A Flash-Frozen View of Condensing Chromatin
For cells to divide, they must first condense their DNA into the X-shape familiar from introductory biology textbooks. While the process of cell division has been studied for over 100 years, how exactly our DNA condenses in three-dimensions within the nucleus remains a mystery. To better understand this condensation process, a new study by Cai et al used a technique called cryo-electron tomography (cryo-ET) to directly visualize nuclei and condensing chromatin in dividing fission yeast cells.
Seemingly closer to science-fiction than science, cryo-electron tomography and a similar technique called cryo-electron microscopy (cryo-EM) have revolutionized the way scientists study biological structures . As the “cryo” part of the name implies, the sample under study is frozen so quickly that ice crystals don’t have time to form, which would disrupt the biological structure of the sample. Instead, this fast freezing process – called vitrification – causes the water molecules in the sample to arrange randomly and preserves the sample in a state that is extremely close to how it exists in nature . The sample is then imaged by electron microscopy, which uses the tiny wavelength of electrons to capture images with atomic-level resolution. Samples are rotated and imaged multiple times from multiple sequential angles. All of the images from different angles are then computationally combined into a 3D model of the structure.
Cai et al performed cryo-ET on nuclei from S. pombe cells in interphase (G2), the step when the cell is preparing for division, or undergoing mitosis (prometaphase), when chromatin has condensed. They found that during interphase, nucleosome-like particles were arranged in irregular clusters and linear formations throughout the nucleus. Regions called “pockets,” which are spaces devoid of structures, and “megacomplexes,” which are large structures like preribosomes and spliceosomes, were also distributed throughout the nucleus.
Surprisingly, they found that nuclei undergoing mitosis actually looked quite similar to those in interphase. The exception lay, however, in regions with condensed chromatin. These regions contained a higher concentration of nucleosomes than in other parts of the nucleus and were also rarely populated with megacomplexes. Using Volta cryo-EM, which has slightly higher contrast than cryo-ET, the researchers also saw that some regions of condensed chromatin had more nucleosomes than others, indicating irregular compaction of the chromatin.
To investigate how chromatin packing changes in the transition from interphase to mitosis, Cai et al performed nearest-neighbor distance (NND) distribution analysis. NND calculates the probability of a point being within a particular distance of another point. If chromatin condenses uniformly during mitosis, then the NND between nucleosomes should decrease because the nucleosomes will move closer together. Intriguingly, the researchers found only a slight decrease in NND between nucleosomes from interphase to mitosis. When they calculated the 10th NND, however, they saw a much larger decrease in distance between nucleosomes. The authors explain that the most likely explanation for this observation is that nucleosomes are forming larger clusters. Nucleosomes that are already close to each other will not move much closer to each other (a small decrease in NND) to form a larger cluster, but a nucleosome that is 10 nucleosomes away, will move considerably more (a larger decrease in 10th NND) to become a part of a larger cluster of nucleosomes.
This compaction of nucleosomes into clusters could lead to a variable overall condensation of chromatin, with some regions packed more loosely or tightly than others. Because chromatin condensation is normally thought to repress gene expression, regions that are packed more loosely may allow for the expression of a subset of genes during mitosis. In fact, a subset of S. pombe genes are known to increase expression during mitosis ; therefore, the researchers asked if the apparent expression of these genes was due to active transcription or the persistence of previously transcribed mRNA. By checking for the presence of a marker for active transcription by immunofluorescence, they confirmed that some transcription was indeed taking place during mitosis in S. pombe cells.
Overall, this study shows that during mitosis, chromatin compacts somewhat unevenly, with nucleosomes likely forming larger clusters of nucleosomes. This irregular compaction allows for the expression of certain genes during mitosis. A deeper understanding of the structural and molecular features of chromosome condensation, often disrupted in certain diseases, will not only support future disease research but will also continue to unravel one of biology’s great mysteries: cell division.
Original article: Cai S, Chen C, Tan ZY, Huang Y, Shi J, Gan L. Cryo-ET reveals the macromolecular reorganization of S. pombe mitotic chromosomes in vivo. Proc Natl Acad Sci U S A, 115(43):10977-10982. DOI: 10.1073/pnas.1720476115.
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