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Arp2/3 complex interactions and actin network turnover in lamellipodia

Cell migration is initiated by lamellipodia—membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin—another prominent Arp2/3 complex regulator—and ADF/cofilin—previously implicated in driving both filament nucleation and disassembly—were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.


Data analysis and presentation
FRAP data were analysed essentially as described (Rabut and Ellenberg, 2005). Best linear fits were calculated using SigmaPlot 10.0 (Scientific Solutions SA, Pully-Lausanne, Switzerland). For treadmilling analysis, the lamellipodium was divided into front and back halves and the respective intensities measured over time (as indicated in Figure 1F). Background fluorescence intensities taken from a region outside the cell (see Supplementary Information, Movie 5) were subtracted from each individual region and frame. Acquisition photobleaching was subtracted as recommended (Rabut and Ellenberg, 2005) and determined using a stable cellular region (usually cytosolic) largely unaffected by experimental bleaching. The average fluorescence intensity as obtained from the lamellipodial regions flanking both sides of the bleached area upon fluorescence recovery was defined as maximum and normalized to 1. For components residing at lamellipodial tips, half time of fluorescence recovery (t 1/2 ) was determined only, whereas proteins localizing to the entire lamellipodium were in addition subjected to treadmilling analysis. Best linear fits for tip components, front-and rear-half as well as entirelamellipodium (not shown) recovery curves corresponded to the following equation: y = a(1-exp(-bx)), except for data obtained from rear-half recovery measurements of actin and Arp2/3-complex, which best fitted to sigmoid curves corresponding to equation: y = y 0 + a/(1 + exp(-(x-x 0 )/b)). t 1/2 values were calculated by solving the corresponding equations with a fluorescence intensity of 50% of the maximum recovery value.
For determining treadmilling factors (TMF), normalized intensities of front-and rear-half lamellipodial regions were plotted over time. The TMF for a given component, expressed in arbitrary units (a.u.), was defined as the average difference in fluorescence recovery between front and back half of the lamellipodium (per second recovery time), and thus calculated as follows: Area under intensity curve for the rear (∫B) was subtracted from that for the front (∫F), and divided by the time (in seconds) required for 100% recovery ( Figure 1F).

Simulation of actin recovery in the lamellipodium
Modelling is exemplified in detail for the model shown in Figure 1D Before simulation of bleaching, the model was run for about 2 min to allow it to settle down to a steady state. In the steady state, the region of high density F-actin extended 3-4µm back from the leading edge, which corresponds well with average lamellipodial dimensions of real cells. Importantly, the transition probability functions of Figure 1E were chosen by trial and error to obtain the best match. Bleaching was simulated by removing all actin molecules from the model in a square region of approximately 14 × 14 µm with one edge situated centrally on the leading edge. The model was then run for a further minute of real time to observe the redistribution of labelled actin after bleaching.
To keep the model as simple and robust as possible, no attempts were made to simulate the forward protrusion of the leading edge or the F-actin density gradient in the lamellipodium.

For obtaining models with variable lamellipodium widths used for the analyses shown in
Supplementary Figure 2b, the same parameters were used except for that the depolymerisation probability profiles (dpp) were changed as shown in Supplementary   Figure 2a. This generated models with a range of widths, which were subsequently bleached as described above. For treadmilling analyses, the measured areas in all models were selected to reflect lamellipodia with equal fluorescence intensity gradients, with an average intensity decay of 80% for the back half as compared to the front half of the lamellipodium. As a result, the lamellipodial widths analysed corresponded to 5µm, 3.5µm, 2.1µm, 1.6µm and 0.7µm for models dpp1 -dpp5, respectively. Treadmilling factors obtained were plotted versus lamellipodium width, and curved-fitted Plotted are averaged data (means and SEMs of means) and best linear fits of 5 independent movies. t 1/2 given was calculated for entire lamellipodium. Although half time of fluorescence recovery was accelerated as compared to construct 1 ( Figure 3A), the TMFs obtained for both constructs were virtually identical, corroborating the conclusion of homogenous recovery of cortactin throughout the lamellipodium. (C) Representative FRAP experiment of B16-F1 cell expressing another EGFP-cortactin construct (construct 3, (Zhu et al., 2007). Note again homogenous recovery throughout the lamellipodium. Time is in seconds; bar = 5µm.