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EFFECTORS OF THE SPINDLE ASSEMBLY CHECKPOINT BUT NOT THE MITOTIC EXIT NETWORK ARE CONFINED WITHIN THE NUCLEUS OF SACCHAROMYCES CEREVISIAE

Lydia R Heasley, Jennifer G DeLuca, Steven M Markus

Preprint posted on June 06, 2018 https://www.biorxiv.org/content/early/2018/06/06/340091

To stay in or go out: The S. cerevisiae nuclear envelope restricts the spindle assembly checkpoint arrest to the nucleus

Selected by Hiral Shah

Context of the study

The spindle assembly checkpoint (SAC) prevents the cell from entering anaphase until all chromosomes are properly positioned and attached to be divided equally between daughter cells. The SAC effectors Mad2, Mad3 and Bub3 form Mitotic checkpoint complexes (MCCs) at unattached kinetochores to prevent the activity of anaphase promoting complex by sequestering away Cdc20. Precise MCC concentrations, regulated by cell volume, ensure a robust checkpoint even in the presence of a single mis-attachment and at the same time allow easy dissipation of the signal for mitotic exit. These checkpoint effectors are conserved between yeasts and metazoans. Interestingly, in metazoans which undergo open mitosis – break down of the nuclear envelope –the checkpoint proteins diffuse throughout the cell. In contrast the budding yeast Saccharomyces cerevisiae shows closed mitosis, where the nuclear envelope remains intact throughtout the cell cycle, only allowing regulated protein transport through nuclear pores. In this preprint, the authors ask whether the presence of an intact nuclear membrane sets different constraints on cell cycle checkpoints? Specifically the authors investigate the mobilization and distribution of SAC effectors and Mitotic exit network (MEN) proteins between the nucleus and cytoplasm.

 

The experimental setup

To study nucleo-cytoplasmic shuttling of cell cycle regulators, the researchers study mitotic progression of two independent spindles in a shared cellular environment. They use binucleate zygotes derived by mating nuclear fusion deficient yeast strains. They reasoned that if effectors are not shared between nuclei, the two independently progressing spindles would show asynchronous anaphase. In contrast if signals were shared, then one nucleus would have the power to trigger or delay the other nucleus and would show synchronous anaphase events.

 

Important findings

Do the MCC components stay in the nucleus or shuttle back and forth? Turns out, they stay in, only affecting the mitotic spindle in their territory. The independent SAC response makes asynchronous anaphase events more common than synchronous ones in binucleate yeast cells. In cells with synchronous anaphase the authors rule out the possibility of a spindle waiting for the other by demonstrating that both spindles show similar mitotic duration and therefore synchrony is a mere coincidence.

How do the effectors mediate this confined nuclear-autonomous response? As expected the spindle arrest catalysts Mad1 and Bub1, tagged with green fluorescent protein (GFP), were exclusively found associated with the nucleus. In contrast Mad3 and Cdc20, the effectors, were seen in the cytoplasm and nucleus, with the nuclear signal intensifying during mitosis. Preferential entry of these effectors into one of the nuclei in binucleate cells, would explain the asynchronous anaphase events. But there was a twist in the tale….

Surprisingly, Mad2, Mad3 and Cdc20, which form the MCCs were imported in all nuclei in early mitosis. To solve this dilemma, the authors studied nuclear-cyctoplasmic exchange of Cdc20, using FLIP (Fluorescence loss in photobleaching)-FRAP (Flourescence recovery after photobleaching). In binucleate cells, Cdc20 fluorescence recovery in one nucleus did not lead to a simultaneous loss of fluorescence in the other nucleus. Thus, Cdc20 complexes are preferentially retained in the nucleus, explaining the independent SAC response. Come to think of it, since the chromosome-spindle attachment is a nuclear phenomenon, it makes sense for the cell to restrict the spindle assembly surveillance system to this compartment.

In contrast, mitotic exit may signal downstream events, not restricted to the nucleus, like cytokinesis, therefore a cell wide distribution of the Mitotic Exit Network (MEN) activators might be more useful. This is exactly what was seen in case of spindle disassembly, which occurred simultaneously in most of the binucleate cells. The first spindle to finish anaphase triggers the Mitotic Exit Network (MEN) leading to a premature mitotic exit in the second spindle often leaving it smaller than the first.

In brief, closed mitosis in the budding yeast Saccharomyces cerevisiae restricts the spindle checkpoint components to the nucleus, ensuring accurate chromosome segregation, before mitotic exit is mediated as a cell wide response.

 

Why I was interested

The beauty of biology lies in the fine tuning that cells and organisms display to suit their specific contexts.  It is clear from diverse studies that the structure, assembly and regulation of even core cellular machinery can spring quite a few surprises across systems. Errors in cell cycle regulation can be fatal in unicellular yeasts where cell volumes and structure often set constraints on protein mobility different from metazoans. This preprint shows nuclear autonomy of the spindle assembly checkpoint in yeasts during nuclear-confined mitosis as compared to the diffusible nature previously reported during open mitosis in metazoans.

 

Future questions

As the authors suggest, the next obvious question is ‘What are the components involved in Nuclear import/export of MCCs?’ It would be interesting to study these checkpoint proteins in fungi that display ‘semi-open’, ‘semi-closed’ and the other types of mitoses that lie between open and closed. What is known about MCC and MEN protein localisation in these fungal groups?

Also read: Heasley, L. R., Markus, S. M. & DeLuca, J. G. (2017) ‘Wait anaphase’ signals are not confined to the mitotic spindle. Molecular Biology of the Cell. 28, 1186-1194.

Tags: cell cycle, checkpoints, mitosis, spindle assembly, yeast

Posted on: 16th July 2018

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