Harvard University - Department of Molecular & Cellular Biology

KEEPING THE FAITH: THE FINAL ACT OF STOPPING CARELESS CELLS

by Derek Lau and Andrew Murray

January 3rd, 2012


(L-R) Derek Lau and Andrew Murray

Faithful chromosome segregation during cell division keeps cells alive and keeps cancer and birth defects at bay. A chromosome’s movements during mitosis are directed by its kinetochore, a specialized multi-protein structure that assembles on a specialized DNA sequence (the centromere) and binds to and moves along microtubules, the stiff filaments that make up the football-shaped spindle. To get the two sister chromosomes to segregate away from each other and end up at opposite ends of the spindle, their sister kinetochores must attach to microtubules from opposite spindle poles. Toward the end of mitosis, an assembly of proteins called the anaphase-promoting complex (APC) and its co-activator Cdc20 triggers anaphase and chromosome separation by catalyzing the ubiquitination and destruction of a protein called securin. In turn, the destruction of securin triggers the cleavage of cohesin, the protein complex that holds sister chromosomes together.


Eukaryotes use a control circuit called the spindle checkpoint to ensure accurate chromosome segregation. Kinetochores that are not attached to microtubules, or are improperly lined up on the spindle, activate the checkpoint, which prevents cell from segregating their chromosomes by targeting the APC and Cdc20 for inhibition. This arrest gives cells an opportunity to correct the attachment errors at the kinetochores, making sure that cells don’t start to segregate their chromosomes until they have all been properly attached to the spindle. Although the major players of the spindle checkpoint are known (which include Mad1, Mad2, Mad3, Bub1, Bub3, Ipl1, and Mps1) and have been studied extensively, we lack a molecular description of how events at the kinetochore are converted into inhibition of the APC.


To ask how the checkpoint components induce metaphase arrest, we fused checkpoint proteins to each other and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic protein interactions that may contribute to checkpoint activation. We found that fusing Mad2 and Mad3, or sticking these two proteins to each other with molecular Velcro (pairs of coiled coils engineered to bind to each other) arrests cells in mitosis and that this arrest is independent of other checkpoint proteins. We believe that combining Mad2 and Mad3 arrests cells because both proteins can bind weakly to Cdc20. When we fuse Mad2 to Mad3, the sum of these two weak bindings creates a hybrid protein that binds tightly to Cdc20, the main target of the spindle checkpoint. We reasoned that if Mad3's job is to make Mad2 bind tightly, artificially tethering Mad2 directly to Cdc20 should also arrest cells in mitosis and this arrest should not depend on any other checkpoint components. Our experiments confirmed these predictions, suggesting that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, that this stable binding is sufficient to inhibit APC activity, and that this reaction is the most downstream event in spindle checkpoint activation. The next step for students of the checkpoint is to find the molecular mechanism that explains how Mad2 binding to Cdc20 inhibits the APC and determine how events at the kinetochore lead to the changes in the conformations of Mad2 and Mad3 that allow them to inhibit Cdc20.

Read more in Current Biology (AOP)

[January 03, 2012]

 

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