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Live-cell Studies Pick Up Pattern in Vesicle Traffic
Clathrin-coated Vesicles Appear Randomly at Cell Membrane, Disappear if No Cargo FoundMany proteins, viruses, and other molecules rely on vesicles to hitch rides into and around cells. A study published in the Sept. 3 Cell from the lab of Tomas Kirchhausen, HMS professor of cell biology at the CBR Institute for Biomedical Research, provides the most comprehensive view to date of how these cellular taxicabs form at the cell's outer membrane and take in passengers from outside.
Molecular movies of endocytosis created by Antoine van Oijen (left, standing), Tomas Kirchhausen, and Marcelo Ehrlich (seated) suggest that clathrin-coated vesicles are constantly forming, only to dissolve if they fail to pick up cargo. (Photo by Leah Gourley)
The research team, led by HMS Dorot fellow Marcelo Ehrlich, shot movies of living cells to capture the dynamics of vesicles as they formed at the membrane. They then applied a mathematical analysis to the moving images to make generalizations about the patterns that emerged. Together, these techniques allowed the team to propose answers to longstanding questions about the traffic patterns at membranes.
Clathrin's MO
When large molecules land on the cell's outer membrane, they are sucked into the cell through endocytosis, in which the membrane envelopes the incoming cargo like quicksand. On the inside surface of the membrane, clathrin, a molecule shaped like a three-pronged pinwheel, forms a cagelike coat around the budding vesicle. Like a hand, the clathrin coat grips the incoming cargo and plucks it off, membrane and all. Then it quickly releases its grip and dissolves, leaving the vesicle to travel into the cell.
| "I think this is a fundamental property in biological systems. You're very close to equilibrium conditions and you're just assembling and disassembling." |
Since the typical coated vesicle is much smaller than the resolution of a light microscope, there is no way of directly measuring the size of the fluorescent spots of clathrin appearing in the images. But the team did have one surrogate for size: intensity of light. Ehrlich isolated fluorescent clathrin-coated vesicles from their cell line and measured their intensity. Most typical coated vesicles are formed from about 60 individual clathrin pinwheels, so this measurement was used to calibrate a scale of intensity that would correspond roughly to the number of clathrin molecules in a structure.
In addition to detecting clathrin coats of the usual size, the team detected many smaller structures--too small to form the full coat. These structures form at sites throughout the membrane and then quickly disappear. The team believes these spots represent partially formed coats, clathrin-coated dimples in the membrane that constantly grow and dissolve.
Adventures of Clathrin
To catch endocytosis in action, the team also used three fluorescent passengers, in three sizes: the tiny protein transferrin, the medium-sized low-density lipoprotein (LDL), and the much bulkier reovirus, thought to use clathrin-coated vesicles to invade cells. When Ehrlich added fluorescent cargo proteins and watched them get swallowed into vesicles, it appeared that none of these aborted coats ever captured a passenger.While the individual steps of vesicle formation have been studied in great detail, it is still unclear how these taxis find their passengers: are they called in or do they show up at the membrane and wait for a passenger to come along? One model had it that the meeting of cargo and coat was a very specific process; the cargo protein would land at its receptor in the membrane and then call in clathrin, like hailing a cab. In another model, sheets of clathrin form a sort of taxi stand at the membrane, waiting for passengers to wander by.
The reality seems more complex. Under the new initiative in cellular and molecular dynamics at HMS, Kirchhausen's team joined forces with Antoine van Oijen, HMS assistant professor of biological chemistry and molecular pharmacology, to analyze how the vesicles were distributed in space. The analysis showed that even though endocytosis does not occur on all surfaces of the cell, there are no specific taxi stands where vesicles prefer to wait. Instead, within the areas where they can form, they seem to emerge in a more random and exploratory way, constantly testing the waters, looking for passengers. If nothing turns up, they dissolve and reform elsewhere.
Kirchhausen calls this dynamic system the "exploratory model" and likens it to the way that microtubules constantly grow and then collapse if they are not stabilized by environmental cues. "I think this is a fundamental property in biological systems," he said. "You're very close to equilibrium conditions and you're just assembling and disassembling."
Ehrlich points out that the ability to see this kind of process is made possible by technology. "The most exciting thing about it is the possibility that we can now see productive and unproductive events," he said. Previous methods always measured an endpoint--purifying fully formed coated vesicles from a sample, for instance. The false starts and retreats get overlooked, unless a technology is sensitive enough to view the whole process.
Mike Roth, the Diane and Hal Brierley chair in biomedical research at the University of Texas Southwestern Medical Center, said that the study brings several lines of research together to provide a far more comprehensive view of endocytosis. "In most of the previous cases the work is like a snapshot," he said. The use of live-cell imaging and mathematical analysis give the study a thoroughness and sophistication that will increasingly become a standard in cell biology.
"We've really gone as far as we can go with people saying, look there are green spots and red spots and now they're yellow," Roth said. Instead, researchers will need to analyze the imaging data they collect to put some weight behind their conclusions.
--Courtney Humphries