Exemplary biophysics

Washingtonians tend to be so focused on their careers that their small talk at parties often and infamously begins with the question, “What do you do?”

If I were a biophysicist, I’d be tempted to describe my own research. But if I wanted to convey the essence of biophysics to an inquiring Washingtonian, I’d tell the story of one famous biophysical experiment: Alan Hodgkin and Andrew Huxley’s investigation in the late 1940s of the physical basis of signal transmission along nerve fibers.

Having learned that squids of the genus Loligo possess axons of exceptional thickness, Hodgkin and Huxley set to work on a series of experiments that entailed painstakingly threading tiny saline-filled capillaries through the axons to use as electrodes. Then, with amplifiers and other equipment that they had designed and made themselves, they recorded the tens of millivolt impulses that zipped along the axons at speeds of 100 meters per second or so.

From their measurements, Hodgkin and Huxley deduced that the signal consists of a wave of depolarization, mediated by ions, that travels along the axon’s outer cell membrane. What’s more, they formulated a set of nonlinear differential equations that approximate the electrical characteristics of nerve cells. Their experiment and model earned the pair the 1963 Nobel Prize in Physiology or Medicine.

Budding yeast

I like the story behind the Hodgkin–Huxley model because it exemplifies two of the main thrusts of biophysical research: devising new experimental techniques for making quantitative measurements and applying the laws of physics to understand those measurements.

Those two main thrusts were also apparent in a talk I heard at the Biophysical Society meeting earlier this month. Theorist Aleksandra Walczak of the École Normale Supérieure in Paris described experiments led by her collaborator Nathalie Dostatni of the Curie Institute, also in Paris. As Walczak put it, the broad aim of their research is to discover how the form and function of organisms are determined by the transcription factors, promoters, morphogens, and other components of gene regulatory networks that are ultimately responsible.

Dostatni’s principal experimental tool was an ingenious technique invented in the late 1990s by Robert Singer of the Albert Einstein College of Medicine in New York City to investigate yeast reproduction. Most of the time, yeast, which is a unicellular fungus, reproduces asexually by forming a bud at one end the cell. A daughter nucleus forms in the bud, which then splits off from the mother cell. But when their food is scarce or when their environment is harsh, yeast cells merge, reproduce, and create spores, which are more robust than yeast cells.

Singer knew that for budding to proceed, a particular protein, ASH1, concentrates at the tip of the bud before the daughter nucleus forms. The genetic instructions for making ASH1 are embodied in strands of messenger RNA (mRNA), which must somehow make their way from the mother nucleus to the bud tip. Knocking out the gene for a transport protein, myosin, prevents budding, which suggests that myosin carries ASH1 mRNA directly to the tip. But myosin’s role in budding could conceivably entail carrying to the bud tip a molecular anchor that ASH1 mRNA subsequently binds to.

To determine which, if either, of the two possibilities was right, Singer and his colleagues created and inserted two constructs into the DNA of their yeast cells. The first encoded green fluorescent protein (GFP) and a protein from the coat of a virus, MS2, which infects the bacterium Escherichia coli. The construct also encoded a sequence that makes sure that any mRNA transcribed from the construct is flagged for retention within a mother nucleus.

To assemble within E. coli, MS2’s coat proteins must recognize at least part of the virus’s RNA. For their second construct, Singer and his colleagues used the part of the ASH1 mRNA sequence that myosin would latch onto if myosin were indeed the ferry that conveys ASH1 mRNA to the bud. They also added six copies of the part of MS2’s RNA sequence that the virus’s coat protein latches onto. (Six to boost the fluorescent signal.)

Both constructs also contained DNA sequences that induced the cell to transcribe the constructs in the first place.

Yeast cells that contained the two constructs manufactured GFP in their mother nuclei. When the nuclei manufactured the mRNA for the second construct, six GFP molecules attached to the strands to form a fluorescent particle. By illuminating the cells with laser light and videoing them under an optical microscope, the researchers could track the particles, which made their way toward the bud tip.

The particles moved along paths and with speeds, 200–440 nm/s, consistent with myosin motors running along rail-like filaments of actin that span the cell. Singer and his colleagues confirmed that observation by creating mutants without the protein that connects the myosin motor to its actin rail. Interestingly, once the particles reached the bud tip, they appeared to wander for a while, suggesting that whereas ASH1, and not an anchor molecule, is transported to the tip, an anchor molecule might still be needed to fix ASH1 to the tip.

The experiment that Walczak described at the Biophysical Society meeting was on fruit fly embryos. Using Singer’s technique, Dostatni and her team tracked the dynamics of two proteins, bicoid and hunchback, that concentrate at the front ends of cells in the early development of embryos. Working sometimes separately and sometimes together, bicoid and hunchback control the transcription of genes for proteins that end up in the fly’s head and other front-end body parts.

In tracking how bicoid and hunchback work together, Dostatni’s experiments revealed rich dynamical behavior. They could identify, for example, when and where bicoid regulates the production of hunchback and when and where it doesn’t.

Like Hodgkin and Huxley before them, Walczak and her collaborators are developing a system of nonlinear equations to describe those dynamics.