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CELL BIOLOGY Structure Derived for Coat of Versatile Protein-Trafficking Vesicle When membrane proteins need to travel through the cell, vesicles are their vehicle of choice. The process of sorting and packaging proteins into vesicles is often carried out by coat proteins, which gather on budding vesicles at the membrane, help select cargo molecules, and launch the vesicle before dissolving. A study led by Tomas Kirchhausen, HMS professor of cell biology at the Center for Blood Research, and published in the Sept. 11 Proceedings of the National Academy of Sciences, offers a model for how some of these coats are assembled. A team led by Tomas Kirchhausen (below) proposed a model for how two components of COP II form a flexible, basketlike coat around vesicles. The rigid, bone-shaped complex Sec23p/24p connects to the vesicle membrane, while the complex Sec13p/31p forms a flexible scaffold. The team favors this arrangement because the crosslinks between the two complexes and head-to-head associations between Sec23p/24p complexes (shown above) allow the coat to expand both laterally and longitudinally (black arrows). A movie showing a full 3-D view of Sec23p/24p created by averaging multiple views of the complex under a high-powered electron microscope is available at the lab's website. Illustration adapted from original by Tomas Kirchhausen Photo by Graham Ramsay Much is known about clathrin coats, which carry specific proteins from the cell membrane and Golgi, but less is known about the other major coats, COP I and COP II. Kirchhausen's team used electron microscopy and a variety of biochemical studies to explain the organization of COP II coats in yeast, which carry all the protein traffic from the endoplasmic reticulum (ER) to the Golgi. What they found is a system that is more flexible than clathrin, allowing COP II to handle the larger loads of proteins bound for secretion. Stitching Form and Function Two years ago, Kirchhausen's lab produced a crystal structure of the main portions of clathrin and developed a model for how the clathrin coat assembles. The molecule, with three arms pinwheeling out from a central hub, forms a distinct, elegant lattice around the vesicle. This cagelike formation is so striking that Kirchhausen has developed sequential pictures of the clathrin coat assembling as an art piece and a movie. But if clathrin was a classical study in symmetry and proportion, COP II is decidedly avant-garde. Unlike clathrin's telltale lattice, COP II coats show up in electron microscopy images as inky rings with no discernible organization. "To make a coat, everything has to be organized," Kirchhausen said, "but we see these coats that are as good as clathrin--even better because they deal with every protein that goes through the pathway--and there is no order." Since micrographs of the coat itself yield no clues, Kirchhausen's group decided to dismantle the puzzle and see if the shape of its pieces would suggest how they were arranged. They chose two components to study: the complex Sec 23p/24p, which is thought to be involved in recognizing and sorting cargo proteins, and Sec13p/31p, thought to form the scaffold of the coat. To determine the structure of the complexes, the researchers collaborated with the lab of Thomas Walz, HMS assistant professor of cell biology, which uses high-resolution electron microscopy to glean information about molecular structure. Though crystal structures of proteins are coveted for their great detail, x-ray crystallography encounters problems capturing many large or flexible proteins or labile complexes of proteins. "In biology, almost no protein works by itself--it works in complex with other proteins," said Walz. High-resolution electron microscopy can provide a view of macromolecular complexes, though the view is usually limited to the outer surface. "It's like molecular skin," Walz said, but the skin can reveal much about what lies beneath. Electron microscopy is also limited by the radiation damage of the specimen it captures. But researchers are pushing these limits by using beams with fewer electrons and compensating for the added noise by taking multiple images and averaging them computationally. Walz's group was able to determine a structure for Sec23p/24p by capturing thousands of images of the complex. Because microscopy only provides projection images, the third dimension had to be restored by obtaining different views of the complex, imaging the specimen at a 60-degree angle. By combining the different views, the group was able to compute a 3-D reconstruction of the entire molecule. Down to the Bone The high-resolution images of Sec 23p/24p revealed a complex with a rather symmetrical shape suggestive of a dog bone. The team had determined biochemically that the complex was a heterodimer, and they were able to find similarities in the amino acid sequences of the two proteins to show that the entire complex was really two symmetrical pieces joined at the middle. In contrast to this rigid form, Sec 13p/31p, which they had determined was a tetramer, appeared in the electron micrograph as a series of five blobs acrobatically rearranged in positions as varying as a straight line, a ring, or a squiggle. Clearly this was a highly flexible molecule, which was surprising to Kirchhausen because Sec 13p/31p, as the main scaffold protein of COP II, is the element most functionally analogous to the rigid clathrin. Using what was known biochemically about the two complexes and their shape, Kirchhausen's group proposed a model for the organization of the coat that is much more flexible than clathrin. "With clathrin, everything locks in place, and as it grows, it's creating curvature," said Kirchhausen. But the curve--and therefore the size of the coat--is limited by the shape of the molecule. In the group's model of COP II, Sec 23p/24p anchors to the membrane and connects to Sec 13p/31p, which shapes the scaffold as do the fibers of a basket. As haphazard as the COP II coat seemed from a distance, Kirchhausen believes it may actually represent an evolutionary improvement to clathrin's cage, because the basketlike construction allows COP II coats to expand to fit the larger and more diverse proteins they carry. --Courtney Humphries