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Interdisciplinary Team Yields High-Res Clathrin Model

Researchers from the Center for Molecular and Cellular Dynamics used electron cryomicroscopy to produce a high-resolution model of the clathrin lattice, shown here complexed with a key regulatory protein, auxilin (in red). (Image courtesy of Nature)

"This result simply would not have been possible if we hadn't had the organization of this center," said Harrison, a Howard Hughes investigator, HMS professor of biological chemistry and molecular pharmacology, and one of four senior co-authors on two papers describing the structure, which appeared on Oct. 24 in Nature online (doi's: 10.1038/nature03079 and 10.1038/nature03078). Noting that the three HMS laboratories involved in this work make up the core of the structure initiative, Harrison added, "We can only do this because we've got a community that's already talking and interacting and can integrate structural technologies and structural thinking at a variety of levels. In particular, the innovative research program established by Tom Walz, with the aid of facilities provided several years ago by the Armenise- Harvard Foundation, and the close interaction of the Walz and Grigorieff labs made the whole project possible."

Portrait of a Lattice

Their model, the most detailed to date of a complete clathrin lattice, shows for the first time the precise molecular contacts responsible for the rapid self-assembly of the transport vesicle.

The effort was headed up by biophysics graduate student Alexander Fotin, who shuttled from lab to lab, preparing protein, taking electron micrographs, and helping write code to transform the raw data into 3-D image reconstructions. Each of the four research groups contributed to pushing the limits of existing techniques in electron microscopy, image processing, and computational science, according to Fotin and Harrison.

With help from Kirchhausen, HMS professor of cell biology at the CBR Institute for Biomedical Research, Fotin prepared purified clathrin trimers whose triskelion shapes then were assembled into complete cage structures. On to the Walz lab, where second author Yifan Cheng, an HMS instructor in cell biology, helped collect electron cryomicrographs of the preparations. At this point, the images, thousands of snapshots of clathrin lattices taken from all different angles, looked like gray fuzz on a foggy background. Using algorithms developed by Walz and Grigorieff and the considerable computing power available through CMCD's grid, set up by co-author Piotr Sliz, the researchers aligned and averaged the individual two-dimensional images to produce a three-dimensional composite of the entire lattice. The last step was to apply knowledge of the atomic structure of individual clathrin molecules from Harrison's and Kirchhausen's previous X-ray crystallographic studies to model the complete structure of the 36 clathrin triskelions in the cage.

The finished model, viewed at 7.9Å, trumps the 21Å resolution of the previous electron cryomicroscopy model. In that view, researchers saw an outline of the legs of each triskelion in polygons resembling the faces of a soccer ball. Now they can see the molecular detail of how nine clathrin proteins from three triskelions interlock at the vertex of each polygon to create the interdigitated lattice structure.

The central contact points, called hubs, consist of a complex set of invariant interactions between incoming clathrin molecules that lock them into the growing cage structure. Clathrin cages come in different sizes, and a look at the different shapes shows that any size could be built without changing the basic structure of the hub, but by adding hubs and flattening the angles between them.

Opening the Cage

"Now we can start to think about the process of disassembly in molecular terms and start to build testable hypotheses of what's happening," explained Kirchhausen. "Before this it was obscure. We knew it happened, but we had no idea how." Understanding how Hsc70 participates in disassembling clathrin coats could shed light on how other protein complexes are assembled or disassembled, for example, during DNA replication or viral uncoating, said Harrison.

Harrison and Kirchhausen are eager to apply the insights gleaned from the clathrin model to their continuing investigations in cell biology and biochemistry of transport systems. According to Harrison, Kirchhausen's recent work on imaging of clathrin vesicle traffic in live cells (see Focus, Sept. 17, 2004) exemplifies the interdisciplinary approach they espouse. "There is a lot of cross-talk between insights gained at the level of live-cell imaging of single clathrin vesicles and the structural work embodied in these two papers," said Harrison. "This ensemble of work represents pretty neatly the whole vision of where we're headed with structural biology at Harvard and, I believe, in the world at large."

--Pat McCaffrey