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Biology, the aerial view
To understand the processes of life we must get past the school buses, says Sandy Simon
Proteins in our bodies’ cells have always been believed to zip around the cytoplasm, wriggling in and out of the nucleus and escaping through the cell membrane as they perform the array of tasks that keep us alive and well. Recently scientists have visualized these dances in the cell by fusing a cellular protein to a fluorescent tag from jellyfish. This has led to many insights into the cell, but comes with many cautionary tales.
“Our eyes catch the brightest star in the heavens — the signals from a cell that shine strongest,” says Sandy Simon, professor and head of the Laboratory of Cellular Biophysics at Rockefeller. It is a classic example of what microscopists call the “yellow school bus” problem.
“Let’s say you’re a spy and you come to America to figure out what is the function of the yellow school bus,” says Simon. “I give you a spy plane, and you fly over the country taking pictures of all the school buses. What you mainly get are pictures of large parking lots filled with school buses. Every so often you’ll see a single school bus on the street, but obviously it’s a rare, stray event. You conclude it’s not significant.”
Scientists studying protein transport in living cells too often have tended to see only the school bus depots — the locations where proteins are synthesized. When they see something that associates with the protein they assume that it is an important co-factor or chaperone when in reality it may a nearby but unrelated structure.
“In biochemistry and genetics, when you look at this macroscopic picture — focusing on 10,000 buses and ignoring two — you’re averaging,” explains Jyoti Kumar Jaiswal, a postdoctoral fellow in the Simon lab. By averaging, Jaiswal says, scientists may be missing important events that are happening inside the cell.
“The way around the conundrum is to observe the single school buses,” says Jaiswal. “It’s the one that is actually performing the function it was built for: picking up and dropping off children. With patience, eventually you’ll see that every bus leaves the depot.”
(Of course, single events can also be misleading. “Each single event you observe is an anecdote,” Simon says. “Let’s say you were an alien spying on Earth, and you happened to pick Adolph Hitler as your subject to follow. The impression you get of the human race might not be the same as if you chose Winston Churchill.”)
Simon’s lab tackles the yellow school bus problem with the biophysicist’s version of the spy plane: a special fluorescence imaging system called TIRFM, or total internal reflection fluorescence microscopy. Using TIRFM, scientists can track a protein of interest with a fluorescent tag and follow it as it travels near the cell membrane. Three important discoveries have recently emerged from the Simon lab’s efforts to focus on a single bus.

How vesicles unload their cargo

A family of proteins called synaptotagmins was widely believed to trigger the release of a cell’s cargo. But after using TIRFM to study the function of a specific synaptotagmin protein, SytVII, Simon, Jaiswal and their colleagues say it actually restricts release.
SytVII is one of 15 members of the synaptotagmin family of fusion proteins, which are associated with vesicles that contain hormones and nerve signaling proteins. In response to a rise in the level of calcium in the cell, synaptotagmins are believed to help trigger fusion of the vesicles with the cell membrane allowing them to release their contents, a process called exocytosis.
Jaiswal expected vesicles in SytVII-deficient cells to fail to release their cargo, or at least to delay it. But the exact opposite occurred: vesicle release was faster and more robust. The absence of SytVII caused the vesicles to release even cargo that would otherwise have been too big to pass through the fusion pore.
“The faster response to calcium argued against the assumption that SytVII was doing its job as a calcium-dependent trigger for fusion,” says Jaiswal.
The findings, published in the August issue of PLoS Biology, help to explain how cells heal potentially lethal tears in their membranes by using cellular structures called lysosomes — regulated in part by SytVII.
“This finding may explain Jyoti’s previous discovery in which he found that when a cell has a rupture in its surface the membrane will try to patch the surface by fusing lysosomes there, but preventing the intermixing of the lysosomal and plasma membrane proteins,” says Simon. “Syt VII may be a scaffold that holds all those membrane proteins together, so they can’t diffuse out, and the cell has a way of putting a Band-Aid on to heal over.”

How SNAREs help membranes fuse

The secretion of insulin by a pancreatic cell, the transmission of a signal by a brain cell, and the growth of all types of cells depend on the proper fusion of cell membranes. In a May issue of Proceedings of the National Academy of Sciences, Simon, Jaiswal and postdoc Marina Fix set out to answer once and for all the question: what role do a class of membrane proteins called SNAREs play in membrane fusion?
According to Simon, cell biologists are divided into two camps: one that has argued that SNAREs are the key proteins necessary for membranes to fuse, and the other countering that SNAREs need to dock with vesicles before fusion can occur.
“To answer this question, we decided to start from scratch, build up a system and ask ‘What does the cell need for fusion to occur,’” says Simon.
First, Fix recreated the plasma membrane by forming a “lipid bilayer” on the coverslip of a microscope slide. She then added two types of SNAREs, vesicle-based (v-SNAREs) and target-membrane (t-SNAREs), and found that they did not fuse to the bilayer. But when she added calcium to the mix, in a concentration designed to mimic what occurs naturally at the synaptic terminal of nerve cells in the body, she watched as individual vesicles released their contents as they fused to the lipid bilayer.
“Marina’s findings are the first solid evidence that SNAREs are the minimal sufficient machinery to get physiologically relevant fusion,” says Simon.
Jaiswal says that the PNAS paper validates the Simon lab’s approach. “Our experiments used reagents that have been used by other researchers in many other experiments; it’s not that we made these things up ourselves. The difference is that other studies were done as an average, in populations. No one looked at single vesicles.”

Watching as tumors glow

Proteins aren’t the only biological structures that can be tracked by using fluorescence. Sandy Simon’s lab was one of the first to use quantum dots, nano-sized fluorescent crystals, to simultaneously track multiple proteins in living cells for days at a time. Now, in research reported in the September issue of Nature Medicine, Simon’s lab extended this technique to track cancer tumor cells in living mice.
Because the glow they emit is bright, long lasting and highly precise, quantum dots overcome some of the obstacles that limit the usefulness of other organic fluorescent molecules. Simply by altering their size, scientists can manufacture them to produce light in any color of the rainbow, and, additionally, only one wavelength of light is required to illuminate all of the different-colored dots. Thus, spectral overlap doesn’t limit the number of colors that can be used at once in an experiment. In addition, quantum dots shine for an average of 1,000 times longer than most known fluorescent dyes.
In the Nature Medicine paper, Simon, Jaiswal and postdoc Evelyn Voura injected melanoma tumor cells tagged with quantum dots into the tail veins of mice. Using a two-photon microscope at Rockefeller’s Bio-Imaging Resource Center, they tracked the tumor cells as they metastasized and traveled to the lungs. The tumor cells labeled with quantum dots were equally proficient as the unlabeled cells in metastasizing and forming tumors, and the cells did not have any detectable effect on the health of the mice.
The Rockefeller researchers say that quantum dot labeling of tumor cells could have an enormous impact on how scientists everywhere investigate the mechanisms of metastasis. “The ability to specifically tag cells with quantum dots can be a crucial tool that could enable oncologists to monitor quantitatively how well cancer cells have been eradicated after experimental treatments,” says Jaiswal.

November 19, 2004



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