Unraveling mitotic chromosome structure using optical tweezers.

Abstract

During mitosis, vertebrate chromosomes compact into X-shapes that are approximately four orders of magnitude shorter than the contour length of the DNA-molecule they consist of. Visualizing the internal structure of these chromosomes is difficult because of their high compaction, though some light has been shed on this problem using (super-resolution) fluorescence microscopy and Hi-C experiments. We developed an optical tweezers method to manipulate mitotic chromosomes and ‘unravel’ them starting from the telomeres, in a way reminiscent of unraveling a woolen sweater by pulling on a thread. The DNA-threads that are pulled from the chromosome are coated by different proteins that are involved in mitotic chromosome architecture (histones, condensins, topoisomerases). Unraveling the chromosome in this way allows us to image its contents without running into problems with the limited resolution of light microscopy, and without resorting to harsh treatments like chromosome fixation. Using immunostaining, we label several different proteins on chromosome threads, and like so, determine their distribution along the chromosomal DNA. While imaging, we measure the force exerted on the chromosome and thread by our optical tweezers. The well-defined overstretching reaction of double-stranded DNA, which occurs at ∼65pN, helps us to determine the number of parallel DNA strands in the unraveled DNA-thread. These DNA-threads show a wide range of thicknesses, with on one end of the spectrum sturdy fiber-like chromatin structures, and on the other end, fragile singular DNA strands. Imaging chromosomal proteins on both these structures will help elucidate the internal structure of mitotic chromosomes. Furthermore, observing the force-extension behavior while unravelling the chromosome gives insight in the folding structure of chromatin in mitotic chromosomes.