Using a novel single-molecule analysis technique, the laboratory of Tom Kirchhausen, professor of cell biology at Harvard Medical School and investigator at the Program in Cellular and Molecular Medicine at Children's Hospital Boston and the Immune Disease Institute (PCMM/IDI), has revealed the chemical dynamics that drive clathrin lattice disassembly, the last step in the formation of clathrin coated vesicles, carriers used to transport nutrients, neurotransmitters, signaling molecules and even viruses to their cellular destinations.
The work, described online Jan 30 in Nature Structural and Molecular Biology, shows not just the mechanism that triggers a clathrin lattice to disassemble, but describes how all of the molecules involved in the mechanism may interact over time. This work opens the door to applying single-molecule visualization to reveal molecular-level details of other cellular processes.
"For the first time, we have been able to track in real time the disassembly of a very large structure that undergoes an inherently complex process that is involved in many cellular functions," said Kirchhausen.
For instance, when a neuron fires, it releases neurotransmitters. When a cell needs nutrients, it carries in transferrin and low-density lipoproteins. Cells take in such cargo by creating a specialized carrier, essentially a bubble, called a vesicle. A spherical clathrin coat resembling a basket forms to protect the bubble. During the last step in the formation of these carriers, the lattice-like coat disassembles, allowing the now naked vesicle to dock with other vesicles or with large, membrane-bound compartments and deliver the cargo.
The coat is made of many proteins organized by a scaffold of clathrin, a three-legged protein. Dozens of clathrin molecules interlock like twisty tinker toys to form a basket around the bubble. Assembly takes from 30 seconds to about a minute. "This speed is remarkable since so many different proteins have to come together in an very organized, synchronized way," said Kirchhausen.
Scientists have known about clathrin coats since the late sixties, when they detected them in electron microscope images. When Kirchhausen began researching these coats in 1979, scientists were just beginning to develop the technology to reveal the atomic details of such tiny structures.
"My dream was to look at clathrin coats at atomic resolution," said Kirchhausen. In collaboration with Stephen Harrison, Giovanni Armenise - Harvard Professor of Basic Biomedical Science at Harvard Medical School, he finally succeeded about seven years ago. But Kirchhausen's ultimate goal is to understand how the coat assembles and disassembles, down to the most exquisite details.
His group devised a live-cell imaging technology to take movies of coated vesicles as they form inside a cell using a fluorescence light microscope. While these movies do not show the details of the clathrin coat, they do reveal how long the lattices take to form, how quickly they fall apart, and they allow tracking of other molecules involved.
In 2010, Kirchhausen and Yi Xing, a post-doctoral fellow in Harrison's group, were able to see, using very high-resolution electron cryomicroscopy, how two tiny molecules, Hsc70 and auxilin, team up to break up the comparatively giant clathrin coat. These tiny molecules, just 70-90 thousand daltons in mass, wedge themselves into the 22 million dalton spherical coat, causing tiny distortions that help break apart the lattice.
But even with high-resolution electron cryomicroscopy snapshots and real-time but comparatively blurry movies, Kirchhausen and his colleagues still couldn't put all the details together. To figure out how a tiny little molecule could take down a giant, stable lattice, they needed another tool.
"The live cell movies we'd taken before were like looking at a field of dandelions as the wind blows their seeds away. We can see the seeds blow, but we can't tell how much wind it takes to blow the seeds off a single flower, or how quickly the seeds detach," said Kirchhausen. "Our big goal was to merge these two approaches, the snapshots and the movies, and ultimately make molecular movies."
To do this, Kirchhausen worked with lead author Till Böcking, a former research fellow at PCMM/IDI now at the University of New South Wales, to devise a new technique to place clathrin coats on a glass slide and observe them with a light microscope as it disassembled with single-molecule sensitivity.
The process begins with the synthesis of recombinant clathrin, Hsc70 and auxilin in the laboratory and outfitting them with fluorescent dyes so they could be seen under a light microscope. The team put the synthesized and colored proteins together in a test tube and confirmed that the lattice forms properly and that adding Hsc70, together with auxilin and ATP, causes the lattice to fall apart.
Böcking then tethered a single fully-assembled spherical clathrin coat onto a glass slide. When he introduced Hsc70, auxilin and ATP, sure enough, the lattice disassembled. The difference is that, this time, he was able to catch the action in a movie. "The new technique is like zooming in on a single flower in the field of dandelions and seeing how different speeds and directions of wind affect the seeds," Kirchhausen said.
Using the new technique, Böcking observed that Hsc70 must accumulate to a certain level before causing the lattice to fall apart. The laboratory's earlier electron cryomicroscopy work showed that an accumulation of Hsc70 in the lattice distorts the structure. Putting these observations together, the team now proposes that each Hsc70 molecule traps a tiny distortion in the lattice. The distortions accumulate until they reach a tipping point and the lattice falls apart.
In some cases, however, the lattice did not fall apart. It turns out that, while Hsc70 does attach to the lattice, it does not stay attached forever. If the concentration of Hsc70 is high, enough of it sticks to the lattice to cause the breakdown. If the concentration is too low, the distortions accumulate, but slowly. As the number creeps up on the tipping point, Hsc70 molecules that have been hanging on for a while, begin to fall off the lattice. The distortions never reach the critical point and the lattice never disassembles.
"The single particle imaging approach we developed can be a powerful tool to study other cellular assembly and disassembly reactions," said Böcking. In fact, Kirchhausen and his team are already visualizing how the spherical lattice surrounding coated vesicle forms, and how several types of viruses manage to enter into cells during the course of infection.
Böcking T, Aguet F, Harrison SC, Kirchhausen T. "Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating." Nat Struct Mol Biol. 2011 Mar;18(3):295-301. Epub 2011 Jan 30.
