Cellular hokey pokey: Excitable actin dynamics at the leading edge [with video]

The extension and retraction of the leading edge of motile cells is important in human development and disease. Researchers from Lehigh University and Tohoku University used fluorescence microscopy and mathematical modeling to show how fluctuations of the leading edge of XTC cells represent an excitable system driven by changes in actin polymerization and depolymerization.

HFSP Program Grant holders Dimitrios Vavylonis and Naoki Watanabe and colleagues
authored on Tue, 05 June 2012

Crawling eukaryotic cells initiate motility by protruding lamellipodia, thin extensions of the cell’s leading edge.  Polymerization of actin monomers into filaments in the lamellipodium generates a meshwork of actin filaments that pushes against the cell membrane, causing protrusion. These actin structures are dynamic and regulated by many other proteins. The control mechanisms in the lamellipodium have been studied extensively. However, because of the complexity of the process and differences among cell types, it has been hard to identify the main mechanisms responsible for protrusion and retraction and quantify them with mathematical models.

Figure: Protrusions and retractions of the leading edge of a cell expressing a fluorescent marker of actin filaments. The contours are drawn at 10 s intervals.

To gain insight into the regulation of actin assembly at the leading edge, Naoki Watanabe used a line of frog cells (XTC cells) that have wide lamellipodia and exhibit a periodic pattern of protrusion and retraction, a quantifiable process suggestive of mathematical models (recently reviewed by Ryan, Watanabe and Vavylonis (2)). Postdoctoral scientist Gillian Ryan fitted active contours to images of the leading edge of cells over time (see figure), which allowed for careful measurement of the cell boundary and of the protrusion and retraction rate. She found that the total amount of actin filaments within 5 µm of the leading edge (measured by the intensity of a fluorescent protein, LifeAct-Cherry), exhibited periodic behavior similar to the leading edge velocity, but the two signals were almost perfectly out of phase. This anti-correlation suggested that leading edge extension is tightly coupled to the total amount of actin filaments near the leading edge. Local wave-like features further demonstrate an activation process of actin polymerization that propagates laterally across the membrane.

Protrusions and retractions can also occur by changes in the rate of retrograde flow, the rate with which the actin filament network flows away from the leading edge. But careful measurement of retrograde flow rates via single molecule fluorescence speckle microscopy, a method extensively developed by the Watanabe group, showed little variation in retrograde flow.

Having identified a cell system where protrusion and retraction are driven by fluctuations in actin polymerization, Ryan and Vavylonis were then able to develop a mathematical model, with the help of undergraduate student Heather Petroccia.  They modeled F-actin dynamics near the leading edge of XTC cells as an excitable system. Such a system typically requires an interaction between an activator and an inhibitor: in an excitation, an activator species self-recruits rapidly; this activator in turn recruits an inhibitor that causes the activator to slowly dissipate. The model assumes a diffusive activator that rapidly generates new actin filament ends near the leading edge in an autocatalytic fashion. Actin filaments polymerize at a rate proportional to the concentration of uncapped ends, and this generates an increase in the amount of actin filaments. The growth of the actin network acts as a delayed inhibitor in the model: the maturation of the actin cytoskeleton restricts the recruitment of the activator, causing the amount of actin filaments to decrease. A new cycle of actin polymerization and eventual decay is triggered by random concentration fluctuations. Computer simulations of this model led to patterns of actin concentration dynamics along the leading edge similar to those from experiment.

A possible candidate for the autocatalytic activation mechanism is the branching of actin filaments by theArp2/3 protein complex. The Arp2/3 complex attaches to the sides of existing filaments and creates new branches off the mother filament, increasing the number of polymerizing barbed ends. The authors examined cells with fluorescent markers for both actin filaments and the Arp2/3 complex and found that Arp2/3 complex accumulates in bursts along the leading edge. These bursts precede maxima of F-actin concentration, similar to the activator dynamics in the model. Thus the Arp2/3 complex may have a direct role in the proposed activation mechanism, which also includes additional diffusive components.

The findings of this study motivate further work to examine the mechanistic details of the basic components of the model such as the autocatalytic activation mechanism for actin assembly and the negative feedback from actin to its activators.

 

 

References

1. Excitable Actin Dynamics in Lamellipodial Protrusion and Retraction. Ryan, G. L., H. M. Petroccia, N. Watanabe, and D. Vavylonis. 2012. Biophys. J. 102:1493-1502.

2. A review of models of fluctuating protrusion and retraction patterns at the leading edge of motile cells.Ryan, G. L., N. Watanabe, and D. Vavylonis. 2012. Cytoskeleton  69:195-206.

Pubmed link 1

Pubmed link 2