of a protein called Ndc1, Mauro observed faster recovery of bleached areas. This suggested that, in the absence of Ndc1, the nuclear pore complex became more mobile.

The researchers wanted to understand the role this protein plays in the early steps of building the nuclear pore after cell division. After cell division, the two daughter cells must reconstruct their nucleus, using membranes to construct the envelope and inserting necessary nuclear pores into that membrane.

The results of the experiment show that Ndc1 is responsible for both the nuclear pore complex density and ultimate nucleus size, alongside other factors.

This paper is part of larger efforts to study and understand the growth and characteristics of the cell nucleus for potential applications in medicine. Both Mauro and Bahmanyar explained that nuclear size is an established diagnostic marker for various cancers.

In addition, a change in nuclear size can affect the developmental change of a cell by disturbing the concentration of certain factors within the cell. For example, a relatively large nucleus will lead to a lower concentration of factors, which can delay or speed up transitions in the cell cycle. Finally, if the nucleus is too small, it may not be positioned correctly within the cell and could impact the cell’s division.

The next step for this research is to better understand how Ndc1 helps to stabilize and incorporate the scaffold proteins of the nuclear pore. Mauro explained that this paper is “part of the puzzle” to better understand what happens during nuclear pore complex assembly and how different factors fit into the process.

Nuclear pore complexes are composed of approximately 30 proteins called nucleoporins.