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CELL BIOLOGY Molecular Movies Catch Mitochondria Dividing Demonstrate the Strides in Imaging Since X-rays Gave Clues to DNA's Double Helix On a winter afternoon 50 years ago, two scientists pored over a photograph that would change modern biology. The picture, an X-ray of DNA taken from calf cells, was one of the clearest images of DNA ever produced. But its maker, Rosalind Franklin, was not satisfied and did not publish it. Maurice Wilkins retrieved and showed the photo to his colleague James Watson, who immediately grasped its significance. Watson and his partner Francis Crick spent the next month furiously working on a cardboard model of the DNA molecule. On Feb. 28, 1953, they announced what Franklin's X-ray had suggested--that DNA is a double helix. "I happen to believe that we as human beings acquire a lot of information visually," said Tomas Kirchhausen, below left behind Ramiro Massol. "Our brain is in some ways amazingly evolved to capture images in three dimensions that are changing in time, and to interpret that information." Their movies show clusters of Dnm1p (green) attached to a site on the mitochondrion (red) that is already constricted (above left), leading to fission (above right). Once the mitochondrion has split apart, the Dnm1p cluster remains attached to one side of the divide. (Photo by Jeff Cleary. Image adapted from original by Ramiro Massol) Scientists' ability to image the inner recesses of the cell, not just with X-rays but also with light, has come a long way since that dramatic afternoon in Cambridge, England. Keener microscopes, along with ingenious dyes and cameras are bringing about an almost giddy renaissance in optical imaging. While Franklin's image was flat and steeped in an almost eerie gray, the images produced by the new paraphernalia are three-dimensional, vivid, and colorful. And they are dynamic, depicting living cells and molecules in living organisms. "We now have molecular movies," said Tomas Kirchhausen, HMS professor of cell biology. "So the first thing you see is that the mitochondrion is not static; it is actually changing in shape and in diameter all the time." --Tomas Kirchhausen Kirchhausen, who is also a senior investigator at the Center for Blood Research, and colleagues Aster Legesse-Miller and Ramiro Massol have recently trained their microscopes on the cells of living yeast with the aim of capturing mitochondria in the act of dividing. Their images, which appear in the Feb. 6 online version of Molecular Biology of the Cell, are remarkable not just from a technical point of view--they are among the first 3-D images of living mitochondria--but also from a scientific perspective. They are helping to rewrite what we know about mitochondria, the main powerhouses of the cell. Mitochondria have captured biologists' attention for centuries and certainly long before the existence of DNA was even suspected. Not only do they perform a critical cellular function, converting sugars and fats into ATP, they also carry their own packet of DNA--the only organelle to do so. Defects in their function are linked to a host of diseases, including Parkinson's, Alzheimer's, cancer, and diabetes. Until recently, mitochondria were thought to be relatively rigid tubular structures, and their division was thought also to be a relatively straightforward affair. Copies of a protein, Dnm1p, would attach to a spot on the tube, tighten, and essentially nip the mitochondrion in two. Organelle Intrigue The Kirchhausen movies reveal mitochondria to be more intriguing in appearance and behavior. In one movie, a mitochondrion painted in green fluorescent protein appears to undergo a series of constrictions and expansions along its whole length, almost as though it is wriggling. "So the first thing you see is that the mitochondrion is not static; it is actually changing in shape and in diameter all the time," said Kirchhausen. Nor do mitochondria appear to divide in the expected way--that is, by the simple attachment of Dnm1p. In another movie, for example, Dnm1p molecules are shown alighting on a patch of a mitochondrion, only to fall away. What is more likely, the movies suggest, is that the mitochondrial tube divides only when Dnm1p attaches to an area that is already constricted. Whether the pairing of Dnm1p and areas of constriction is merely a matter of being at the right place at the right time or the result of some kind of signaling is not clear. "We do not have a molecular mechanism yet," said Kirchhausen. He is working to put the images together with other data to tell a coherent story about mitochondrial division. But the new movies should be of interest to more than just fans of mitochondria. All kinds of organelles --Golgi apparatus, endoplasmic reticulum--must replicate as cells divide, and there is a good chance that some of these organelles may obey similar principles in dividing. Provocative Science Like the best of what Hollywood has to offer, the mitochondrion movies could be provocative. "My hope is by providing this new tool of imaging we will be able to generate new ideas, models, and hypotheses, and test them as we have done for the mitochondria, and also do the integration between general concepts and molecular mechanisms," Kirchhausen said. Little in the script of Kirchhausen's career pointed to him making a movie about mitochondria. Until recently, he had trained his sights squarely on an altogether different molecule, clathrin, which is known to form a coat around certain vesicles. Kirchhausen suspected that clathrin interacts with the cell membrane, eventually enclosing portions of it into a vesicle, though the exact details of how this happens are still unclear. He also knew that the vesicle is pinched off from the rest of the cell membrane and that a protein, dynamin, is involved in this process. But exploring dynamin's role in clathrin-coated-vesicle formation was difficult for technical reasons. So the researchers turned to mitochondria. "We knew that mitochondria divide and that a dynamin-like protein, Dnm1p, is necessary," said Kirchhausen. Working in yeast, Legesse-Miller, until recently a Dorit research fellow in cell biology at HMS, designed proteins that would enter the mitochondrion carrying fluorescent tags. The researchers labeled the Dnm1p molecules with a different fluorescent marker. Using wide field epifluorescence microscopy, Massol, a research fellow in cell biology, was able to focus light at various depths in the yeast cell, activating the fluorescent tags. He then snapped pictures--15 photos, 100 milliseconds apart. "In a few seconds we were able to collect data coming from the complete volume of the yeast cell," said Kirchhausen. Ten seconds later, Massol would shoot another 15 pictures, creating a time-lapse movie. Computers performed mathematical algorithms to remove what was out of focus in the consecutive stacks. "Now you can see over time what is going on in the structure of the mitochondrion," Kirchhausen said (see http://cbr.med.harvard.edu/investigators/kirchhausen/lab/research.html). These observations were surprising, especially that the mitochondrial tube must already be constricted for Dnm1p to bring about fission. But Kirchhausen had suspected that the prevailing view that Dnm1p on its own could make the cut was too simplistic. "What bothered me is that the tube linking the donor membrane with a vesicle ready to pinch is only about 100 angstroms in diameter, whereas a mitochondrial tube is 30 times larger," he said. "Something was not completely obvious to me." He is still not sure about how Dnm1p and the constricted mitochondrial tube conspire to bring about division. "Maybe there is a sensor system associated with Dnm1p that is telling when the constriction is reaching some size which is appropriate to then continue into fission," he said. At that point, Dnm1p would act as a molecular scissor, cutting the tube. And the findings raise additional puzzles. Originally, it was thought that a ring of Dnm1p molecules falls apart after making the cut. But the cluster actually clings intact to one of the cut ends after fission. "Nature is cleverer than we are," Kirchhausen said. "She is always finding something new for us to solve." --Misia Landau