The role of Ki-67 in mitotic cell division has been a mystery. Extensive imaging reveals that this highly positively charged protein coats chromosomes to prevent them from coalescing. See Letter p.308
DNA is often imagined as an abstract string of genetic information, but in fact it is folded up by proteins into chromosomes, which are reminiscent of the dense polymeric materials that are often studied in physics and chemistry. The physical and chemical properties of this material are particularly crucial during cell division (mitosis), when the cell's chromosomes are disentangled from one another and compacted into tidy packages that segregate into two daughter cells. But after more than a century of study, the compaction and individualization of mitotic chromosomes remains poorly understood. On page 308, Cuylen et al.1 report that a protein called Ki-67 coats the surface of chromosomes, providing a barrier that keeps them apart during mitosis.
During prophase, the initial stage of mitosis, replicated chromosomes are compacted into thick fibres. The membrane that surrounds the nucleus subsequently breaks down and the chromosomes separate from one another in the cytoplasm, eventually aligning along the centre of the cell during metaphase. This alignment is organized by a microtubule-based structure called the mitotic spindle, to which the chromosomes are attached. At the end of mitosis, the spindle pulls a set of chromosomes to each pole of the dividing cell.
One might expect chromosomes to coalesce rather than separate after the breakdown of the nuclear membrane, because the molecules responsible for chromosome compaction are by themselves unable to distinguish different chromosomes. Many other polymeric assemblies in cells — including RNA–protein (RNP) bodies such as P granules and nucleoli — do exhibit liquid-like coalescence on contact with each other2. Cuylen et al. questioned what mechanisms the cell may use to keep chromosomes from sticking together when they are released into the cytoplasm.
The authors used automated live-cell imaging to analyse the effect of removing different proteins from cells. Out of more than 1,000 candidates tested, only cells lacking Ki-67 showed a severe defect in chromosome separation — their chromosomes were no longer visible as distinct cylindrical units, but were instead stuck together in an amorphous mass. The resulting glob of chromosomes was relatively impenetrable to the growing mitotic spindle, hampering progression through mitosis.
Ki-67 is a large protein that has long been known to associate with the surface of chromosomes3. It is used as a marker of proliferation in cell biology and to assess the growth of tumour cells during cancer diagnostics. What is Ki-67 doing to keep chromosomes distinct during mitosis? Cuylen et al. point out that Ki-67 is a highly positively charged protein and is predicted to be mostly structurally disordered. The authors report that amino-acid substitutions or deletions in the middle or amino-terminal regions of Ki-67 had little or no effect on chromosome separation. However, the carboxy-terminal end of the protein was essential for its function.
In a set of simple but well-designed imaging experiments, Cuylen and colleagues showed that Ki-67 sits on the chromosome surface with the C-terminal end binding the chromosome and the other end sticking out into the cytoplasm, rather like bristles on a brush (Fig. 1). The authors observed a direct correlation between the amount of Ki-67 on the chromosome surface and the average spacing between chromosomes, which increased to around one micrometre when Ki-67 was at its highest levels. Thus, Ki-67 forms a brush-like arrangement on the surface of chromosomes, apparently providing a physical and electrostatic barrier that keeps chromosomes apart.
The behaviour of Ki-67 is reminiscent of that of a class of molecules known as surfactants, which are named after their 'surface-active' behaviour. As with Ki-67, surfactant molecules have ends with different affinities. Amphiphilic phospholipids are a ubiquitous example in biology — their charged ends prefer water, whereas their hydrocarbon tails prefer oil. The tails can also stick to themselves, leading to the formation of phospholipid bilayers, such as those that make up cell membranes. More generally, surfactants assemble at interfaces, and are often used to keep droplets or particles from aggregating4.
Protein surfactants have arisen before in biology: for example, lipoprotein complexes keep the alveoli in lungs from sticking together and collapsing5. Cuylen and colleagues' work indicates that proteins also act as surfactants in the cell by stabilizing large macromolecular assemblies. Because there are many other types of membrane-less body in the cell (for example, RNP bodies and large signalling assemblies), it is possible that protein surfactants have a similar organizational role in these other structures. Between cell divisions, Ki-67 is located in nucleoli, in which the cell's protein-synthesizing machinery is assembled; perhaps it has a yet-to-be-discovered surfactant role there, helping to tune the surface tensions of the nucleolus and thus facilitate subcompartmental organization6.
Cuylen and colleagues' work opens up several avenues for investigation. It remains unclear why Ki-67 comes into play only when chromosomes are in the cytoplasm and not during prophase — how are the mechanisms that underlie prophase chromosome compaction and individualization7 integrated with Ki-67's assembly and function? The brush-like self-assembly of Ki-67 is particularly intriguing because chromosomal DNA should be negatively charged and so attract positively charged proteins, and yet Ki-67 is oriented away from the chromosome surface. And given the nanometre-scale distances over which electrostatic interactions occur, it will be interesting to define exactly how increasing the amount of Ki-67 increases interchromosome separation by up to a micrometre. These and other sticky chromosome questions are no doubt surfacing in many researchers' minds.Footnote 1
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Brangwynne, C., Marko, J. A sticky problem for chromosomes. Nature 535, 234–235 (2016). https://doi.org/10.1038/nature18904