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Study sheds light on chromosome separation in dividing cells
Improper separation is a hallmark of many cancers
MADISON, Wis. — A more detailed understanding of how dividing cells ensure they receive the correct amount of DNA is the focus of a new study by University of Wisconsin Carbone Cancer Center researchers. The study sheds light on how the process occurs, how it can go awry and how it can be therapeutically targeted in cancers.
“Cancers often arise because the genome of a cell gets all mixed up, and ultimately we’re trying to understand how and why that happens,” said Dr. Mark Burkard, associate professor of medicine at UWCCC and senior author of the study. “One way to look at that is to learn how normal cells preserve their genome when they are copying DNA and dividing.”
When cells divide, they first make a copy of all their chromosomes, which contain all the DNA for a cell. The chromosome copies are lined up at the center of the cell via a tiny structure called the kinetochore. Proteins in the kinetochore send signals that the chromosomes are ready and one of each copy moves into a new cell. In normal growth and development, this separation of identical chromosomes happens flawlessly; in cancer, some cells may end up with multiple or incomplete copies, a feature known as chromosome instability that is a hallmark of many cancers. The kinetochore may provide a key to how this happens.
“We have a rough map of how things are laid out in the kinetochore, but what we don’t understand is where these signals are being made and how they move around in the kinetochore,” said Dr. Rob Lera, a postdoctoral fellow in Burkard’s lab and lead author of the study. “If we can understand those signals, it can help us figure out why cancer is mixed up in its ability to move chromosomes around the proper way and identify which signals we should inhibit with drugs.”
To answer the questions of ‘where’ and ‘how’ signaling occurs in the kinetochore, Burkard and his team focused on one of the signaling proteins, Plk1, that is required for the cell to divide chromosomes correctly. They wanted to fine-tune the map of where and how Plk1 was functioning in the kinetochore, a structure that is 500 times smaller than a human hair.
“In the past, researchers have seen Plk1 in a region of the kinetochore and they assume its signal reaches throughout the whole structure,” Burkard said. “The problem is, this structure is smaller than light itself, so typical microscopes cannot resolve Plk1’s location well enough. We did not know if Plk1 was found in one location and spreading its signal throughout the kinetochore, or if it is found in distinct pockets with localized signaling.”
They designed a special cell line where they could let the cells grow normally, then chemically shut off most of the Plk1 while leaving some of it at targeted regions within the kinetochore. Then they used those cells for two sets of experiments.
First, they looked at the ability of cells to divide when Plk1 was directed to a specific region of the kinetochore and asked if diffuse or location-targeted Plk1 allowed the cells to divide correctly. Second, they collaborated with UWCCC member Dr. Josh Coon, professor of chemistry and biomolecular chemistry, and one of his graduate students, Gregory Potts, to use a technique known as mass spectrometry to find which molecular signals from Plk1 occurred depending on where it was located. With Coon’s expertise, they could determine which proteins Plk1 was sending its signal through, in the form of small chemical changes on those downstream proteins. Together, the results from the two approaches led them to some unique insight into Plk1’s role in cell division.
“What we discovered was that the Plk1 was operating at distinct regions within the kinetochore, and primarily in a completely different region than where most people have been studying it,” Lera said. “This is a huge opportunity to discover how these signals, including Plk1 and other signaling proteins, work in a tiny structure and how they are dysregulated in cancer.”
Other collaborators include Dr. Aussie Suzuki and Dr. Ted Salmon, both of the University of North Carolina-Chapel Hill, who helped provide super-resolution microscopic maps of the kinetochore. Super-resolution microscopy was the subject of the 2014 Nobel Prize in Chemistry, and uses special tricks to ‘see’ structures smaller than light itself. Burkard says his group is using a powerful new super-resolution microscope acquired by UW for an improved view of Plk1 distribution, and trying to determine what role Plk1 plays in this previously unstudied location within the kinetochore.
