Volume 2, Issue 16 November 2005
Seeing Cellular Machinery
Eva Nogales is also a staff scientist at the Lawrence Berkeley National Laboratory.
A cell is perhaps the most complex factory in the world. The basic structural and functional unit of all life, cells convert nutrients to energy, perform highly specialized tasks based on instructions stored in their DNA, and reproduce themselves. How are these feats accomplished though? UC Berkeley biologist Eva Nogales is using electron microscopy to watch some of these cellular mechanisms in action.
"Traditional techniques of structural biology break a cell's complex assemblies down into small units," says Nogales, associate professor in the Department of Molecular and Cell Biology and Howard Hughes Medical Institute investigator.. "But we want to look at them closely as they exist in conditions where they're still fully functional."
A ribbon diagram based on the an image of tubulin generated by cryo-electron microscopy.
Several years ago, Nogales was part of a group at Lawrence Berkeley National Laboratory that for the first time unveiled the structure of tubulin, a protein in the cell that's essential to cell division and other cellular processes. In a living cell, tubulins chemically come together, or polymerize, into microtubules, fibers that make up the cytoskeleton to give shape to the cell and organize its contents. The magic of the microtubules is that during cell division, they disassemble and reassemble to organize chromosomal material and shuttle it to the daughter cells. Using cryo-electron microscopy, the researchers generated a high-resolution 3D image of the protein. For Nogales though, that groundbreaking picture was just the beginning.
"Obtaining the structure doesn't tell you much about its dynamics, how it does what it does," she says. "It's like looking at a piece of Lego without having any idea of all the things that can be built when you combine pieces together."
In these stills from a computer animation representing the work of Eva Nogales and Hong-Wei Wang, a sheet of tubulin transitions into a closed cylinder. Full-length video available here.
Nogales is particularly interested in how the mictoubules can shift from growth to shrinkage and back again during cell division. For a cell to divide properly, it must first duplicate its chromosomes. Pairs of connected chromosomes attach to the microtubles through structures called kinetochores. The kinetochores keep the chromosomes lined up along the ends of the microtubule fibers. When the fibers disassemble, the chromosome pairs are literally pulled apart to the opposite ends of the cell.
Since solving the atomic structure of tubulin, Nogales has been investigating how the proteins assemble and disassemble into the microtubules. Unlike the way DNA self-assembles one nucleotide at a time in a helical growth pattern, the microtubules proceed through several distinct states as they form and break down. It's a reaction called polymerization, where many small molecules come together to form macromolecules. By capturing electron microscopy images of the microtubules in various polymerized states, Nogales and postdoctoral researcher Hong-Wei Wang have been able to create a computer animation of the assembly and disassembly process. For example, the tubulins form a sheet that closes itself up into a tube. And during disassembly, the microtubule doesn't simply fall apart. Rather, the chains of tubulins peel back like a banana.
In these animation stills, protein caps fall off the ends of the microtubles spurring it to peel apart. Full-length video available here.
The latest work has already provided insight into an essential mechanism of cell division. In a collaboration with Molecular and Cell Biology professors Georjana Barnes and David Drubin, and their postdoctoral researcher Stefan Westermann, Nogales has helped reveal how a kinetochore "collar" moves down the microtubule. As the fiber peels apart, the kinetochore ring is pushed along to the appropriate daughter cell.
Even as the mechanisms of cell division slowly become clear, Nogales says, more questions arise. The researchers are now beginning a "cell-wide search" to identify the cellular factors that bind to specific tubulin assemblies during the assembly and disassembly of the microtubules. The structures they've already characterized will serve as the bait on this fishing trip, hopefully attracting the molecules they're seeking.
In this electron microscope image generated by the Nogales Lab, kinetochore rings are visibly bound to the microtubules.
In her earlier work, Nogales looked at how the anti-cancer drug Taxol interferes with the flexibility of tubulins. Deepening our understanding of how the microtubules form, she says, could someday lead to a more effective cancer treatment that targets only the tubulins of diseased cells.
"We are very from being able to build structures that have the flexibility and incredible robustness of tubulin," Nogales says. "But nature does it beautifully."