Living cells maintain a constant buzz of molecular activity. Molecular motors power the shuttling of proteins to their specific destinations, the curling and twisting of enzymes as they burrow into their substrates, and the unwinding and copying of DNA. Using magnetic beads, an atomic force microscope, and laser tweezers, Carlos Bustamante has developed innovative ways to investigate the physical forces that drive the movement of individual molecules within cells. His discoveries are revealing a deeper understanding of the inner workings of the cell.
Bustamante's enthusiasm for tinkering with machines in the laboratory has its roots in his childhood. As a boy in his native Peru, he spent hours taking apart and rebuilding toy cars and propelling homemade rockets with an explosive combination of chemicals. Then, as a young teen, he became inspired by the story of Santiago Ramón y Cajal, a Spanish neuroscientist who won the Nobel Prize in Physiology or Medicine in 1906 for his work on the structure of the nervous system. "I kept telling myself, here is a scientist who has a name that sounds like mine," said Bustamante.
Reading about Cajal's research motivated Bustamante to purchase his first microscope and chemistry set. By age 16, he was working in his own home laboratory, studying the behavior of paramecia. And by his mid-20s, he was an international Fulbright scholar at the University of California, Berkeley, with a background in math and physics and a lasting love of microscopes.
Bustamante first became interested in the physical forces at work in DNA replication in the late 1980s, while at the University of New Mexico. He and his colleagues used new DNA fluorescence staining techniques and wire electrodes to coax DNA to move under a microscope. To their amazement, the DNA molecules demonstrated remarkable elasticity, inching along like caterpillars. Powered by his curiosity, Bustamante wondered how much force it would take to extend a molecule of DNA, like a rubber band. To accomplish this, he anchored one end of a DNA molecule to glass and attached magnetic beads of known mass to the other end. The weight of the beads stretched the DNA, allowing the force required to extend DNA from its resting state to be precisely measured for the first time. Perhaps more importantly, this experiment showed that it is possible to study and manipulate individual molecules.
Until this time, scientists had only studied the behavior of molecular machines in massive populations. While this approach is not without benefit, it reveals little about the way large molecules work inside the cell or the forces they are capable of generating. "Inside the cell, many of these fundamental processes are carried out by only a few molecules at a time," Bustamante notes. "I believe we'll get a more realistic view of the cell's inner workings if we can follow the behavior of each molecule individually."
He and his colleagues later used laser tweezers to capture, move, and stretch DNA molecules, enabling them to determine the difference in elasticity between single- and double-stranded DNA and to measure the torsional, or twisting, elasticity of DNA. They have also introduced the enzyme DNA polymerase to a single strand of DNA and measured its motion as it helped the strand rebuild itself into double-stranded DNA, a process that occurs naturally during DNA replication.
As for his next project, Bustamante has set his sights on building a living cell, using mitochondria, the cell's "power plants," to generate cells. This idea has its origins in evolution. Around two billion years ago, as oxygen in the atmosphere was increasing, bacteria that had learned to use oxygen as an energy source were engulfed by primitive cells with a nucleus. Over time, the engulfed cells relinquished most—but not all—of their DNA to the nucleus, and evolved into modern mitochondria. Today, mitochondria still contain their own DNA, as well as protein-producing ribosomes. By inserting additional genes back into mitochondria, Bustamante hopes to orchestrate the construction of a simple, minimal cell. At the same time, he plans to learn a great deal about the underlying design principles of living cells. "It's a crazy idea," he acknowledges, "but I like crazy ideas."
Dr. Bustamante is also Professor of Molecular Cell Biology, Physics, and Chemistry, and the Raymond & Beverley Sackler Chair of Biophysics at the University of California, Berkeley.
RESEARCH ABSTRACT SUMMARY:
Carlos Bustamante uses novel methods of single-molecule visualization, such as scanning force microscopy, to study the structure and function of nucleoprotein assemblies. His laboratory is developing methods of single-molecule manipulation, such as optical and magnetic tweezers, to characterize the elasticity of DNA, to induce the mechanical unfolding of individual protein and RNA molecules, and to investigate the machine-like behavior of molecular motors.
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Photo: Ben Ailes