LSE Logo MBoC Logo

Formins specify membrane patterns generated by propagating actin waves

    Published Online:https://doi.org/10.1091/mbc.E19-08-0460

    Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territoriy and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape, using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave, ForG the inner territory, whereas ForA, ForE and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains prior to differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

    Video S1: Dynamics of the network of actin bundles in the external area of the wave pattern. This video shows the full image sequence from which the frames in Figure 1C were taken. In the TIRF images of the large cell, GFP-filamin-decorated network structures are seen in the external area. The 23-s frame in the video corresponds to the 0-s frame in Figure 1C. The images have been processed through a super-resolution radial fluctuations (SRRF) algorithm. Frame-to-frame interval is 1 s. Bar, 10 μm.
    Video S2: A large cell expressing YFP-ForG-N (green) together with mRFP-LimEΔ as an actin label (red). This video displays the same labels as Figure 3, but represents another recording. The cell shows expansion, fusion, retraction, and splitting of actin waves. ForG is localized in the form of micropatches to the inner territory surrounded by a wave. In addition, the ForG is enriched at the dynamic fronts of the actin waves. Frame-to-frame interval is 2 s. Bar, 10 μm.
    Video S3: Fusion and mutual extinction of colliding actin waves. This video shows another example than Figure 3B. The large cell expressed mRFP-LimEΔ for F-actin (red) and YFP-ForG-N (green). In the images on the left, the position of the line scans of fluorescence intensities is indicated. These scans are shown on the right. When the waves collide, the low ForG decoration of the external area turns into the high decoration of the inner territory. Frame-to-frame interval is 2 s. Bar, 10 μm.
    Video S4: Continued propagation of an actin wave upon collision with another one. This video shows another example than Figure 3C. The large cell expressed YFP-ForG-N (green) and mRFPLimEΔ as an actin label (red). In the images on the left, the position of the line scans of fluorescence intensities is indicated. These scans are shown on the right. Propagation of the actin wave is followed by occupancy of the expanding inner territory with ForG. Frame-to-frame interval is 2 s. Bar, 10 μm.
    Video S5: A wave-forming cell that expressed GFP-ForAΔDAD (green) and mRFP-LimEΔ for Factin. This video shows the same recording as Figure 4A. At the beginning, the membrane is densely decorated with highly dynamic micro-patches of ForA. Subsequently, an actin wave is initiated. During expansion of this wave, the ForA decoration decreases. Finally, the wave retracts and ForA re-enters the membrane area from the right cell border. Frame-to-frame interval is 1 s. The 41-s frame in the video corresponds to the 0-s frame in Figure 4A. Bar, 10 μm.
    Video S6: ForB fluctuations preceding the insertion of trailing waves. This video shows the same recording as Figure 6. The large cell expressed GFP-ForBΔDAD (green) and mRFP-LimEΔ as an actin label (red). Frame-to-frame interval is 2 s. The 0-s frame in the video corresponds to the 0-s frame in Figure 6. Bar, 10 μm.
    Video S7: Illustration of the fluctuations of ForB along a line scan. This video shows in full-length the recording from which Figure 8A displays a sub-sequence corresponding to the 444-s to 599-s frames in the video. The large cell expressed GFP-ForBΔDAD (green) and mRFP-LimEΔ as an actin label (red). Fluorescence intensities were scanned along the line demarcated in the left panel and displayed in the kymograph of the right panel. Frame-to-frame interval is 1 s. Bar, 10 μm.
    Video S8: Gain and loss of ForB fluorescence intensities in consecutive frames of a cell forming a U-shaped trailing wave. The large cell expressed GFP-ForBΔDAD. Left panel: Fluorescence intensities in the first of two consecutive frames are shown in green and in the second frame in red. Persisting fluorescence intensities appear in yellow. In this way, the incipient trailing wave is clearly recognizable. Right panel: The fluorescence intensities in the first of two consecutive frames are subtracted from that in the second frame, and positive values (gain) are displayed in yellow, negative values (loss) in cyan. Frame-to-frame interval is 2 s. Bar, 10 μm.
    Video S9: Inhibition of wave formation by 1 μM SMIFH2. This video shows the same recording as Figure 9A. A cell expressing GFP-PHcrac as a label for PIP3 and mRFP-LimEΔ for filamentous actin was exposed to the formin inhibitor in a flow chamber. The fluorescence in the extracellular space of the left panel marks arrival and removal of the inhibitor. Recovery of wave formation after removal shows reversibility of the inhibition. In the presence of the inhibitor, the cell continues to form small actin patches, which in Figure 9B are shown to be associated with clathrincoated pits, and to form large actin assemblies while the synthesis of PIP3 is inhibited. Frame-toframe interval is 1 s. Bar, 10 μm.
    Video S10: Shape changes of a cell in 10 μM SMIFH2. This video shows the same recording as Figure 9C. The cell expressed mRFP-LimEΔ for F-actin and GFP-dajumin for membranes of the contractile vacuole system. The inhibitor causes the cell to bleb and to form long extensions that contain actin in their cortex. Vacuoles are still contracting, indicating that the cell is alive and the osmo-regulatory system working. Frame-to-frame interval is 2.08 s. Bar, 10 μm.