The images you see define my internship experience at this point in time. I have been working with a drug known as Calyculin A which is a phosphatase inhibitor which condenses chromosomes. The reason I am trying to condense chromosomes is to be able to clearly see any mis-incorporation of the histone variant, CENP-A. Tubular chromosomes give an easy visual in order to see where the point at which the centromere location at which CENP-A should be. If CENP-A is not at the correct location and supposedly at a chromosome arm, then chromosomes can break and segregate unequally which has dire consequences. However, optimizing this drug has not been easy.
There are two types of fixation methods that basically preserve cells (and in my case) on slides. Fixation is necessary for things like immunofluorescence which makes it possible to visual proteins that you are looking for. The two potential methods are paraformaldehyde fixation or methanol fixation. One the sides are pictures of JG3 cells both treated with Calyculin A. However, the picture on the right is methanol fixed whereas the left on is paraformaldehyde fixed. See the difference? One does not really have condensed tubular chromosomes that we want. Now you might be wondering why it matters since methanol fixation worked. A problem with methanol fixed cells is that when performing immunofluorescence on the cells, the most fluorescence gets destroyed by methanol. (The above images are stained with something called DAPI which intercalates with DNA and causes the blue color and doesn’t mind methanol).
Since we need the fluorescence of the antibody in order to locate CENP-A, optimization of the Calyculin A treatment with the paraformaldehyde fixation is necessary. Therefore, I will be looking towards different ways to make the chromosomes look more like the methanol fixation method. One of the possibilities would be make the cell membrane and nuclear membrane of the cells more permeable in order for the chromosomes to come out more. We believe (based on other images not shown) that Calyculin A is working to condense chromosomes in all cases, but in the paraformaldehyde condition, the chromosomes are not coming out of the membrane. We have thought about using detergents such as Triton-X or NP-40 in order to get rid of cell membrane.
It has been very interesting to learn all this within a short time of coming here. To be able to learn all these techniques from great mentors who guide me every step of the way has been more than an honor for me. I hope we can continue to get the results we are looking for.