Microtubules play a number of different critical roles in cells, ranging from structure to intracellular transport. These little tubulin polymers also play a key role in mitosis, forming mitotic spindles that pull apart the chromosomes during cell division to ensure that the resulting cells get identical copies of the genetic material. Past research suggested that a ubiquitin ligase, anaphase promoting complex (APC), was the master regulator of the mitotic process, yet how it orchestrated the precise timing of mitotic spindle activity was not known. Michael Rape, a molecular biologist at the University of California, Berkeley, wondered if microtubules might be involved.
Rape and colleagues used a multi-pronged method to study the role of the microtubules biochemically and mechanistically. After reconstituting the substrates in vitro with purified components, the researchers added microtubules to see if they could stop the APC from degrading spindle assembly factors (SAFs).
“We developed a technique years ago where you basically break open mitotic cells,” he said. “If you do it in the right way, the cellular contents, or extracts, recapitulate many aspects of cell division, except now it’s in a test tube and you can experiment.”
They quickly learned that the addition of microtubules blocked degradation—and when they depolymerized microtubules, that stabilization could be reversed. “These microtubules generate a protective environment. And it makes sense because a lot of different proteins bind to these fibers during mitosis to help separate the chromosomes,” he said. “They work together to create a sort of protective shield to make sure that once the kinetochore fiber has been established, it won’t be degraded too early during cell division. It’s a very elegant way to make sure that cell division is robust and accurate.” This demonstrates that microtubules play an important role in regulating the timing of spindle assembly factor turnover.
“What’s good about this approach is that we could go from something very reductionist—reconstitution in the test tube with purified components—all the way to the cellular approach, where we could follow what was happening during cell division,” said Rape. “The ability to go back and forth like that helps inform us what is really happening in the cell, and when it is happening, with these different components.”
Rape said this is the first study to demonstrate that microtubules directly affect spindle protein turnover, regulating the process in a selective manner. But there is still much to learn about the mechanisms involved in this process before it might be targeted for therapeutic purposes.
“We don’t know the specific mechanistic steps yet. We know that the microtubules interfere in this degradation event, but it’s not clear how they do it,” he said. “If we want to be able to target this process therapeutically, we’ll need to learn more about the biochemical mechanism. And that’s what we hope to learn next.”
Song L, Craney A, Rape M. Microtubule-dependent regulation of mitotic protein degradation. Mol Cell. 2014 Jan 23;53(2):179-92.