Dani Bodor, PhD candidate at Jansen's laboratory and first author of this study, explains the context of this study: "We knew the CENP-A protein was playing a crucial role in the formation of centromeres. Previous studies showed that without this protein, cells failed to divide properly, with consequences in the number of chromosomes transmitted to the daughter cells. But exactly how much CENP-A was required to form a centromere? We needed to find a way to count CENP-A molecules, that have a size in the order of nanometers (1.000.000 times smaller than 1 millimeter)."
The research team set to develop tools that allow for such a measurement. Using modern genetic engineering they fused a gene that codes for a fluorescent protein to the CENP-A gene. By using this genetic trick, all CENP-A proteins produced by cells became fluorescent. Next, the researchers observed these cells under the microscope, and were able to quantify the total amount of fluorescence present in the cell and the fraction of fluorescence at centromeres. Ultimately, these measurements allowed them to determine that approximately 400 molecules of CENP-A are present on the centromeres of human cells.
Dani Bodor says: "We were inspired by a methodology used in yeast. But until now, no one had used it to measure molecules in more 'complex' cells. Yeast cells have more or less the same shape and volume, but human cells differ in shapes and volumes which increases the degree of complexity when this kind of techniques are used."
To confirm their calculations were accurate, the researchers used two other techniques. Their results showed that independently of the technique used they would always reach a number around 400.
Lars Jansen says: "Centromeres need to be very stable structures t
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Instituto Gulbenkian de Ciencia