Feb. 24, 2003 ANN ARBOR, Mich. -- A path-breaking collaborative effort of University of Michigan researchers will attempt to capture never-before-seen views of the chemical activity inside living cells in real time and 3-D.
The three-year project brings together public health, engineering, chemistry, biology, physics and statistics with support from a $1.5 million grant from the W.M. Keck Foundation in Los Angeles. The University of Michigan has committed an additional $500,000 to the project.
The U-M team will be using synthetic nanoprobes small enough to fit inside a cell without interrupting its normal functions to measure the activity of crucial metal ions like zinc and copper as the cell works. Sophisticated statistical modeling programs will be used to interpret data that looks something like a swarm of fast-moving fruit flies zinging around a bowl of fruit.
Trafficking metal ions in and out of the cell is crucial to basic functions like muscle contraction and the nervous system. But science has been unable to measure this dynamic process in real time.
"Only by combining several fields of science can this exploration even be attempted," said U-M President Mary Sue Coleman. "This is an innovative, cross-cutting, collaborative project that represents the best of what the University of Michigan has to offer." She noted that four deans---engineering, public health, medicine, and literature, science, and the arts---and the provost's office provided support for the Keck Foundation grant.
The study will look for patterns in the motion of ions to determine when and how individual molecules in the swarm might trigger the cell to act in a certain way at a particular time. Biochemists in the group will provide proteins that bind specifically to zinc and copper ions to help the nanoprobes do their work.
"In this project, the biochemists are the device guys and we engineers are the hypothesis-testers," said Ann Marie Sastry, the project co-leader, and associate professor of mechanical and biomedical engineering. "It's usually the other way around."
"The key is to model the experiment beforehand to design a probe that won't be too aggressive about capturing ions, or too passive," Sastry said. "The simulations are also used to figure how and where to deliver the probes to the cell. A supercomputer crunches through millions of different scenarios to help the scientists later determine which actions were random, and which had meaning.
"If we don't do this," Sastry said, "we have no way of interpreting the richness of data provided by the probes."
"We're creating a seamless connection between analytical chemistry, experimental cell biology and these mathematical models," said Martin Philbert, associate professor of toxicology in the School of Public Health, the other project co-leader. "For the first time, we have a real shot at looking at the function of these low-abundance metal ions which we know are so critical for cell function."
Biologists have long known that ions like calcium, zinc, sodium, potassium and copper are critical to cell function, but they've never been able to see the individual buying and selling of ions that each cell does, nor where those ions go inside the cell. In fact, an enormous amount of the literature on cell function concerns the mere presence or absence of an ion, or its average abundance. But that's like a two-dimensional snapshot in time, not the dynamic, three-dimensional process that actually occurs.
"The biologists have been discarding the statistical noise, but the noise is where you see the speed and degree of ion exchange in the cell," said Sastry, whose group has developed software to model swarms of ion-containing probes moving through the complex intracellular space.
In a test of the modeling software, Sastry's group simulated zinc binding with parameters provided by U-M biochemist Carol Fierke, professor of chemistry and a charter faculty member of the Life Sciences Institute, and found that significant noise results from rapid binding and unbinding of zinc ions within the cell. "We showed that the intracellular zinc concentrations are probably higher than previously thought, by analyzing this 'noise'," Sastry said, "but we need to track the actions of individual atoms to be certain."
Christian Lastoskie, associate professor of civil and environmental engineering, is the team's specialist in simulating the atom-by-atom interactions between ions and protein binders, providing the binding sites and velocities of individual ions in the cell. "It's pretty well understood how ions exit and enter the cell, but less well-understood how they move about once they're inside the cell," said Lastoskie. "Our simulations model how ions bind and move from site to site."
Zinc ions are one of the targets of this study because they are known to be important players in many neurological diseases and conditions, including Alzheimer's and brain injuries, but they are notoriously difficult to measure. Fierke estimates that the understanding of zinc signals is about 20 years behind what we know about calcium signals. "In the zinc field, we are just beginning to learn how to think about the complexity of ion exchange," Fierke said.
The nanoprobes that will help make these measurements were developed by Philbert and Raoul Kopelman, the Kasimir Fajans Collegiate Professor of Chemistry, Physics and Applied Physics. They can be made from a variety of synthetic materials, including plastics, and tailored for a variety of uses, including exploding on cue as a smart-bomb against individual cancer cells. Kopelman and Philbert call them PEBBLEs, or Probes Encapsulated By Biologically Localized Embedding. The probes---which are as small as 20 nanometers---can be made to emit light when an ion, such as zinc, binds to a specific site within the sensor.
To get some sense of how small these probes are, if the cell were the size of a football stadium, the PEBBLE would be about the size of an offensive lineman. So it and thousands of its colleagues are able to move around without disturbing the cell too much.
Fierke's earlier discovery and description of several proteins that bind selectively to zinc will enable Philbert and Kopelman to make the PEBBLEs smaller. Dennis Thiele, professor of biological chemistry, also will be providing a complete catalog of the proteins he has discovered that bind copper.
Each of the technologies being applied to this project has developed a pretty good track record on its own. But by bringing them together in a new way, this approach to cell-by-cell diagnostics should be able to see healthy and diseased cells in action and determine how they operate differently from one another.
"The applications for this kind of technology are going to be as wild as the imagination of the people we are training in our labs," Philbert said.
The W. M. Keck Foundation was established in 1954 by William Myron Keck, founder of The Superior Oil Company. The foundation's grantmaking is focused primarily on the areas of science, engineering, medical research and higher education.
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