How membranes get the bends

In cells, as in people, flexibility is important. To move, communicate, divide, or shuttle cargo about their interiors, cells must shape membranes—the fatty sheets that form their outer boundaries and the borders of their internal organelles—into tubes, spheres, and other curved structures. Such shape-shifting of cell membranes is crucial to all life on Earth. In humans, impairments of mechanisms involved in membrane curvature are thought to be associated with several diseases, including muscle disorders, epilepsy, and mental retardation.

In a paper published in March 2008 in the journal Cell, a School of Medicine team led by Vinzenz M. Unger, Ph.D., associate professor of molecular biophysics and biochemistry, gave researchers a clear new view of this process and answered some of the unresolved questions in the field. That paper was recently selected by the journal Nature as one of the most significant scientific contributions of 2008.

Over the past several years, scientists around the world, including Pietro De Camilli, Ph.D., the Higgins Trust Professor of Cell Biology, have used molecular biology, electron microscopy (EM), and X-ray crystallography to determine that banana-shaped protein modules called BAR domains help membranes assume tubular or spherical shapes.

But the precise role played by BARs in the transformation of flat membranes to curved structures was unknown. Some scientists proposed a scaffolding model, in which attractive forces acting between membranes and the curved face of BAR domains create tubes and spheres in a passive manner. Other researchers, including De Camilli—whose lab first established the curvature-generating properties of proteins containing BAR domains—found that a type of BARs known as n-BARs include, or are flanked by, a molecular “wedge.” It was suggested that this wedge is inserted into the membrane, causing the membrane to buckle and bind to the BAR domains’ curvature.

While each of these processes may contribute to membrane bending to some extent, it has been difficult to appreciate the sequence of events that lead to membrane deformation, because no one had ever directly visualized BAR domains at work. “In structural biology, there is a complete black box at the interface between the membrane and water,” says Unger. “But it’s there that molecules come together, forming complexes of different compositions, and it’s those dynamic events that make a lot of biology happen.”

Unger and his colleagues are breaking open that black box with a technique known as cryoelectron microscopy (cryoEM), which preserves the biological integrity of membranes and their associated proteins. In cryoEM, a biological structure of interest is preserved in a near-native aqueous environment by plunging the sample into liquid ethane, which cools the water at the rate of 100,000 degrees Celsius per second and suspends the structure at minus 172 degrees Celsius in a protective, utterly transparent ice-like solid.

To get a close-up view of how BARs might operate in cells, Adam Frost, M.D., Ph.D., a student in Unger’s lab who began a postdoctoral fellowship at the University of California–San Francisco in April, created artificial membranes that closely mimic those found in cells and then added protein domains in the BAR family known as f-BARs. “It was a little less than a year of biochemistry to get a sample that generated a great image,” Frost explains. “Then it was another two years of taking images and analyzing them.”

In the paper lauded by Nature, on which Frost was first author, one of the most surprising things those images revealed is that BARs can bind to membranes while lying on their sides, like individual bananas sitting on a kitchen counter. Previous experiments had not identified any membrane-binding regions in this part of BAR proteins, but the cryoEM images clearly showed sidewise binding.

The team also examined BARs on tubular membranes. (The smallest were less than 600 angstroms across. By comparison, a sheet of paper is about 1 million angstroms thick.) Images of these showed that abundant BAR proteins had formed a dense outer coat on the tubes by binding on their curved surfaces and by interacting with one another at sites on their sides and at their tips; complementary experiments performed in De Camilli’s lab showed that mutations in these lateral or tip-to-tip binding sites disrupt tubule formation in cells.

Finally, the micrographs showed that the angle at which BARs sidle up to one another when forming a tube’s coat helps to determine the tube’s diameter.

Based on these combined results, the authors propose that, on flat membranes, BARs accumulate on their sides, nesting within one another until attractive forces at their lateral surfaces cause them to turn onto their tips en masse, pulling the membrane into a rounded shape as the binding regions on their curved surfaces come into play. The BARs then interact with one another to provide a stabilizing coat and to determine the diameter of tubular structures.

As the authors write in Cell, at least in the case of F-BARs, the work demonstrates that “tubule formation … results through a shape-based scaffolding system that is amplified by the self-assembly of a helical coat,” with no apparent contribution of molecular wedges.

Some of the most important cellular processes, including many involved in human disease, take place at membranes, but Unger says that the limitations of most imaging methods in this realm mean that cell biology textbooks have so far had to rely on “cartoons”—artists’ renderings largely based on inference. As Nature’s top paper designation indicates, however, scientists are increasingly turning to cryoEM to get a truer picture of these interactions. “We’ll never stand a chance of targeting any of these molecules for drug development,” Unger says, “if we don’t use imaging to replace those cartoons with the real thing.”