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CELL BIOLOGY What's Behind Cellular Morphing? Research on Cytoskeleton and Signal Transduction Join to Reveal How Cells Move Cell biology does not usually remind one of stargazing. But as Le Ma, in a darkened room, mixes two pinhead-size drops of clear liquid onto a coverslip, a view quite like the night sky opens up under the microscope, complete with stars and comets. Comets? Yes, luminous tails appear against a black background dotted with bright points, and successive pictures taken over the course of some 10 minutes actually show those tails shooting along in curved trajectories (see image). Image by Le Ma The "comet tails" shown in this series of video images are not celestial bodies. About one-thousandth of an inch long, they are made of actin, a protein component of every cell's inner skeleton. In this test tube experiment, the comets move out of, or into, the visual field as millions of actin proteins get assembled in a series of biochemical reactions. These reactions are started by the tiny sphere made of cell membranes that sits atop each comet tail. The growing tail propels the vesicle along like a rocket engine. The images are stills from a video, taken at two-minute intervals.     This observation—enjoyed in various forms by biologists for seven years—exemplifies how seemingly bizarre findings in one area of research can literally propel another one forward.     Microbiologists curious about how food-poisoning bacteria crisscross infected host cells sparked a line of intense study that is now producing a molecular understanding of a fundamental question in cell biology, namely, how cells move in response to changes occurring outside their membrane. Shaping the Cell Scientists have long been fascinated by the question of what controls a cell's inner skeleton. This medley of individual proteins can polymerize into weight-bearing threads at one moment and dissolve into a soft gel the next. One such protein, actin, forms a filament network running just underneath the cell's outer membrane. The actin cytoskeleton is like a scaffold that is constantly being built and taken apart in ways tightly controlled in space and time, depending on whether the cell changes its shape, moves, or divides.     Scientists in this burgeoning field—the past year alone saw more than 900 published papers mentioning the actin cytoskeleton—have spent the last four years trying to understand the precise molecular channels of communication that link a specific outside stimulus to the assembly of actin filaments. Though they have identified myriad molecular players in these signal transduction pathways and glimpsed partial connections between sets of two or three of them, complete chains of command remained elusive—until now.     Recently four reports have begun to fill this gap, two of them by Harvard scientists. In the April 16 Cell, Marc Kirschner, the Carl W. Walter professor of cell biology, graduate student Le Ma, and others describe how they staged in the test tube a series of signaling events that lead from specialized membrane lipids all the way to polymerization of actin filaments. Rong Li, HMS assistant professor of cell biology, and her colleagues report in the April 26 Current Biology a similar link in live yeast cells.     Across Harvard, many scientists study this question in cell types ranging from the epithelial cells involved in breast cancer, to blood and immune cells (see sidebar, p.7), to neurons (Focus, March 5, 1999). The breadth of the field is unsurprising, Kirschner says. "We could spend the next 20 minutes listing biological problems that occur through regulated changes in the actin cytoskeleton, from development to angiogenesis and metastasis, to phagocytosis and inflammation."     The problem is also a knotty one. Central dogmas in biology—the DNA, RNA, and protein sequences—are essentially linear. Not encoded in the DNA, however, is the assembly of proteins into intricate 3-D architectures, which differ among hundreds of cell types and organisms, and change with time. "This is one of the great, least understood problems in biology," Kirschner says. Rocket Science Kirschner's current research grew out of work by others, including Tim Mitchison, HMS professor of cell biology, who was then at the University of California, San Francisco. Studying how pathogenic bacteria called Listeria monocytogenes managed to travel through cells—an unusual feat for bacteria, which generally live in the spaces between, not inside, host cells—the scientists found in 1992 that listeria propelled itself sitting atop a tail of actin filaments. Stationary themselves, the filaments continuously grew just underneath the bug.     Mitchison suspected that the pathogen might hijack a normal, cellular mechanism for making actin filaments. He found that listeria's propulsive mechanism also worked in extracts of frog eggs, an experimental system much simpler to study than infected cells.     This work was most significant, Kirschner says, for providing a way to study the initial steps of the de novo assembly of filaments from individual actin proteins, a mysterious process called actin nucleation. Previous work in the field had focused on ways of manipulating existing filaments, such as severing or bundling them, mostly because the nucleation of a new filament is a fleeting event hard to capture in an experiment.     In 1997, the Mitchison lab described the function of what has become a key player in all signaling chains controlling actin polymerization. Called the Arp2/3 complex, this set of seven proteins is thought somehow to enable three individual actin proteins to overcome energetic barriers and form a germinal filament, onto which additional actin proteins can settle easily.     The key question in the field then became: how does this complex connect back up to the membrane? This is where the current set of papers comes in.   Stephen Moskowitz, Advanced Medical Graphics This diagram depicts how a signal arriving at the outside of a cell (far left) gets passed on inside through a series of protein­protein interactions, which ultimately translate the original signal into changes in the cell's inner skeleton (far right). If staged in vitro, this series gives rise to moving "comet tails" that form as individual actin proteins (red) assemble into cross-linked threads. In a cell, these actin threads push into the membrane, causing it to extend the tiny spikes that typically appear as the cell stirs in response to the signal. See story for molecular details. Mapping Signals As often happens in signal transduction research, Kirschner's study ran into the Arp2/3 complex by coming from the opposite direction. He and Ma began by working with Lewis Cantley, HMS professor of cell biology at Beth Israel Deaconess Medical Center. Over the past 15 years, Cantley and others had found that certain lipids in the cell's outer membrane were not simply structural components of that membrane but helped convey signals to the cytoskeleton, among other places.     On this project, the researchers initially dropped bits of real cell membrane into frog egg extracts. Presto! Comet tails of actin appeared behind the vesicles, telling the researchers that some component of the membrane started the process. To find out which one, the scientists embedded into artificial lipid spheres PIP2, a twice-phosphorylated lipid known to somehow modulate the cytoskeleton. Again, comet tails corkscrewed these spheres about under the microscope, the scientists reported last year.     Kirschner, Ma, and others went on to isolate biochemically the components that lay between PIP2 and the actin tails. With them, they then reconstructed the chain of events in a completely purified system. These other players are a protein called cdc42, and N-WASP, a variant of WASP, the protein mutated in an immune disorder marked by cytoskeletal defects (see sidebar). Both had already been implicated in actin remodeling, but never in the context of a complete pathway. (For example, Tomas Kirchhausen, HMS associate professor of cell biology at the Center for Blood Research, and others at Harvard reported in 1996 that cdc42 and WASP interact.)     How can one transfer Kirschner's in vitro data back into a real live context? Consider this simplified example: The ligand platelet-derived growth factor, emanating from a blood clot plugging a wound, docks onto the receptor on a nearby fibroblast, telling it to repair the damage.     In response, several enzymes tack phosphate groups onto the water-loving end of inositide lipids floating in the fibroblast's membrane, creating PIP2 (see diagram, p.5). These phosphate groups fit like plugs into sockets called PH domains. The N-WASP protein has such a domain and, with it, anchors itself to the membrane. Once activated by yet other proteins called exchange factors, cdc42 binds N-WASP, which sticks to the membrane by holding on to cdc42 and PIP2. This allows N-WASP, a protein that is usually clamped shut, to spring open and bind the Arp2/3 complex.     WASP does two things, Kirschner says. Its binding sites for many different proteins make it a hub that integrates several lines of upstream information. But it then also works with the Arp2/3 complex in somehow lining up those first few actin monomers needed to make a filament.     Once that happens, actin filaments grow, align themselves perpendicularly to the membrane, and push into it. In this way, they gradually generate one of the cell's finger-like spikes, a process much like sticking a finger out from inside a balloon, Cantley says. The comet tails generated in vitro by lab-made lipids may simulate that.     And on the hypothetical fibroblast, the spikes represent the first stirrings towards moving into the wound.     Many questions remain, however. The Kirschner paper only presents a pathway that can nucleate actin in vitro. It does not capture the myriad variations that will be found in different cell types in vivo, or even prove that this ever occurs in a living cell.     Rong Li's study is the only one of the current set to have tested this pathway in vivo. Her lab found that the yeast version of WASP indeed activates the Arp2/3 complex to build the actin structures that help yeast grow. But she already has data suggesting that yet other proteins work with Arp2/3, too. "The story does not end there," she says. Stay tuned.   Seen Through Lens of Cytoskeleton, Immune Disorders' Symptoms Start Making Sense Raif Geha has treated patients with Wiskott–Aldrich Syndrome (WAS) for 25 years, but just recently has begun to understand their panoply of problems much more clearly. The Prince Turki Bin Abdul Aziz Al-Saud professor of pediatrics at Children's Hospital, he says that soon after the underlying gene defect was first described five years ago by Stanford scientists, basic cell biological research converged with clinical observations to explain this rare immune system disorder. Symptoms of WAS include bleeding, immune deficiency, eczema, inflammatory bowel disease, and lymphomas. Considered independent at first, they now appear linked through a flaw in controlling a cell's inner skeleton. The affected protein, WASP, turned out to play a central role in coordinating incoming signals into the proper cytoskeletal change. WAS can sometimes be cured by bone marrow transplants, but better treatments are still needed. Researchers at Harvard and elsewhere are studying gene therapy approaches. T cells, monocytes, and mast cells all share aspects of a central pathway of signal transduction (described in the main story), says Geha. Yet cell-specific variations are important. For instance, Geha studies SLP-76, a protein active at the beginning of this pathway. It connects signals coming in through the cell-specific receptor, such as the T cell receptor, and going to an exchange factor called Vav, which activates cdc42. From then on, immune cells use the common pathway. Geha's lab also has discovered a protein that functions at the end of the pathway. Called WIP, it is thought to cooperate with WASP and actin monomers in making filaments and turning them into the little spikes extended by active cells. In WAS patients this pathway is disrupted, suggesting a reason why their platelets cannot stop bleeding quickly and why T cells cannot mount adequate immune responses. The cytoskeleton is one part of the explanation, says Geha; the other lies in the fact that the signals that ultimately reshape the cytoskeleton also branch off to the nucleus, where they control growth. Geha suspects that the cytoskeleton might also play a role in the eczema and inflamed intestine often seen in WAS. T cells' hampered motility might trap those that somehow enter the skin and the intestine, though evidence for this remains scant. The pathway keeps pointing out unexpected connections. In this month's Journal of Clinical Investigation, Geha's group reports that mice lacking SLP-76 cannot release granules from their mast cells. Known to require cytoskeletal changes, this process underlies common seasonal allergies. "When we started work on these proteins, we had no idea they were going to act in the same pathway," recalls Geha. But, he adds, in this bench-to-bedside research, "projects eventually come together, and not always by design." —Gabrielle Strobel Back to Top