Gone in 80 Seconds

By Eric Sauter

In the words of Gaudenz Danuser, an associate professor in the Scripps Research Institute's Department of Cell Biology, cell movement is a multi-parameter universe, a vast series of complex molecular processes and forces, some known, some not, all managed in perfect balance to keep the cell moving inexorably forward.

The process starts with the protrusion of the cell edge, the bulging out of the plasma membrane that is critical to cell migration, not to mention the pathological first act of metastatic cancer. Cell protrusion itself is made possible by growing actin filaments (F-actin) that extend the front of the cell through several complicated interactions, working in harmony with various adhesion complexes that bind the actin filaments to the extracellular matrix, the structural web that provides support to all cells. Actin, a wildly ubiquitous and abundant protein, combines with other binding proteins to create a number of different cell structures.

But the process of cell migration doesn't stop there. Effective cellular movement also depends on retrograde or contraction forces that pull the edge of the cell back to center so that the process can begin again.

Understanding this orchestrated complex of signaling pathways, intracellular forces, and feedback mechanisms that help make it all possible has been problematic, primarily because the methods used to discern its various components have been so clunky. Problematic, that is, until the publication of Danuser's latest study, "Fluctuations of intracellular forces during cell protrusion" in the December issue of the journal Nature Cell Biology (Volume 10; number 12).

"The key impact of our study for the field of cell biology and for the science of cell migration," Danuser said, "is that for the first time we can begin to understand force transmission together with the modulation of actin filament assembly and disassembly in a fast process like cell protrusion across an entire cell front. Until now, only the portion of cellular forces that is coupled to the extracellular substrate have been measured, and those at much lower resolution or even only in a single point on the cell. With our approach, you can finally see how the forces play out across the whole cell edge and, more importantly, how they change as other parameters of the protrusion machinery change."

"Cell migration is key to normal development, the maintenance of tissues, and to processes such as wound healing and cancer metastasis," said James Deatherage, who oversees cell migration grants at the National Institutes of Health's National Institute of General Medical Sciences, which funded the work. "This combination of state-of-the-art microscopy, image analysis, and mechanical modeling is an important new approach to analyzing the forces and actin flows within cells that enable them to move."

Establishing a New Sequence

Danuser and his Scripps Research colleagues, Lin Ji and James Lim, devised a mechanical model that relates variations in F-actin flow to variations in intracellular force levels, and tracked cells' progress all with fluorescent speckle microscopy, a technology that marks macromolecular assemblies with fluorescent clusters called speckles. The technology, which was developed by cell biologist Clare Waterman-Storer, formerly of Scripps Research, has become critical to the advanced understanding of cell structures and cell processes in general.

Danuser and his colleagues have developed their own software for the quantitative analysis of cytoskeleton dynamics from data acquired by the technology as well as statistical models to extract this information from large sets of speckles tracked over their entire lifetime.

"In the field of cell migration, the generally accepted idea is that this movement is totally mediated by actin-filaments, so the textbook model is fairly linear—the more F-actin you assemble, the more the cell will protrude," said Danuser. "But that's not what our study showed."

The study established a new and surprising fast-paced sequence of events during cell protrusion, where the remarkably short interval between feedback activation of F-actin at low plasma membrane tension to feedback inhibition of it under membrane high tension sets the timescale of the entire cell protrusion cycle.

Contrary to the currently accepted model where protrusion rates are directly related to actin assembly rates, relatively low assembly rates at the onset of the protrusion cycle are enough to rapidly push forward the plasma-membrane. The rate of F-actin polymerization increases as the plasma membrane expands, probably mediated by tension-feedback. Twenty seconds after peak protrusion, tension reaches a threshold level beyond which the efficiencies of feedback and/or F-actin assembly begin to decay. During this same period, boundary and adhesion forces begin to rise, indicating a tension increase in the expanding plasma-membrane; boundary and adhesion forces reach their maximum 20 seconds later. Forty seconds after peak protrusion, the resistance of the plasma membrane is too high to allow further advancement.

"It had been thought that you have signals that increase filament assembly and increase protrusion," Danuser said. "But now we see that you need even more signals that lead to this reinforcement or anchoring process, which is the key finding of this study. You assemble and protrude fast and the membrane pushes forward. But to reinforce the protrusion gains, you have to increase the rate of assembly. Apparently, protrusion is limited by plasma membrane tension. We don't show that directly, but it's pretty clear that this must be case. So now we need to think about these processes in a much more physical way and begin to better understand the mechanics of membranes."

The entire time span of this intensely complicated corps de ballet movement is around 80 seconds, Danuser said, a very fast process.

Putting the Pieces Together

The other potential value of Danuser's new study may lie somewhere in the future, as Danuser and his colleagues take one more leap into the multiparameter cellular universe, searching for the chemical signals that trigger the process.

Because even with the new study, the molecular details of feedback between membrane tension and F-actin assembly still remain unknown, although the findings suggest several possible candidates, including curvature-dependent activation and/or scaffolding of signaling molecules within the plasma membrane.

Unraveling these connections will depend on in situ measurements of the timing between forces, feedback signals and cytoskeleton dynamics, Danuser said. But the force reconstruction presented in the new study already provides an unprecedented source of high-resolution data to achieve this goal.

One final interesting note about the new study is that Danuser first noticed this odd stutter in F-actin assembly and cell protrusion several years ago but really didn't think much of it.

"We observed this assembly delay about three years ago but we were mostly just puzzled by it," he said. "We really didn't think about the physics of it very much. But now that we've done that and mapped out all the mechanics around it, it's turned into a kind of classic example of how the pieces of the science puzzle come together."

For more information on the study, see http://www.nature.com/ncb/journal/vaop/ncurrent/abs/ncb1797.html.

Send comments to: mikaono[at]scripps.edu

"With our approach, you can finally see how the forces play out across the whole cell edge and, more importantly, how they change as other parameters of the protrusion machinery change," says Associate Professor Gaudenz Danuser.