Chromosome segregation (mitosis and meiosis) is one of the most fascinating biological processes known.
Chromosomes are segregated by a conserved machine called the mitotic spindle, a microtubule-based structure that captures chromosomes and guides them to opposite poles. The spindle microtubules connect to chromosomes at the proteinaceous kinetochores and uses a system of “checkpoints” and error-correction mechanisms to ensure that each daughter cell receives exactly one set of chromosomes. Unfortunately, chromosome mis-segregation does occasionally happen, resulting in birth defects and cancer progression.
Even though chromosome segregation has been studied for more than a century, we do not fully understand its molecular details. We seek to determine how hundreds of different proteins work together to segregate chromosomes. To do this, we will use state-of-the-artelectron tomography to generate “tomograms” to map out how key protein complexes are organized in their native context inside a cell.
Such tomograms can show the 3-D spatial relationships of the macromolecular complexes over length scales from nanometers to micrometers. In the long term, 3-D structures and other biochemical and biophysical results will feed into advanced simulations that can generate hypotheses about how chromosome segregation can go wrong, and how to potentially artificially correct or mitigate these errors.
The high-end imaging is done at the NUS Centre for Bioimaging Sciences, which is equipped with state-of-the-art transmission electron microscopes. The CBIS Titan Krios, for example, is a 300-keV electron cryomicroscope (cryo-EM) optimized for high-resolution, high-throughput electron cryoto-mography (cryo-ET). Using the Krios, we will be able to image mitotic cells in a “life-like”, frozen-hydrated state. Such cellular cryotomograms will reveal the organization of spindles, kinetochores, and chromosomes.
Electron cryotomographic analysis of picoplankton
Picoplankton are the smallest known eukaryotes, measuring less than 2µm across. These unicellular plants are significant contributors to the oceans’ primary production. Picoplankton are excellent models for cell biology because they have a simplified ultrastructure: each cell typically has one nucleus, one chloroplast, one mitochondrion, and one Golgi body. Remarkably, picoplankton pack tens of chromosomes into their tiny nuclei. We recently began studying the details of mitosis in the picoplankton Ostreococcus tauri, a cosmopolitan marine algae that can be found all over the world. O. tauri cells must segregate their 40 mitotic chromosomes in the confines of a nucleus < 700 nm wide.
Our recent results show that the O. tauri spindle has ~ 10 microtubules. We would now like to determine how such a spindle can segregate 40 chromosomes. Clues are likely to be found in the kinetochores, which might form oligomers.
Spindles in other single-celled eukaryotes
Unicellular eukaryotes have diverse spindle architectures, yet they share the same underlying checkpoint and error-correction mechanisms. By exploring the organization of other unicellular eukaryotes – including those with features resembling animal cells – we will learn how different organisms have adapted their mitotic machinery to carry out the same function. These adaptations include the density of chromosome packing, the mechanism of kinetochore-microtubule attachment, and the overall organization the spindle. Each organism will likely contribute clues to our overall understanding of mitotic control systems. For example, the structure of budding yeast “point” kinetochores will be needed to model how a fundamental kinetochore “subunits” pack into the huge kinetochores found in human cells.
Applications of electron tomography to cell biology
While our core interest is mitosis, we are open to collaborations on other cell-biological problems, specifically when our experience in electron tomography can be brought to bear. Electron tomography in its present form is suited to determine structures of “unique” objects (asymmetric protein complexes, organelles, cells) to ~ 4 nm resolution. Please visit our other website for examples.
Cai, S., Chen, C., Tan, Z.Y., Huang, Y., Shi, J., and Gan, L. (2018), Cryo-ET reveals nucleosome reorganisation in condensed mitotic chromosomes in vivo.
Cai, S., Song, Y., Chen, C., Shi, J., and Gan, L. (2017), Natural chromatin is heterogeneous and self-associates in vitro.
Chen, C., Lim, H.H., Shi, J., Tamura, S., Maeshima, K., Surana, U., and Gan, L. (2016), Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo. Mol Biol Cell 27: 3357-3368
Gan, L., Ladinsky, M.S. and Jensen, G.J. (2013), Chromatin in a marine picoeukaryote is a disordered assemblage of nucleosomes. Chromosoma 122: 377-386