In a study that answers a question few had thought to ask, researchers have discovered what happens to the excess membrane when a cell shrinks itself into a round ball before dividing.
A sphere is nature’s most efficient storage container. It requires the least amount of surface material to enclose a given volume. When it rounds up to divide, a normally flat and sprawling cell needs far less membrane.
Yet a mitotic cell does not have a lot of sagging membrane flopping about. Anyone who has ever eyeballed one through a microscope knows this. The orb appears taut as a bubble when its duplicated chromosomes line up and split apart.
So where does the extra membrane go? Endocytosis carries on, but exocytosis takes a holiday, report postdoctoral fellow Emmanuel Boucrot and Tom Kirchhausen, HMS professor of cell biology and senior investigator at the Immune Disease Institute (formerly the CBR Institute for Biomedical Research). Their findings appear in the May 8 Proceedings of the National Academy of Sciences.
In their single-cell imaging studies, Boucrot and Kirchhausen found that tiny membrane vesicles continue to pinch off and plunge into cells with their cargoes of iron and other molecules from the surface. In the rounded dividing cells, the vesicles that normally empty and fuse back into the surface membrane instead loiter and accrue.
Only when the cellular contents are divvied up between the daughter cells do the vesicles rush back out to the membrane. The researchers’ molecular movies show the released vesicles literally bubbling out in the transient blisters known as blebs. In minutes, the daughter cells recover the total membrane area of the mother cell from the mitotic vesicle reservoir.
The research refutes a belief so ingrained that the paper took a year to publish. “The main hang-up was that people thought the basic membrane trafficking mechanisms were shut down during mitosis,” said Marc Kirschner, head of systems biology at HMS, who was not a co-author on the paper but who submitted it to PNAS. “It’s been hard for people to accept that was wrong.”
The belief dates back to a study in the early 1980s. The authors apparently misinterpreted the lack of transferrin uptake into mitotic cells as a halt in endocytosis. In hindsight, it’s likely that the available receptors for the iron-carrying blood protein were all captured inside the cell and unable to cart in more transferrin because of the never-ending endocytosis, Kirchhausen said. These days, scientists who specialize in studying these processes routinely correct their results for the amount of receptor on the cell surface, he said.
But that kind of mistake is likely to happen again. “Science is so complex today,” Kirchhausen said. “You keep repeating experiments that were validated before you, and you don’t question anymore whether the procedure is right or wrong.”
Boucrot was beginning an unrelated project when he made the initial observation that led to this paper. He wanted to follow the activity of clathrin inside cells using a new three-dimensional dynamic imaging technique for live cells. The protein shapes divots in the surface membrane, which form fatty transport vesicles that account for at least half of cellular endocytosis.
Another group found clathrin on the mitotic spindle, possibly adding structural support there. In a fluorescently labeled lab cell line, Boucrot verified clathrin at the spindle. Dividing cells, with their distinctive dark bunches of separating chromosomes, are easy to find under the microscope. But Boucrot became distracted by the bright blips at the plasma membrane and adjusted the microscope focal plane to investigate.
When he tried imaging with another labeled protein, AP-2, crucial for clathrin to form the surface membrane pits, it still looked like clathrin-mediated endocytosis. But it couldn’t be, he thought. “I knew that endocytosis was supposed to stop, as it is written in any cell biology textbook and which I learned during my biochemistry master’s in Geneva,” Boucrot said.
When Kirchhausen saw the molecular movies, he was startled that they defied the dogma about membrane traffic halting for mitosis. But he also saw it was a perfect opportunity to launch a pet project—a simple visual method to measure cell size—which he had been unable to convince anyone in his lab to do until now. All in all, it was a good time to reevaluate the validity of the 25-year-old assumption.
The results were dramatic. In cells rounded up for metaphase, the total surface area shrank by half in a common human cell line and by about six to eight times in green monkey kidney cells.
Geometry has barely begun to make its impact felt on biology, said Joel Hass, a mathematician at the University of California, Davis. “These kinds of questions are just at the edge,” he said.
Eventually, no matter what new tools and techniques reveal, biologists must get down to the detail work of figuring out the mechanism. Boucrot and Kirchhausen found a continuous process of endocytosis and two distinct phases of exocytosis—a sharp fall, then an abrupt rise.
Their experiments show that blocking endocytosis by chemical or genetic means prevents the cells from shrinking into round balls. Interfering with exocytosis prevents cells from finishing cell division, resulting in multinucleated cells. Disrupting the Golgi apparatus, which manufactures new membrane, has no apparent effect on the stop and go of membrane trafficking.
The results seem to extend to other cell types, including a normally round immune cell model line and a three-dimensional breast lumen cell culture used to study breast cancer in Joan Brugge’s lab at HMS.
The new model, in which exocytic traffic controls cell area during cell division, raises many questions. For example, how do cells coordinate the process with mitosis? For now, the researchers know three new things, Kirchhausen said: cells shrink and expand back during the cell cycle; endocytosis is constant during cell division; and exocytosis and membrane recycling regulate cell size.
