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FEATURES: Cells on the Move
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In a 2011 follow-up PLoS Biology study, Theriot quantified every move a keratocyte makes on “sticky” versus smoother surfaces—how fast actin filaments form and dissolve, how much traction the cell gets, how its shape changes—so she could calculate cell speed in various microenvironments. “Cells of the immune system may travel through the bloodstream, inflammatory environments, or layers of epithelial cells where things could get stickier,” she says. Knowing how to calculate speed through different tissues could come in handy when devising ways to speed up cells or stop them in their tracks.
March of the Growth Cones
Orkun Akin started his career playing soccer with the actin cytoskeleton. As a UCSF graduate student working with R. Dyche Mullins, Akin used a cell-free system to analyze motility by tweaking concentrations of actin, Arp 2/3 complex, and other actin-binding proteins. Without the boundaries of a cell, he measured “motility” based on how well the “motility mixture” kicked around a polystyrene bead in a dish, similar to propulsion of Listeria. His observations, published in Cell in 2008, suggest another mechanism of actin branching.
“It was a big surprise that we could look through a fly pupa and see R8 cells developing in synchrony. When I saw that, I knew we had something unique.”
Now a postdoctoral fellow in the lab of HHMI investigator Larry Zipursky at the University of California, Los Angeles (UCLA), Akin is imaging the cytoskeletal machines guiding nerve cell axons, to form synapses—neural connections—between R8 photoreceptors in the Drosophila eye and the fly’s brain. During synapse formation those machines, called growth cones, creep forward, seeking the right target.
A slice through the optic lobe of the fruit fly brain reveals a ring of R8 photoreceptor growth cones (white) parked on top of the dome-shaped medulla neuropil―the area of the medulla where synaptic connections are made. A few hours after this image was acquired, these growth cones reached down into the medulla―beyond the plane of the image―and past the projections of the Dm3 class of neurons, shown in red. Image: Orkun Akin and Larry Zipursky
Zipursky’s group and others are making headway in understanding the cell surface receptors, signaling molecules, and cytoskeletal proteins that regulate growth cone movement in the fruit fly. Much of the work on the developing visual system has drawn on the power of the fly model, which allows scientists to genetically manipulate specific neuronal cell types. A major limitation to linking gene function to growth cone motility, however, has been the lack of a robust system for visualizing growth cone movement in live animals.
To address this challenge, Akin teamed with UCLA neurobiologist Joshua Trachtenberg to build a two-photon microscope to follow development of R8 growth cones in flies. The work led to the creation of a remarkable video of R8 growth cones forming connections in the intact animal. First, some 750 amorphous R8 photoreceptor growth cones glowing green with actin filaments hover like a fleet of Close Encounters spacecraft over the optic lobe landing pad. Some hours later, each R8 growth cone extends a spiky, fluorescent finger-like extension, a filopodium, into the region of the optic lobe where it will make synapses. This action is followed by extension of the rest of the axon to the same region.
This time-lapse image sequence shows a row of R8 growth cones (white) extending projections into the medulla region of the optic lobe (red) where they will make synapses. The movie starts approximately 46 hours into pupal development and covers the following 14 hours. Video: Orkun Akin and Larry Zipursky
“It was a big surprise that we could look through a fly pupa and see R8 cells developing in synchrony,” says Zipursky. “When I saw that, I knew we had something unique.”
Akin will now determine how that choreography is disrupted in flies with mutations in various signaling proteins. “Growth cones go from a stalled morphology abruptly to a moving state and then stop again,” says Akin. “We know the signal that activates the movement and its receptor, and now we want to know what role actin dynamics plays in this transition and how signaling factors regulate that process.”
Clinical applications of the photoreceptor work are far off, but Zipursky sees obvious relevance to stem cell–based replacement therapies aimed at regeneration. “Knowing how to wire neurons up properly will require knowing what’s going on biochemically inside a growth cone,” he says.
And the success of the multiple sclerosis drug fingolimod, which alters cell motility, suggests that this goal is not unrealistic. The factors that drive the cytoskeletal engine are not, in fact, undruggable. Engine components themselves, like coronins and fascins, could be next on the list.
Akin, buoyed by the youthful optimism that drove him to build a microscope, agrees. “The more we focus on specific cell types, the more insight we may gain about whether cells in any disease model have a cytoskeletal Achilles heel.”