Stain a neuron for actin and you see a colored tangle of threads. As an essential component of the neuron’s skeleton, actin shows up as filaments in the main cell body and a crowded knot at the tips of axons: the long, skinny projections that transmit electrical impulses to other neurons. But in the axon shaft itself, it seems as if there’s little, diffuse actin to see. “Your eye goes to the exciting, brighter things in the cell,” says Subhojit Roy, a neuronal cell biologist at the University of Wisconsin–Madison. “Why would you look at something that’s just faint, and dim, and you can barely see it?”

In nerve cells such as this dorsal root ganglion neuron, biologists studying the cytoskeleton easily see actin (red) in the cell body and axon tips, and microtubules (green) throughout the axons. But recently scientists have found that actin also has important roles in the shaft of the axons. Image courtesy of Cell Image Library/Kate Nobes and Mark Shipman.

As it turns out, there’s good reason to take notice. A suite of recent studies suggests that actin in the shaft of the neuron could play a vital role in neural processes, such as maturation and synapse function (1). One research group reported that evenly placed actin rings encircle the shaft, perhaps shoring up its structure—something like ribs in the chest—to help prevent axons from fragmenting (2). “Hotspots” in the center of the axon shaft, where actin fibers sprout and shrink, might help maintain proper actin concentrations, Roy speculates (3). And new insights provide clues as to how actin moves in waves to transport itself and how it collects in patches to allow axon branching (4, 5).

“Just seeing the structure is the very first step,” says Roy. “To be honest with you, we still don’t know what the actin in the axon is doing.” Elsewhere in the neuron, actin is known to be crucial for growth, establishment of proper cell shape, transport of key cellular cargoes, and stabilization of the synapses.

Defects that affect actin and its regulators, particularly at synapses, have already been linked to conditions such as intellectual disability and autism, points out Zhen Yan, a neuroscientist at the State University of New York at Buffalo. “I would be interested to see whether this actin in the shaft is important for the delivery of certain cargoes,” she says. If so, then faulty transport might cause disease. Defects in axonal actin that impact the shape or arrangement of neurons or their ability to transmit information could also interfere with the workings of the brain and spinal cord. Indeed, actin-regulating proteins involved in disease at different locations in the neuron might, Yan suggests, make good drug targets.

Taking Shape To understand how actin makes its way to the axon tips—where it promotes extension in a structure called the growth cone—Roy and colleagues wanted to catch the process in action. They labeled actin filaments and performed live imaging on neurons in a dish. A key trick was to label only the actin polymer, not the soluble subunits, which normally provide bright, diffuse background and obscure the assembled threads. Once they eliminated that subunit background, the researchers saw actin filaments doing something they had not expected. Every three to four microns along the axon Roy and his colleagues observed the hotspots, from which actin filaments contracted and extended. Actin polymers were rapidly assembling and disassembling in a phenomenon the researchers referred to as “actin trails” (3). What good is this frequent cycling? “We haven’t really figured out how this weird mechanism that we are describing can lead to axonal transport [of actin],” admits Roy, although the trails did seem to provide actin to synapses. Roy suspects the hotspots might act as a sort of storage depot: stockpiling actin to provide a steady supply for cellular process that require it, such as the formation of patches or rings. But Erika Holzbaur, a neuronal cell biologist at the University of Pennsylvania in Philadelphia, speculates that the actin trails might help move or remodel organelles. In some cases, actin tails can even propel a small organelle or bacterium, she notes. Roy says one way to test his hypothesis might be to interfere with the molecules that control hotspots and trails, then look for downstream effects on other processes, such as ring formation or axon branching. First, he’ll have to identify those controlling factors. Roy is also exploring the impact of hotspots and trails on actin trafficking by collaborating with researchers to make an in silico model of the axon, with evenly spaced hotspots. Actin rings appear on axons at regular intervals, as shown in this 3D image. Reproduced from ref. 2, with permission from AAAS.

Mysterious Movements Roy’s trails may only be part of the story; a wave of sorts may actually propel actin to axon tips. In 1998, scientists first observed that actin travels along axons, at about three microns per minute, in what they termed “waves” (6). Those waves didn’t garner much attention at the time, says cell biologist and neurobiologist Naoyuki Inagaki at the Nara Institute of Science and Technology in Japan, perhaps because researchers thought they were just a characteristic of cells in a dish and not a real neuron phenomenon. In more recent years, scientists have seen the waves in tissues, too (4, 7). Inagaki’s studies of how the waves travel could, as with Roy’s work, help elucidate how actin reaches the places where it’s needed. Using multiple microscopy techniques, Inagaki’s group found that a wave consists of chain of actin, trundling down the axon by adding subunits in the front, and depolymerizing in the back. This chain attaches itself to the plasma membrane as it crawls, and totes various actin-binding proteins along with it. When the researchers halted the waves, the axon could not grow, indicating they waves are essential for neurons to reach out to other cells (4). Waves are also required to establish the polarity of neurons, with an axon on one side and dendrites on the other, without which they could not transmit signals throughout the brain. “It is a new mechanism of intracellular transport system,” says Inagaki. “This migration supplies actin and actin-related proteins to the growth cone.” And actin at the growth cone helps the axon grow toward other neurons. Inagaki can’t be sure his observations explain all forms of actin transport to growth cones, however. He’s only seen the waves early in neuron development—when the growth cone needs a plentiful actin supply—and not later, when less actin is trafficked to axon tips. The growth cone isn’t the only place actin helps cellular projections grow. In 2006, at Temple University in Philadelphia, Gianluca Gallo noticed what he termed “patches” on the axon shaft (8). Most simply disappeared, but some sprouted a projection called a filopodium, which sometimes transformed into axon branches, allowing one neuron to contact multiple areas of the brain. Now, Gallo’s group is working out the signals and proteins involved in making a patch into a filopodium, and a filopodium into a mature axon branch (9, 10).