By colliding two laser beams head-on, scientists at the University of Illinois (Champaign, IL) can measure the movement of chromatin (tiny packets of DNA) in the nucleus of a living cell.
“DNA, in the form of chromatin, plays a key role in several important chemical reactions that occur in living cells,” said Christopher Bardeen, a UI professor of chemistry. “Understanding how chromatin motility affects reactions, like the transcription of DNA into RNA for the production of proteins, is essential to extending our knowledge in such areas as cell reproduction, embryology and genetic engineering.”
While scientists understand how chemical reactions work in a simple test tube, the dense environment in a living cell presents a far more complicated system. “A living cell is a very complex reaction vessel, crowded with proteins and other large molecules that must move around and interact,” Bardeen said. “If we try to take a cell apart and examine its constituents, we find they no longer behave as they do in intact, living cells.”
To non-invasively measure chromatin movement in a live frog skin cell, Bardeen and graduate students Sara Davis and Andrew Stout combine a two-photon laser fluorescence technique with a standing-wave, counter-propagating geometry. First, the cell is treated with a harmless fluorescent dye that selectively labels the DNA. Then, two counter-propagating, near-infrared laser beams are used to create a standing-wave interference pattern in the cell and excite fluorescence through a two-photon transition. Following this, the researchers turn up the laser power briefly, thereby bleaching some of the dye and creating a distinctive signal pattern. As the DNA wiggles around, this pattern gradually washes out and the fluorescence signal recovers.
“If the DNA wasn't moving, we could bleach a pattern and it would remain frozen in the interference signal forever,“ Bardeen said. “By monitoring the decay of the bleached pattern, we can tell that the DNA is moving, and we can measure that movement to a precision of about 20 nm.”
Preliminary measurements have hinted at the occurrence of subdiffusion within the cell nucleus. According to Bardeen, “The chromatin is wobbling around, apparently bumping into neighboring molecules and not moving as far as it should have in the time elapsed.”
Bardeen and colleagues believe that this indicates that molecular crowding is extremely important at the nanometer length scale, and suggests a major difference between life and death. “When a cell is dead,”, noted Bardeen, “we don't see any diffusion occurring. In fact, we don't see any movement in the cell at all.”
Cellular motion is not just a simple mechanical operation. “Motion is somehow connected with life itself,” continued Bardeen. “It's one of the things that differentiates a living cell from a lump of DNA.”
The researchers described their experimental technique and presented preliminary data on chromatin movement at the American Chemical Society meeting, held August 26 to 30 in Chicago.