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Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

Nuria Ferrandiz, Laura Downie, Georgina P. Starling, Stephen J. Royle

Preprint posted on April 23, 2021 https://www.biorxiv.org/content/10.1101/2021.04.23.441091v1

Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202203021

Chromosomes want to break free: endomembranes can envelop misaligned chromosomes in a new pathway for aneuploidy

Selected by Leonor Keating

Background

In mitosis, errors in chromosome segregation may cause cells to inherit the wrong number of chromosomes. Aneuploidy – the incorrect number of chromosomes in a cell – is associated with cancer.

For accurate chromosome segregation, sister chromatids must correctly bind spindle microtubules from opposite spindle poles during metaphase, which is a challenging task for the cell. There are many ways through which chromosome segregation can go wrong, including spindle defects, errors in kinetochore-microtubule attachments, loss of cohesion between sister chromatids, erroneous cytokinesis, or defects in the checkpoints that regulate these processes. If cell division occurs in any of these perturbed conditions, chromosomes may segregate wrongly. Understanding the causes of aneuploidy is one of the main goals of fundamental cancer research, and most studies to date have focused on the interaction between the chromosomes and the mitotic spindle.

In this preprint, the authors look at chromosome segregation from a different angle and focus instead on what happens outside the spindle. During mitosis, organelles such as the endoplasmic reticulum (ER) and the Golgi apparatus disperse. Their remnants, globally called endomembranes, organize to the cell’s periphery, leaving the spindle and chromosomes in a membrane exclusion zone, somewhat separated from the rest of the cell contents. This separation seems to be required for correct spindle function. The authors of this study wonder what would happen to a chromosome that finds itself in the membrane-packed cell periphery and end up describing a novel pathway for aneuploidy.

Key findings

Misaligned chromosomes outside the exclusion zone can become enveloped in endomembranes.

Mitotic cells are packed with endomembranes, except for an exclusion zone where the spindle and chromosomes prepare for anaphase. This can be beautifully observed in Figure 1 with the ER marker GFP-Sec61b under a confocal microscope. To study the behavior of chromosomes that escape the exclusion zone, the authors artificially increase the rate of chromosome misalignment. They do this mainly by inhibiting the motor protein CENP-E, essential for chromosome alignment, in RPE-1 cells. Electron microscopy reveals that chromosomes beyond the exclusion zone become enveloped in several layers of endomembranes but not fully enclosed in any. They call these chromosomes “ensheathed” (Figure 1).

Figure 1 from the preprint (selected panels). (a) mitotic cell illustrating the clear separation between the endomembrane-rich and the membrane-exclusion zones. (b) mitotic cell with a visible chromosome outside the exclusion zone. (c) 3D rendering of an ensheathed chromosome acquired with serial block face-scanning electron microscopy (violet: endomembranes; red: DNA)

Chromosome ensheathing negatively affects cell fate

The authors next sought to understand what happens to cells with ensheathed chromosomes. They found that two thirds of these cells do not exit mitosis normally, with the most common defect being micronucleus formation. Micronuclei are known markers of chromosomal instability that may drive tumor progression.

Closer observation of these micronuclei revealed that the initial recruitment of nuclear envelope occurs with the same kinetics, whether they come from ensheathed chromosomes or not. However, the micronuclei formed around ensheathed chromosomes have abnormally high levels of proteins with roles in nuclear envelope formation (BAF and LBR; figure 3C). The authors argue that the integrity of the micronuclei is disrupted but do not speculate on the impact of this disruption.

Figure 3C from the preprint (adapted panel). Confocal image of cells presenting micronuclei. Disrupted micronuclei have higher levels of nuclear envelope markers (red). The presence of ER staining (green) in these micronuclei suggests that they formed around ensheathed chromosomes.

Ensheathed chromosomes fail to make efficient microtubule attachments

The spindle assembly checkpoint (SAC) detects incorrectly attached kinetochores and halts the cell cycle until all errors are corrected and chromosomes are aligned at the metaphase plate. The layers of endomembranes around ensheathed kinetochores do not appear to affect SAC activation. This can be seen by the recruitment of SAC proteins Mad2 and Bub1 to these chromosomes. Naturally, this causes a delay in metaphase-anaphase transition, but surprisingly this delay surpasses that of having chromosomes misaligned but “free” from endomembranes.

One possible explanation comes from the observation that ensheathed chromosomes form inefficient microtubule contacts. Inefficient attachments might not be strong enough to quickly pull the chromosomes back to the metaphase plate. Another proposed hypothesis is that traveling through the endomembrane-packed territory impedes chromosome movement and thus delays anaphase.

Expanding the endomembrane exclusion zone rescues ensheathed chromosomes

In the crown-piece experiment of this paper, the authors “trap” the ER to the cell cortex to see if taking it out of the way is enough to rescue ensheathed chromosomes. They express an ER-associated “hook” (FKBP) that efficiently binds to a plasma membrane “anchor” (stargazin) when rapamycin is added to the cells (Figure 5). Within 15 minutes (median time), almost 90% of the ER has been cleared to the cell periphery. The alignment of ensheathed chromosomes is almost fully rescued (Figure 5). This result provides a causal link between chromosomes ensheathing and aneuploidy.

Figure 5 from the preprint (selected panels). (a) Diagram of the ER clearance strategy. (b) and (c) First and last frames of a live-cell imaging experiment where ensheathed misaligned chromosomes fail to align in control conditions (b) or are rescued by ER clearance (c). Note how the ER, in green, is packed against the plasma membrane in the second case.

Why the work is important and what I like about the preprint

In this work, the authors find that misaligned chromosomes can become enveloped in endomembranes and establish a causal link between this intriguing phenomenon and aneuploidy in mitotic cells. This is a novel concept, that changes the way we look at misaligned chromosomes.

Their results are presented very clearly with great care to confirm reproducibility in different cell lines and different systems to induce missegregation. Besides the original work, the images obtained by several microscopy methods are convincing and simply beautiful to look at.

Questions for the authors

  1. How would you explain that cells with ensheathed chromosomes have a longer anaphase delay than cells with “free” misaligned chromosomes? Do you expect that it would be solely due to slower congression caused by the inefficient microtubule attachments?
  2. I understand that ensheathed chromosomes can rarely re-join the metaphase plate. Since these chromosomes can activate the SAC, wouldn’t we expect a cell cycle arrest instead of a delay? Could we imagine that the endomembranes somehow dampen the SAC signal, for example, perturbing Mitotic Checkpoint Complex (MCC) diffusion?
  3. You describe micronuclei having disrupted integrity if formed from ensheathed chromosomes. What would the impact of this disruption be? Do you expect disrupted micronuclei to be more dangerous for the cell’s fate than “regular” micronuclei?
  4. How often do you see chromosomes missegregate because of ensheathing in control conditions? Do you think certain cell types will be more susceptible to this effect than others (maybe accounting for the prevalence of certain types of cancer)?

Tags: aneuploidy, chromosome alignment, endomembranes

Posted on: 29th May 2021

doi: https://doi.org/10.1242/prelights.29178

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Author's response

Nuria Ferrandiz and Stephen J. Royle shared

Thank you for highlighting our work!

 

  1. How would you explain that cells with ensheathed chromosomes have a longer anaphase delay than cells with “free” misaligned chromosomes? Do you expect that it would be solely due to slower congression caused by the inefficient microtubule attachments?

The delay in the anaphase onset is due to prolonged SAC activation. Our results show that the cells with misaligned chromosomes designated “free” (of endomembranes) have a better chance of achieving congression, silencing SAC and progressing to anaphase. The spatial position of free chromosomes gives them more options for lateral microtubule (MT) interactions and to convert those to end-on attachments, thereby allowing the mitotic progression.

However, the ensheathed chromosomes have a physical barrier by being surrounded by endomembranes. We think this makes potential contact with axial MTs less likely or perhaps less productive. In turn, this means the SAC stays active, and anaphase is delayed. The relocation of endomembranes to cell membranes releases this obstacle, and those chromosomes are rescued, suggesting increasing MT attachments. It is possible that chromosomes make proper contact with the spindle but that endomembranes prevent normal congression by slowing the chromosome down.

  1. I understand that ensheathed chromosomes can rarely re-join the metaphase plate. Since these chromosomes can activate the SAC, wouldn’t we expect a cell cycle arrest instead of a delay? Could we imagine that the endomembranes somehow dampen the SAC signal, for example, perturbing Mitotic Checkpoint Complex (MCC) diffusion?

We see a range of fates in cells with ensheathed chromosomes. Most often though, the cells progress to anaphase, albeit with a delay and making mistakes. Why the cell cycle is not arrested at a higher frequency is unclear to us.  A simple explanation is the exhaustion of the SAC but, it could also be that the endomembranes or the position of kinetochores beyond the exclusion zone mean that SAC signaling is “smothered”. It would be very interesting, but technically challenging, to monitor SAC signaling during the delay to anaphase onset. We have not looked at Cdc20, but it would be very interesting to observe diffusion relative to APC/c activation and anaphase onset.

  1. You describe micronuclei having disrupted integrity if formed from ensheathed chromosomes. What would the impact of this disruption be? Do you expect disrupted micronuclei to be more dangerous for the cell’s fate than “regular” micronuclei?

Our prediction is that they are more dangerous. We have not evaluated the impact of this disruption on genome integrity, but it has been shown that disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signaling. In addition, spontaneous nuclear envelope disruption is linked to chromosome fragmentation and ultimately chromothripsis, which may drive tumor evolution. However, further work is needed before classifying them as “good” or “bad” micronuclei.

  1. How often do you see chromosomes missegregation because of ensheathing in control conditions? Do you think certain cell types will be more susceptible to this effect than others (maybe accounting for the prevalence of certain types of cancer)?

In the paper, we mainly used a stable diploid cell line (RPE-1) which has very low rates of missegregation. We artificially increased misalignment using pre-treatment with a CENP-E inhibitor. Other cell lines such as HeLa missegregate at much higher rates and we could see chromosome ensheathing quite readily in this line. So, it is possible to see under normal conditions, but the frequency depends on the cell line. Whether this occurs more readily in some cell types compared to others is a very interesting question. We don’t know the answer! We do know that aneuploid cancer cells have higher rates of chromosome instability and it could be that an increase in chromosome number makes ensheathing more likely.

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