Should I Stay or Should I Go?

Mechanisms Found to Activate or Arrest Final Stage of Cell Division

Ask any cell biologist: breaking up is hard to do. Most dividing cells refuse to separate until they can ensure a precise division of genetic assets. Fatal mistakes can happen any time, but the real cliffhanger in the cell cycle saga comes just before the final split of genetic material.

Shannon Turley
Photos by Graham Ramsay
Frank Stegmeir

Independent projects in biochemistry from (at left) Sashank Reddy (right) and Michael Rape (middle) in the lab of Marc Kirschner (left) and, in genetics, from Frank Stegmeier (above) in the lab of Stephen Elledge in collaboration with Wade Harper converged on a fruitful collaboration that revealed key details of the final checkpoint in dividing cells.

It has been a focal point of scientific suspense as well. How, exactly, does the cell put the brakes on cell division? And then how does it rev up its mitotic engine from a full stop to a fast finish?

New findings from two teams of HMS researchers help explain the longstanding mystery of the mechanics of this final checkpoint in the cell cycle. The studies are published in a pair of papers in the April 19 Nature.

“They address one of the key problems in all of cell biology—how the cell knows when it is time to segregate its chromosomes,” said Jan-Michael Peters at the Research Institute of Molecular Pathology in Vienna, who wrote an accompanying commentary in the same issue.

To divide into two daughter cells with complete genomes, a cell first copies all its DNA and then lines up the duplicated chromosome pairs, the chromatids. The action grinds to a halt while the cell rigs up the mitotic spindle and throws out its tow lines. When the last thread attaches properly, one to each chromosome’s midsection, or kinetochore, this penultimate pause suddenly leads to the denouement. Within minutes, the chromosomes fly apart as the nascent daughter cells reel in their full genetic legacy.

If not lassoed and reeled in, a stray chromosome could be swept into the wrong daughter cell. A grossly abnormal number of chromosomes usually means death for the daughter cells. More subtle aneuploidy is a hallmark of many solid tumors—either as a cause or consequence or possibly both.

The new papers advance understanding of the spindle assembly checkpoint, a pivotal inspection mechanism in the deceptively routine process that drives all normal growth and life. The findings also spotlight ubiquitin’s less familiar job of disassembling protein complexes in contrast to its well-characterized role in tagging individual proteins for destruction. Finally, these new details may help scientists determine the biology behind the misallocated chromosomes in cancer cells and perhaps lead to cancer drugs that target more specific molecules in mitosis.

Point of No Return
The papers center on a cluster of a dozen proteins collectively known as the anaphase-promoting complex (APC), or cyclosome, co-discovered 12 years ago by two HMS teams working independently in frog and clam eggs and a third lab working in yeast. After the long strings of DNA are duplicated, the sister chromatids condense into gene-stuffed sausages joined at their midsections. When it comes time for them to separate, the APC helps to dissolve the protein glue holding them together. The checkpoint’s main mission is to restrain the APC until the spindle apparatus is in place.

“It is the defining moment of mitosis,” said Marc Kirschner, head of the Department of Systems Biology at HMS. (Kirschner’s group discovered the APC machinery at the same time as Joan Ruderman, the Marion V. Nelson professor of cell biology, working in collaboration with Avram Hershko.) “It is the last step, from the point of view of regulation. Once chromosomes separate, it is no longer possible to go backward.”

“They address one of the key problems in all of cell biology—how the cell knows when it is time to segregate its chromosomes.”

The two research teams that published the current papers, one from Kirschner’s lab, began their investigations independently and finished in close collaboration. Their work shows a surprisingly dynamic checkpoint control of the APC by enzymes that put ubiquitin molecules on its molecular partner, Cdc20, and other enzymes that pluck ubiquitin off.

Scientists knew that kinetochores of untethered sister chromatids send checkpoint proteins to shut down the APC. The new model shows that the APC itself (with a little enzymatic help) continually shakes off its molecular oppressors by ubiquitinating Cdc20, according to the team from Kirschner’s lab, led by Sashank Reddy and Michael Rape. When the last kinetochore is tethered, the APC and its partner—ubiquitinated but not destroyed—can finally free themselves from inhibition, dissolve the protein glue binding the chromatids, and let fly the chromosomes into the daughter cells.

A reinforcement system guards the restraints on the willful APC while the cell is in its holding pattern, reports the other study, led by postdoctoral fellow Frank Stegmeier from Stephen Elledge’s lab and in collaboration with the labs of Kirschner and Wade Harper. They discovered an enzyme, dubbed USP44, or protectin, that hustles to remove activating ubiquitin from Cdc20, while the newly assembled spindle is still reaching out to the chromosomes.

The Model Holds Water
Reddy and Rape (pronounced Rappa) approached the problem from a biochemical perspective. Rape, now on the faculty of the University of California, Berkeley, had observed that cells expressing high amounts of the ubiquitin-conjugating enzyme, UbcH10, blow through the spindle checkpoint instead of stopping. They investigated why this happens. Reddy and Rape found that UbcH10 with help from another protein, p31-comet, floods the APC’s partner with ubiquitin, apparently faster than USP44 can swab it away. The imbalance rapidly accelerates the cells back into mitosis from the standing stop of the spindle checkpoint, even if all kinetochores are not attached to the spindle.

spindle assemby
Adapted from original courtesy Nature

Getting ducks in a row. A dynamic balance of ubiquitination and de-ubiquitination regulates the final safeguard in cell division, according to a new model of the spindle-assembly checkpoint. The mitotic spindle must lasso each sister chromatid for secure delivery of a complete genome to new daughter cells. Until then, untethered kinetochores continuously send checkpoint proteins Mad2 and BubR1 to restrain the anaphase-promoting complex/cyclosome (APC/C) and its molecular partner Cdc20 from dissolving the glue binding the sister chromatids. The APC frees itself by adding ubiquitin to its partner, with the help of the UbcH10 enzyme. These ubiquitin tags are plucked off by USP44 while the cell is in its holding pattern. When the last kinetochore is attached, the APC and Cdc20 can move full speed ahead, unencumbered by the checkpoint proteins.

Meanwhile, Stegmeier had identified USP44 through an RNAi screen in mammalian cells and pursued its biological functions. The new checkpoint player came from a customized screen of the extensive short hairpin RNA library targeting more than 800 human genes in the ubiquitin–proteasome pathway system developed with Harper, the Bert and Natalie Vallee professor of molecular pathology at HMS. Intriguingly, the protein was required for checkpoint function, but did not work at the kinetochore like most previously discovered checkpoint proteins. Instead, this de-ubiquitinating protein reinforced the checkpoint proteins binding to Cdc20, acting as a safeguard to sustain the checkpoint in the face of the powerful APC push to move ahead. “We were only able to fully understand this mode of regulation by combined approaches,” said Stegmeier, now at Novartis. “The problem of biochemistry is Who are the players and Is it true in vivo? The problem with genetics is What is the mechanism? We started with a genetic discovery. Using biochemical approaches, we were able to develop a mechanism of action. Finally, we confirmed our biochemically derived model with genetic techniques in vivo.”

Kirschner likens the molecular action at the checkpoint to a bathtub holding water from the running tap with the drain only partially plugged. The antagonistic activities of adding ubiquitin and removing ubiquitin are under separate control and set the thresholds for moving in and out of the checkpoint. When the last kinetochore is attached, there are no more sites to send out APC-inhibiting checkpoint proteins. With that spigot turned off, the bathtub quickly drains. Whether or not there is another signal is unclear.

Recently, other researchers have found high levels of UbcH10 in many cancer cells. The new molecular understanding of the spindle checkpoint provides two potential new targets to test in cancer cells, speculate Reddy and Kirschner.