Microscopy method determines how centromere structure forms

Scientists at the Stowers Institute of Medical Research (Kansas City, MO) have developed a microscopy method—pairing fluorescence correlation spectroscopy (FCS) with calibrated imaging—to count the number of fluorescent molecules in a cluster, and then determine how DNA twists into a unique chromosomal structure called the centromere. This information helps to explain how cells navigate cell division and avoid ending up with the wrong number of chromosomes.

Centromeres, which sit at the cross point of the “X” used to represent duplicated chromosomes, are DNA structures that link those duplicated strands when cells are poised to divide. As division starts, a complex cellular machine drags each chromosome to opposite poles of the cell by grabbing ontocentromeres and pulling each arm of the “X” into what will become a daughter cell.

Researchers had known that a nucleosome—a short coil of DNA twisted around a core of proteins—forms at each centromere. Within the core is a protein, called Cse4 in yeast, that is found only at that location. But the overall architecture of that nucleosome was unknown. Now, Stowers associate investigator Jennifer Gerton, Ph.D., has used live cell imaging to reveal constituents of the centromeric protein core.

“Understanding centromeres is critical because of the role they play in maintaining genomic integrity,” says Gerton. “Losing a chromosome is catastrophic for any cell. And if it happens in sperm or egg cells, it is associated with conditions like Down’s Syndrome.”

Gerton, whose lab uses both the yeast Saccharomyces cerevisiae and mammalian cells to study the mechanics of cell division, says that, previously, people had proposed at least six different centromere structures. “What we found is that centromeric nucleosomes change their structure during cell division,” she says. ”That explained why people had observed different structures. They had likely been looking at different phases of the cell cycle.”

"By demonstrating a new method for monitoring the composition of centromeric nucleosomes in living cells, this work helps to resolve some of the controversies surrounding the architecture of the centromere," said Anthony Carter, Ph.D., of the National Institutes of Health's National Institute of General Medical Sciences, which partially funded the research. "The findings have important implications for understanding chromosome segregation, and may lead to insights on how the process goes awry in certain genetic diseases."

Aiding the effort were Stowers research advisors Jay Unruh, Ph.D., and Brian Slaughter, Ph.D., who used the microscopy method to probe yeast cells engineered to express Cse4 hooked to a green fluorescent protein (GFP) tag. The approach allowed them to track and then count in a living cell the number of Cse4 molecules in a centromeric nucleosome. 

A novel microscopy approach—fluorescence correlation spectroscopy coupled with calibrated imaging—revealed the number of Cse4 molecules within single centromeric nucleosomes over the course of the cell cycle
A novel microscopy approach—fluorescence correlation spectroscopy coupled with calibrated imaging—revealed the number of Cse4 molecules within single centromeric nucleosomes over the course of the cell cycle. (Image courtesy of the Stowers Institute for Medical Research)

“To our surprise, we quickly realized that we observed 16 Cse4-GFP molecules early in the cell cycle, and then 32 Cse4-GFP molecules in anaphase,” says Slaughter. “That meant the composition of the complex was changing.” Further analysis indicated that as cells moved into anaphase a component of the centromeric nucleosome got booted out of the core complex and was replaced by an extra molecule of Cse4, changing both the shape and size of the centromere.

Gerton’s team, led by the study’s first author Manjunatha Shivaraju, Ph.D., confirmed these findings using additional approaches. They found evidence that two molecules of Cse4 were interacting at the centromere in anaphase, but these interactions were not present during the rest of the cell cycle.

Also, Shivaraju explains that because most cancer cells are aneuploid (a condition in which cells exhibit abnormal numbers of chromosomes), “knowing that centromeres undergo this structural oscillation could tell us how aneuploidy occurs at a molecular level.”

Gerton concurs, but also sees the work as reinforcing the utility of yeast as a model organism. ”The fact that nucleosome structure is conserved between humans and yeast shows that yeast is a fantastic model for studying molecular mechanisms underlying cell division,” she says.  “We will continue to use yeast to understand factors that trigger structural changes we see in centromeric nucleosomes.”

The work appears in the journal Cell; for more information, please visit http://www.cell.com/abstract/S0092-8674%2812%2900704-0.

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