James Bear studies wound healing to learn how external signals are coordinated with cytoskeletal rearrangement in migrating fibroblasts. Julie Theriot focuses on fish scale keratocytes—highly motile cells that rapidly repair skin lesions.

The primary constituent of the scaffold is the protein actin—a molecule that never sits still. As the cytoskeleton extends, cells spin single actin molecules into long chains, or polymers, aided by a stew of molecular bundlers, cross-linkers, and branchers. The actin scaffold supports whatever protrusion a cell needs to crawl or pry its way through tissue. Disrupt the balance of construction and demolition and the cellular healers are going nowhere. Neither are metastasizing cells.

Given its importance, the cytoskeleton seems an obvious target for drug discovery. But the very ubiquity of cytoskeletal proteins has raised doubts about whether actin or any of its handlers could serve as pharmaceutical targets. “One prejudice has been that because cytoskeletal proteins are inside cells and abundant, they are undruggable,” says Joan Massagué, cancer researcher and HHMI investigator at Memorial Sloan-Kettering Cancer Center.

But recent findings by HHMI scientists and others reveal that the cytoskeletal architecture differs significantly from cell to cell. “Immune cells and neurons put their Legos together in completely different patterns,” says HHMI investigator Julie Theriot, who studies cell motility at Stanford University. If cells display specialized cytoskeletal structures, researchers have options for speeding the rescuers or blocking the invaders in a targeted way. In other words, the “undruggable” dogma is crumbling.

Moving Forward

Topping the “Greatest Hits” page of Theriot’s lab website is a video of disease-causing Listeria monocytogenes whirling around inside canine kidney cells. The cells are engineered to express fluorescent actin, and the bacteria inside them appear to stream a glowing “comet tail.” But the tail actually represents dissolving actin filaments constructed by the host cell, whose cytoskeleton has been coopted by the bacteria to propel themselves through infected tissue.

“The comet tail video shows that the cytoskeleton is a powerful machine constantly running, poised to push things around,” says Theriot. “All a bacterium needed was to figure how to tap into it.” Her group discovered that a single surface protein expressed by Listeria was sufficient for “tapping into” the dynamic actin cytoskeleton and could generate comet tails when inserted into unrelated bacteria, or even plastic beads. The bacterial protein works by latching onto a host cell actin-binding protein complex called Arp 2/3, an actin “brancher.” Once that happens, the Arp 2/3 complex stimulates growth of a new actin filament from the side of an existing filament, generating a branched structure that first pushes Listeria forward and then disintegrates in its wake.

Rather than conspire with bacteria, the primary purpose of the actin engine in a human cell is to move that cell to a specific location where it is needed. HHMI early career scientist James Bear at the University of North Carolina at Chapel Hill is trying to figure out what controls the migration by studying connective tissue cells called fibroblasts.

After an injury, chemical cues emanating from a wound lure reparative fibroblasts in a process called chemotaxis. Cells migrate toward the wound guided by a flat, foot-like structure known as the lamellipodium, from “lamella” (thin sheet) plus “podium” (foot). Lamellipodia constantly probe forward, advancing a cell by means of the persistent cytoskeletal engine as it assembles and dismantles actin branches. Until recently, many investigators believed that lamellipodia might also interpret chemical signals released from wounded tissue. But a study from the Bear lab published March 2, 2012, in Cell shows it’s not that simple.

Bear genetically engineered fibroblasts without lamellipodia by depleting cells of the Arp 2/3 complex, blocking their ability to make highly branched actin. He then exposed the cells to traces of a growth factor “lure” normally secreted from wounds. Even though their primary means of locomotion had been cut out from under them, the cells were able to move toward the growth factor using less efficient protrusions.

“This was a surprise—everybody in the field assumed Arp 2/3 was essential for chemotaxis,” says Bear. On the other hand, the researchers reported, the loss of Arp 2/3 did adversely affect the ability of the cells to sense and respond to the surface they crawled over.

A fibroblast cell that has been depleted of several subunits of the Arp 2/3 complex and microinjected with purified Arp 2/3 complex to rescue the fan-shaped lamellipodia.
Video: Elizabeth Haynes / Bear Lab

Photos: Bear: Paul Fetters; Theriot: George Nikitin / AP, ©HHMI

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