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CELL BIOLOGY Cell Veil Lifted on Actin ActivitySpeckle Method Reveals Individual Molecules in Action At various points in its life, a cell weaves one or more gauzy protrusions along its membrane. Unfurled in times of need--for example, when a neuron must make its way across the brain or when an immune cell has to chase down bacteria--these gossamer ruffles hold the secret to a central question in biology: how do cells move? Like many filmy structures, the lamellipodia hide more than they reveal.
Actin fibres, seen as fluorescent lines (top left and middle), were thought to grow only at the very edge of the lamellopodium. By tracking individual actin molecules (top right), Tim Mitichison and Naoki Watanabe (shown below) discovered actin polymerization throughout the lamellopodium. Curiously, newly formed actin filaments appear to move backward (videos top middle and right). "The cell tries to go forward, but the overall sense is a backward movment," said Mitchison. Rong Li believes that this apparent retrograde movement is due to the fact that the lamellopodia's leading edge is moving forward faster than the actin filaments, which are fixed to the substrate. The filaments look as though they are going backward, when in fact they are stationary. Images above courtesy of Naoki Watanabe. Photo below by Pam Murray.
For instance, it is known that lamellipodia are knit from filaments of the protein actin that essentially pull the cell along. But how do cells weave these sturdy diaphanous skirts? Do the actin fibers, which are spun together from single molecules, take shape at the hem or assemble throughout the length of the lamellipodia? The popular scientific response has been that the actin molecules are joined together at the leading edge. In the Feb. 8 Science, HMS researchers Naoki Watanabe and Tim Mitchison offer a contradictory view. Using a method that allows them to trace individual fluorescing actin molecules, they have observed actin fibers forming throughout the lamellipodia. Cell FeatIt is an observation that does not quite make sense. Lamellipodia lie uniformly flat as they grow. Yet if new actin filaments are being produced throughout, the lamellipodia should become rounded in areas and bunch up. "I am puzzled--it's not the neat answer," said Mitchison, the Hasib Sabbagh professor of cell biology. "It says we don't understand the biochemistry as much as we might think. How does the lamellipodium maintain a constant thickness?"What makes it even more puzzling is that earlier work by Mitchison and also by HMS colleagues Rong Li and Marc Kirschner has shown that key molecules needed to polymerize actin lie at the cell membrane. As is often the case, confusion is provoking new scenarios. "It's making people think and that is good," said Li, HMS associate professor of cell biology. "There are always lots of stories about molecules and what they do, and Tim's is more a story about trying to understand how a whole ensemble of things functions," said Kirschner, the Carl W. Walter professor of cell biology. In fact, Mitchison has told his share of molecular stories. His research style is to alternate between taking a bird's eye view, mostly by imaging the cell as he does in his latest piece, and diving into its molecular trenches. His first big molecular story on actin came in 1997, when he discovered in listeria a key protein required for actin polymerization, Arp2/3. Hive of ActivitySuspecting that lamellipodia growth might also depend on Arp2/3, Li and other researchers began searching for homologs. "All heck broke loose," Mitchison said. Within a short time, Li found the yeast homolog. Intriguingly, it appeared to interact with Bee1, a protein that turned out to be the yeast counterpart of the protein that listeria uses to activate Arp2/3. Activators of Arp2/3 were soon revealed in a wide variety of organisms. The next step was to find the proteins that triggered the activators. Marc Kirschner found two such proteins, Cdc42 and PIP2, both of which occur at the cell membrane.Mitchison and Watanabe, HMS research fellow in cell biology, set out on a different path. Rather than look at what triggers actin polymerization, they wanted to understand what happens once polymerization occurs. "We wanted to know what actually makes listeria move after polymerization," said Mitchison. To do that they needed to resolve a more basic question--where exactly does actin polymerize? The ideal approach would be to watch the behavior of individual actin molecules, but this had never been done, even with recent imaging techniques. One of the most useful of these techniques--appending the gene for green fluorescent protein (GFP) to a gene of interest--produces high levels of stain, making it impossible to distinguish individual actin particles. To dim the light enough to show up discrete molecules, Watanabe tried damaging the promoter that controls GFP-actin expression, and the strategy worked. "Once we knew we were looking at single molecules, we could do measures of actin turnover that hadn't been possible before," Mitchison said. By following each fluorescent spot, seeing when and where it flickered on and off, they were able to infer that actin was polymerizing throughout the lamellipodia. "I feel I can wave this data in people's faces and say, 'Look, this is what is going on,'" said Mitchison. Unraveling an EnigmaHe is much less certain about how and why it is occurring. His gut feeling is that in the flattened structure of the lamellipodia, actin is never too far away from the cell membrane and its polymerization-triggering proteins. But that still leaves the question: how do the lamellipodia stay flat in the first place? Kirschner suggests that actin filaments could be pushing the cell membrane from the inside out, keeping the lamellipodium flat.It is also possible that the actin fibers being formed inside the lamellipodia are unraveling as fast as they are being spun, leading to a steady state and a flat profile, said Li. This unraveling, or depolymerization, creates a supply of actin molecules that are then recycled to the leading edge. In fact, she thinks that actin polymerization at the tip and inside the lamellipodia are two different things. "Tim's research shows there is actin polymerization, but that does not say it is the same as actin polymerization at the leading edge," Li said. The actin activity observed by Watanabe could be due to the knitting together of broken actin fibers. "There are lots of barbed ends in the middle. Once you create a barbed end, it has a tendency to grow. That's very different from polymerization at the leading edge. Tim's data do not agree or disagree with that. They just say it happens, not how it happens." Mitchison plans to get to the how. He and Watanabe are planning to track other proteins, such as Arp2/3, using their speckle method. Another fellow in Mitchison's lab, Bill Breiher, is using biochemical methods to purify the proteins that, with actin, actually make listeria move. Though work here has been slower than in the imaging arena, Mitchison hopes to get to a point where he can link his two approaches. For example, using small protein inhibitors, which Kirschner and he are developing, Mitchison intends to link the biochemistry of movement proteins with actual observations of movement. "Our aim is to film under a microscope as you knock out proteins on a scale of seconds," he said. "Once you know the pure components, you can really start to go to town on the system." --Misia Landau
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