Cell Cycle Protein Has Surprising Secondary Function

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Dividing cells separate their duplicated chromosomes normally to two daughter cells (left, blue arrow). Cells expressing a mutated form of CDT1 are defective in binding to microtubules and exhibit severe delays in accomplishing this task (right, yellow arrows).

A protein involved in DNA replication was found to also play a role in microtubule binding during mitosis, an unexpected secondary function, according to a Northwestern Medicine study published in the Journal of Cell Biology.

These findings could inform more effective cancer treatments and help answer larger questions about molecular mechanisms, according to Dileep Varma, PhD, assistant professor of Cell and Molecular Biologyand senior author of the study.

“It’s very surprising for a protein involved in DNA replication to be also binding microtubules,” said Varma, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. “Whatever we find in the future will be very interesting.”

The protein, CDT1, helps facilitate DNA replication during the initial part of the cell cycle, called interphase, during which DNA is replicated and checked for errors, before being separated into two daughter cells in the latter part of the cell cycle, called mitosis.

During mitosis, microtubules pull the replicated chromosomes into two separate strands, one for each daughter cell. These microtubules manipulate the chromosomes using another protein complex called Ndc80 as a connecting structure. Ndc80 is located at specialized structures referred to as kinetochores located on the chromosomes, where the complex binds to microtubules.

It was previously known that CDT1 was present in high concentrations during the early part of interphase, corresponding to its replication function, after which the protein is degraded. However, it was recently discovered that levels of CDT1 also rose during mitosis, suggesting it had an additional, unknown function at this stage.

Towards exploring this discrepancy, the investigators created cells with dysfunctional CDT1, and found that these mutant cells stalled during the mitosis phase and never correctly divided in two. Further analysis showed that CDT1 was required to physically connect Ndc80 and the microtubules, linking the two structures together efficiently, according to Varma.

Dileep Varma, PhD, assistant professor of Cell and Molecular Biology, was the senior author of a study published in the Journal of Cell Biology.

“Apart from making a more robust connection, we think it enables better modulation of the Ndc80-microtubule connection,” Varma said. “If there are three microtubule binding sites, it might attach at two sites at some times, and with all three sites at other times.”

This newly discovered function of CDT1 has potential implications for both patient care and basic science. According to Varma, cancer drugs targeting CDT1 could improve on current therapies because CDT1 is active in two separate phases of the cell cycle.

“Traditionally, some types of drugs try to inhibit mitosis in cancer cells, but normally the number of dividing cells in tissue is very small,” Varma said. “If we can target CDT1, we can target two different mechanisms that cause genomic instability and eventually cell death.”

On a larger scale, a single protein performing both DNA replication and microtubule binding is peculiar, and may hint at a common evolutionary origin for both mechanisms, according to Varma.

“Both processes come down to negatively charged interactions,” Varma said. “It’s possible that DNA and microtubule binding proteins have retained similar mechanisms as a backup, of sorts.”

Other Northwestern Medicine authors include Shivangi Agarwal, PhD, postdoctoral fellow in the Varma laboratory and Kyle Smith, a sixth-year graduate student in the Driskill Graduate Program for the Life Sciences.

This work was supported by National Cancer Institute grant R00CA178188 and an American Cancer Society Institutional Research Grant.