Unequal segregation of chromosomes during cell division is called aneuploidy and can contribute to cancer (left). Aussie Suzuki uses his optimized microscopy method to map out the kinetochore proteins that have an important role in the dividing process (right).
Dr. Aussie Suzuki, a new faculty member in the McArdle Laboratory, is utilizing an optimized method of microscopy known as 3-dimensional (3D) fluorescence co-localization that allows accurate measurement at the scale of 10 nanometers.
This novel method of microscopy allows for measurements in the x, y, and z planes, making it three dimensional and extremely helpful in studying cell division errors that lead to cancers. Moreover, the technique has broad research applications for studying any subcellular structure within the cell, he said.
His research primarily focuses on understanding the molecular mechanisms that ensure faithful chromosome segregation. Cancer can be caused by errors during chromosome segregation in mitosis, a process of cell division.
An unequal division of chromosomes in mitosis is known as aneuploidy, and roughly 90% of solid tumors and 50% of blood tumors contain aneuploid cells. Additionally, aneuploidy is one of the main causes of developmental diseases such as trisomy 21, also known as Down syndrome, and miscarriages.
Correct separation of replicated sister chromatids requires long protein filaments known as microtubules to attach to each kinetochore. A kinetochore is the mega-protein complex built at the primary constriction of each sister chromatid.
The microtubules attach to individual kinetochores and pull the sister chromatids to opposite sides of the cell to undergo accurate division. If microtubules from opposite spindle poles attach to a single kinetochore, the chromosome lags, resulting in an uneven split of genetic material between daughter cells.
Fluorescence microscopy is one of the most powerful tools in biological and biomedical research, said Suzuki. However, the optical limitation of many fluorescence microscopes is on the scale of only ~250 nm laterally and ~500 nm axially without applying special techniques and mathematical image processing.
A kinetochore is made up of at least 25 different core-structural proteins and each protein has 70~250 copies. So, despite being only about 200 nm wide, it is an intricate structure and can not be studied using traditional fluorescence microscopy techniques alone, said Suzuki.
“It is surprising that this tiny protein architecture (the kinetochore) on each chromosome is responsible for proper chromosome segregation machinery including microtubule assembly, monitoring errors, repairing errors, and generating force for chromosome movements,” he added.
It is a daunting challenge to study kinetochore deformation since the kinetochore is smaller than the diffraction limit of microscopy, said Suzuki.
The kinetochore protein architecture can deform during mitosis and this structural change is thought to be important for proper chromosome segregation.
Another limitation of fluorescence microscopy is chromatic aberration, which means if someone is trying to assess two or more structures in the cell using multiple fluorescent labels, the colors are sometimes shifted due to optical reasons. This shift makes is difficult to obtain precise measurements. Specifically, each color has a different wavelength, and the microscope lens alters what color the viewer sees because each wavelength has a different focal point through the lens.
Therefore, to measure the separation between green and red fluorescently labeled proteins, chromatic aberrations must be corrected for. Chromatic aberration is not uniform, which makes optimizing a method a challenge, said Suzuki.
To solve this issue and obtain accurate measurements of subcellular structures, such as the kinetochore, Suzuki developed the optimized 3D fluorescence localization method.
The 3D fluorescence co-localization method “is an amazing technique due to the 5 nm resolution for 2D measurements and ~10 nm accuracy for 3D measurements, which is better than any expensive super-resolution microscopes,” said Suzuki. With his optimized method, Suzuki has mapped ~25 different core-kinetochore protein’s 3D positions at metaphase, a stage of mitosis.
“We now have a tool to study the mechanisms of how the kinetochore architecture is deformed, and what the function of this deformation is in mitotic checkpoint control and proper chromosome segregation.”
The loss of kinetochore structural integrity may be involved in carcinogenesis. He is excited to further examine kinetochore function and move research forward in understanding the cancer-causing process, he added.
By Dominique Barthel (firstname.lastname@example.org)