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The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division.1,2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability."3-7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho.7,8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation.9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics.10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo.7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.
Figure 1. An artificial cortex supports the self-organization of excitable waves of active Rho and F-actin
(A) Schematic of cell-free reconstitution of cortical dynamics. Interphase Xenopus egg extract was added to a supported lipid bilayer atop a glass coverslip and imaged using TIRF microscopy. Active Rho (cyan) and F-actin (magenta) associated with the bilayer were visualized using recombinant GFP-tagged active Rho probe (rGBD) and Alexa Fluor 647-labeled calponin homology domain of utrophin (UtrCH).
(B) Image of the custom aluminum coverslip holder with wells attached.
(C) Micrographs (30 s difference subtraction) of traveling excitable waves of active Rho (cyan) and F-actin (magenta) that originate from discrete maxima (white arrows). Yellow arrowheads indicate wave fronts that annihilate on collision. Time is in minutes:seconds. Dashed line represents region used to generate kymograph.
(D) Kymograph of excitable waves of active Rho (cyan) and F-actin (magenta) generated from a 10-pixel-wide line shown in (C).
(E) Normalized time series of intensities of active Rho and F-actin spatially averaged over a representative 20 × 20-pixel box selected from the field of view (see Figure S1A). Moving time average of three frames.
(F) Temporal cross correlation between active Rho and F-actin intensities. Dashed line indicates the peak time shift of 15 s.
(G) Excitable wave velocity, 17.6 ± 1.6 μm/minute (mean ± SD), was measured over 50–60 s for 151 wave fronts across five experiments. Each dot represents the average velocity for one experiment.
(H) Full width at half maximum, 12.4 ± 2.6 μm (mean ± SD), was measured for 678 wave fronts across five experiments. Each dot represents the average full width at half maximum for one experiment.
See also Figure S1 and Video S1.
Figure 2Active Rho and F-actin coherently oscillate within an artificial cortex.
(A) Reconstituted oscillatory dynamics of active Rho (cyan) and F-actin (magenta). Time is indicated in minutes:seconds. Dashed line represents the region used to generate kymographs.
(B) Kymographs of oscillatory dynamics of active Rho (cyan) and F-actin (magenta) generated from a 10-pixel-wide line shown in (A).
(C) Normalized time series of intensities of active Rho and F-actin spatially averaged over a representative 20 × 20-pixel box selected from the field of view (see Figure S1A). Moving time average of three frames.
(D) Morlet power spectra showing the time-dependent periodicity of the active Rho and F-actin dynamics computed for the same 20 × 20-pixel box as shown in (C). Dashed lines indicate dominant oscillatory periods.
(E) Morlet power spectra of the active Rho and F-actin dynamics averaged over all boxes constituting the whole field of view shown in (A). Dashed lines indicate dominant oscillatory periods.
(F) Temporal cross correlation between active Rho and F-actin fluorescence signals. Dashed line indicates the time shift of 25 s between the two oscillations.
See also Figure S2 and Video S2.
Figure 3. Supported lipid bilayer fluidity enables excitable waves.
(A) Schematic of SLB transfer experiment. Interphase egg extract containing probes for active Rho and F-actin was added to a well with a fresh SLB, and an excitable wave was imaged to completion. The same extract was then transferred to a new SLB to determine if it could produce a second excitable wave.
(B) Interphase egg extract can produce a second excitable wave after transfer to a new SLB. Active Rho (cyan) and F-actin (magenta). Time is indicated in minutes:seconds. Dashed lines represent the regions used to generate kymographs.
(C) Kymographs of excitable waves of active Rho (cyan) and F-actin (magenta) generated in the same extract on two different SLBs.
(D) Kymographs of active Rho, F-actin, and phosphatidylcholine (PC) co-imaged after addition to a SLB.
(E) Plot of the % fluorescence recovery after photobleaching (FRAP) of Cy5-PC in the SLB before and 30 min after extract addition (mean ± S.E.M., before extract: n = 6 bleach regions from two independent experiments, after extract: n = 9 bleach regions from three independent experiments). Before extract, mobile fraction = 76.85 ± 0.82%, t1/2 = 33.21 ± 2.08 s (mean ± SD). After extract addition, the mobile fraction and t1/2 could not be calculated due to slow recovery.
See also Video S3.
Figure 4. Oscillations in the reconstituted cortex require Rho activity and F-actin polymerization.
(A) Active Rho (cyan) and F-actin (magenta) dynamics in a control cortex (top) or following treatment with the Rho inhibitor C3 transferase (33 ug/mL or 100 ug/mL). Time is in minutes:seconds.
(B) Quantification of mean fluorescence intensity of active Rho (cyan) and F-actin (magenta) averaged over the whole field of view in control and C3 transferase-treated extract.
(C) Active Rho and F-actin dynamics before and after addition of DMSO (vehicle control) or 15 μM Latrunculin B. Time is in minutes:seconds. Yellow bars represent the time of DMSO/Latrunculin B addition (30 min). Dashed lines represent regions used to generate kymographs.
(D) Kymographs of active Rho (cyan) and F-actin (magenta) generated from a 10-pixel-wide line, as indicated in (C). Yellow arrowheads represent the time of DMSO or 15 μM Latrunculin B addition.
(E) Mean whole field fluorescence intensity of Active Rho (cyan) and F-actin (magenta) over time in extract treated with DMSO or 15 μM Latrunculin B. Yellow arrowheads indicate time of DMSO or 15 μM Latrunculin B addition.
See also Figures S3 and S4 and Video S4.
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