Schwarz GJ et al. (2026), Membrane composition-dependent patterning of Rh...

XB-ART-61748

Mol Biol Cell 2026 Feb 01;374:ar30. doi: 10.1091/mbc.E25-07-0362.

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Membrane composition-dependent patterning of Rho and F-actin in an artificial cell cortex.

Schwarz GJ, Suber JR, Andhare D, Ragusa MJ, Landino J.

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Cortical excitability, a phenomenon in which the cell cortex is dynamically patterned with waves of F-actin assembly, has been described in a variety of model systems, including embryos of mammals, flies, frogs, and echinoderms, as well as a variety of cultured cells. While the cortical F-actin network is closely linked with the plasma membrane, it is not known if membrane composition or fluidity regulates dynamic cytokinetic patterning. Phospholipids partition within the plasma membrane during cytokinesis and phosphoinositides play a key regulatory role in other excitable systems, suggesting a role for membrane-dependent regulation of cytokinetic patterning. Here, we use an artificial cell cortex comprised of Xenopus laevis egg extract and supported lipid bilayers (SLBs) to show that membrane composition regulates self-organized cortical patterning. We find that manipulating the levels of candidate lipids, including phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, sphingomyelin, and cholesterol, changes the dynamics of traveling waves and standing oscillations of active Rho and F-actin, as well as the kinetics of Rho activation and F-actin assembly on SLBs. Our findings demonstrate that membrane composition regulates the assembly of cortical F-actin, as well as emergent active Rho and F-actin patterning.

???displayArticle.pubmedLink??? 41705942
???displayArticle.pmcLink??? PMC13008262
???displayArticle.link??? Mol Biol Cell
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Species referenced: Xenopus laevis
GO keywords: cell cortex [+]

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	FIGURE 1: PE SLBs alter active Rho and F-actin dynamics. A) Traveling waves on SLBs with 0% or 20% PE. Arrowheads mark merging wave fronts. Dashed lines mark regions used for kymographs. Yellow dashed boxes mark regions used for intensity traces. Time is shown in minutes:seconds. B) Kymographs of traveling waves. Arrowheads indicate consecutive wave fronts. C) Intensity over time traces of traveling waves. D) Traveling wave velocity, mean ± 95% confidence interval (CI) is shown. Control (0% PE), n = 20, 4, 3, 3, 20% PE, n = 26, 5, 4, 4 for wave fronts, videos, extract preps, experimental days, respectively, **p ≤ 0.001. E) Quantification of active Rho dynamics in traveling waves. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± standard deviation (SD). Control, n = 3083, 4, 3, 3, 20% PE, n = 5288, 5, 5, 4 for boxes, videos, extract preps, experimental days, respectively, p ≤ 0.05. F) Standing oscillations on SLBs with 0% or 20% PE. Time is shown in seconds. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Yellow dashed boxes mark regions used for intensity traces. G) Kymographs of standing oscillations. H) Intensity over time traces of standing oscillations. I) Quantification of the active Rho and F-actin dynamics in standing oscillations. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. Control, n = 7415, 6, 5, 4, 20% PE, n = 9754, 6, 4, 2, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, *p ≤ 0.01.
	Still from Movie S1: Traveling waves and standing oscillations on SLBs containing 0 or 20% PE. Related to Figure 1. Time is shown in minutes:seconds. Acquisition interval is 10 seconds. Playback is 10 frames per second (fps).
	FIGURE 2: Active Rho and F-actin self-organize in the artificial cortex independent of intracellular organelles. A) Schematic of low-speed supernatant (LSS) or membrane-depleted extract (MDE) preparation. 15 min centrifugation to generate LSS, and subsequent 90 and 30 min centrifugation steps to generate MDE. B) Normalized whole-field intensity of active Rho and F-actin over time in LSS (black) and MDE (blue). Mean (solid line) ± SD (shading) is shown. For LSS, n = 7, 4, 3, for MDE, n = 13, 9, 6, for videos, extract preps, experimental days, respectively. C) Standing oscillations in the artificial cortex in LSS or MDE. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Time is indicated in seconds after the start of oscillatory dynamics. D) Kymographs of standing oscillations in LSS or MDE. E) Quantification of oscillatory dynamics in LSS (black) or MDE (blue). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For LSS, n = 6352, 6 videos, 4, 3, for MDE, n = 4753, 6, 4, 3 for boxes, videos, extract preps, experimental days, respectively.
	Still from Movie S2: Standing oscillations in low-speed supernatant (LSS) and membrane-depleted extract (MDE). Related to Figure 2. Time is shown in minutes:seconds. Acquisition interval is 10 seconds for LSS and 5 seconds for MDE. Playback is 10 fps.
	FIGURE 3: PIP2 supports F-actin assembly on SLBs. A) Quantification F-actin assembly PIP2 SLBs after 10 min. SLBs are comprised of 0% PIP2 (60% PC, 30% PS, 10% PI), or 0.1% PIP2 (60% PC, 30% PS, 9.9% PI, 0.1% PIP2) or 1% PIP2 (60% PC, 30% PS, 9% PI, 1% PIP2) or 5% PIP2 (60% PC, 30% PS, 5% PI, 5% PIP2). For 0% n = 12, 11, 6, For 0.1% n = 12, 11, 6, for 1% n = 11, 10, 5, for 5% n = 10, 10, 5 for videos, extract preps, experimental days, respectively. B) Fluorescence intensity of PIP2 biosensor (GFP-C1-PLCδ-PH) over time 0% (left) or 0.1% (right) PIP2 SLBs in the absence (buffer) or presence of MDE. Mean (solid line) ± standard error of the mean (shading) is shown. C) Relative change in fluorescence intensity of a PIP2 biosensor after 20 min of incubation with buffer (−) or MDE (+). Mean ± SD is shown. For buffer, n = 3 experimental replicates on one experimental day, for MDE n = 5 videos, 5 extract preps, 2 experimental days p ≤ 0.05, p ≤ 0.005. D) Normalized active Rho (left) or F-actin (right) intensity over time after treatment with DMSO or inhibitors of PIP2 synthesis (30 µM PI4KIIIɑ inhibitor [GSK-A1] + 20 µM PIP5K1α inhibitor [ISA-2011B]). Extract was incubated with PIP2 synthesis inhibitors for 10 min at room temperature before adding the extract to the SLB. Time = 0 indicates time of extract addition to the SLB. Mean (solid line) ± SD (shading) is shown. For both n = 3 videos, 3 extract preps, 2 experimental days. E) Standing oscillations in MDE on PIP2 SLBs at time = 0 s. Dashed lines mark regions used for kymographs. F) Kymographs of standing oscillations on PIP2 SLBs. G) Oscillatory dynamics in MDE on PIP2 SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PIP2, n = 4554, 4, 4, 3, for 0.1% PIP2, n = 6773, 6 videos, 4, 3, for 1% PIP2, n = 9190, 8, 6, 4, for 5% PIP2, n = 9209, 8, 5, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	Still from Movie S3: Standing oscillations in MDE on SLBs containing 0 or 0.1% PIP2 and on SLBs containing 0, 10, 20, or 30% PE. Related to Figures 3 and 4. Time is shown in minutes:seconds. Acquisition interval is 10 seconds. Playback is 10 fps.
	FIGURE 4: Cortical oscillation amplitude increases in MDE on PE SLBs. A) Standing oscillations in MDE on PE SLBs. SLBs are comprised of 0% PE (60% PC, 30% PS, 10% PI), or 10% PE (50% PC, 10% PE, 30% PS, 10% PI) or 20% PE (40% PC, 20% PE, 30% PS, 10% PI) or 30% PE (30% PC, 30% PE, 30% PS, 10% PI). Time is shown in seconds (s). Dashed lines marks regions used for kymographs. B) Kymographs of standing oscillations in MDE on PE SLBs. C) Quantification of oscillatory dynamics in MDE on PE SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 3872, 4, 3, 3, for 10% PE, n = 5099, 5, 3, 3, for 20% PE, n = 3354, 3, 3, 2, for 30% PE, n = 8222, 4, 4, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Quantification of oscillatory dynamics in MDE on PE SLBs with PIP2. SLBs are comprised of 0% PE (60% PC, 30% PS, 9.9% PI, 0.1% PIP2), or 10% PE (50% PC, 10% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 20% PE (40% PC, 20% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 30% PE (30% PC, 30% PE, 30% PS, 9.9% PI, 0.1% PIP2). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 5338, 4, 3, 3, for 10% PE, n = 4832, 5, 4, 4, for 20% PE, n = 4671, 5, 5, 4, for 30% PE, n = 5552, 5, 4, 4, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 5: Standing oscillations are altered on sphingomyelin and cholesterol SLBs. A) Standing oscillations on control, sphingomyelin, and cholesterol SLBs. White dashed lines indicate regions used for kymographs. Right: kymographs of standing oscillations on control, sphingomyelin, and cholesterol SLBs. B) Quantification of oscillatory dynamics in MDE on control, sphingomyelin, and cholesterol SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For control, n = 5623, 6, 3, 4, for sphingomyelin, n = 6606, 6, 3, 4, for cholesterol, n = 6647, 8, 6, 3 for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. C) Left: active Rho on control, cholesterol and sphingomyelin SLBs and masks used to determine spatial Rho activity. Right: percent area of increasing active Rho at each time point and average active Rho cluster size. Violin plot = all time points, dots = mean of each experiment, bars = total mean ± SD. For control, n = 1000, 6, 3, 4, for cholesterol, n = 991, 8, 6, 3, for sphingomyelin, n = 868, 6, 3, 4 time points, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Kymographs of Rho activation and F-actin association (arrowheads) with control, sphingomyelin, and cholesterol SLBs. E) Representative normalized intensity curve of active Rho and F-actin after extract addition on a control SLB. Dotted lines indicate best fit sigmoidal curve. F) Quantification of the t1/2 and the temporal phase shift between active Rho and F-actin on control, sphingomyelin, and cholesterol SLBs. Mean ± SD is shown. For all, n = 3 videos, 3 extract preps, 2 experimental days. *p ≤ 0.05.
	Still from Movie S4: Standing oscillations and cortical self-assembly in MDE on SLBs containing cholesterol or sphingomyelin. Related to Figure 5. Time is shown in seconds. For oscillations, acquisition interval is 10 seconds. For cortical assembly, acquisition interval is 2 seconds. Playback is 10 fps.
	FIGURE 1:. PE SLBs alter active Rho and F-actin dynamics. A) Traveling waves on SLBs with 0% or 20% PE. Arrowheads mark merging wave fronts. Dashed lines mark regions used for kymographs. Yellow dashed boxes mark regions used for intensity traces. Time is shown in minutes:seconds. B) Kymographs of traveling waves. Arrowheads indicate consecutive wave fronts. C) Intensity over time traces of traveling waves. D) Traveling wave velocity, mean ± 95% confidence interval (CI) is shown. Control (0% PE), n = 20, 4, 3, 3, 20% PE, n = 26, 5, 4, 4 for wave fronts, videos, extract preps, experimental days, respectively, **p ≤ 0.001. E) Quantification of active Rho dynamics in traveling waves. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± standard deviation (SD). Control, n = 3083, 4, 3, 3, 20% PE, n = 5288, 5, 5, 4 for boxes, videos, extract preps, experimental days, respectively, p ≤ 0.05. F) Standing oscillations on SLBs with 0% or 20% PE. Time is shown in seconds. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Yellow dashed boxes mark regions used for intensity traces. G) Kymographs of standing oscillations. H) Intensity over time traces of standing oscillations. I) Quantification of the active Rho and F-actin dynamics in standing oscillations. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. Control, n = 7415, 6, 5, 4, 20% PE, n = 9754, 6, 4, 2, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, *p ≤ 0.01.
	FIGURE 2:. Active Rho and F-actin self-organize in the artificial cortex independent of intracellular organelles. A) Schematic of low-speed supernatant (LSS) or membrane-depleted extract (MDE) preparation. 15 min centrifugation to generate LSS, and subsequent 90 and 30 min centrifugation steps to generate MDE. B) Normalized whole-field intensity of active Rho and F-actin over time in LSS (black) and MDE (blue). Mean (solid line) ± SD (shading) is shown. For LSS, n = 7, 4, 3, for MDE, n = 13, 9, 6, for videos, extract preps, experimental days, respectively. C) Standing oscillations in the artificial cortex in LSS or MDE. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Time is indicated in seconds after the start of oscillatory dynamics. D) Kymographs of standing oscillations in LSS or MDE. E) Quantification of oscillatory dynamics in LSS (black) or MDE (blue). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For LSS, n = 6352, 6 videos, 4, 3, for MDE, n = 4753, 6, 4, 3 for boxes, videos, extract preps, experimental days, respectively.
	FIGURE 3:. PIP2 supports F-actin assembly on SLBs. A) Quantification F-actin assembly PIP2 SLBs after 10 min. SLBs are comprised of 0% PIP2 (60% PC, 30% PS, 10% PI), or 0.1% PIP2 (60% PC, 30% PS, 9.9% PI, 0.1% PIP2) or 1% PIP2 (60% PC, 30% PS, 9% PI, 1% PIP2) or 5% PIP2 (60% PC, 30% PS, 5% PI, 5% PIP2). For 0% n = 12, 11, 6, For 0.1% n = 12, 11, 6, for 1% n = 11, 10, 5, for 5% n = 10, 10, 5 for videos, extract preps, experimental days, respectively. B) Fluorescence intensity of PIP2 biosensor (GFP-C1-PLCδ-PH) over time 0% (left) or 0.1% (right) PIP2 SLBs in the absence (buffer) or presence of MDE. Mean (solid line) ± standard error of the mean (shading) is shown. C) Relative change in fluorescence intensity of a PIP2 biosensor after 20 min of incubation with buffer (−) or MDE (+). Mean ± SD is shown. For buffer, n = 3 experimental replicates on one experimental day, for MDE n = 5 videos, 5 extract preps, 2 experimental days p ≤ 0.05, p ≤ 0.005. D) Normalized active Rho (left) or F-actin (right) intensity over time after treatment with DMSO or inhibitors of PIP2 synthesis (30 µM PI4KIIIɑ inhibitor [GSK-A1] + 20 µM PIP5K1α inhibitor [ISA-2011B]). Extract was incubated with PIP2 synthesis inhibitors for 10 min at room temperature before adding the extract to the SLB. Time = 0 indicates time of extract addition to the SLB. Mean (solid line) ± SD (shading) is shown. For both n = 3 videos, 3 extract preps, 2 experimental days. E) Standing oscillations in MDE on PIP2 SLBs at time = 0 s. Dashed lines mark regions used for kymographs. F) Kymographs of standing oscillations on PIP2 SLBs. G) Oscillatory dynamics in MDE on PIP2 SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PIP2, n = 4554, 4, 4, 3, for 0.1% PIP2, n = 6773, 6 videos, 4, 3, for 1% PIP2, n = 9190, 8, 6, 4, for 5% PIP2, n = 9209, 8, 5, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 4:. Cortical oscillation amplitude increases in MDE on PE SLBs. A) Standing oscillations in MDE on PE SLBs. SLBs are comprised of 0% PE (60% PC, 30% PS, 10% PI), or 10% PE (50% PC, 10% PE, 30% PS, 10% PI) or 20% PE (40% PC, 20% PE, 30% PS, 10% PI) or 30% PE (30% PC, 30% PE, 30% PS, 10% PI). Time is shown in seconds (s). Dashed lines marks regions used for kymographs. B) Kymographs of standing oscillations in MDE on PE SLBs. C) Quantification of oscillatory dynamics in MDE on PE SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 3872, 4, 3, 3, for 10% PE, n = 5099, 5, 3, 3, for 20% PE, n = 3354, 3, 3, 2, for 30% PE, n = 8222, 4, 4, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Quantification of oscillatory dynamics in MDE on PE SLBs with PIP2. SLBs are comprised of 0% PE (60% PC, 30% PS, 9.9% PI, 0.1% PIP2), or 10% PE (50% PC, 10% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 20% PE (40% PC, 20% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 30% PE (30% PC, 30% PE, 30% PS, 9.9% PI, 0.1% PIP2). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 5338, 4, 3, 3, for 10% PE, n = 4832, 5, 4, 4, for 20% PE, n = 4671, 5, 5, 4, for 30% PE, n = 5552, 5, 4, 4, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 5:. Standing oscillations are altered on sphingomyelin and cholesterol SLBs. A) Standing oscillations on control, sphingomyelin, and cholesterol SLBs. White dashed lines indicate regions used for kymographs. Right: kymographs of standing oscillations on control, sphingomyelin, and cholesterol SLBs. B) Quantification of oscillatory dynamics in MDE on control, sphingomyelin, and cholesterol SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For control, n = 5623, 6, 3, 4, for sphingomyelin, n = 6606, 6, 3, 4, for cholesterol, n = 6647, 8, 6, 3 for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. C) Left: active Rho on control, cholesterol and sphingomyelin SLBs and masks used to determine spatial Rho activity. Right: percent area of increasing active Rho at each time point and average active Rho cluster size. Violin plot = all time points, dots = mean of each experiment, bars = total mean ± SD. For control, n = 1000, 6, 3, 4, for cholesterol, n = 991, 8, 6, 3, for sphingomyelin, n = 868, 6, 3, 4 time points, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Kymographs of Rho activation and F-actin association (arrowheads) with control, sphingomyelin, and cholesterol SLBs. E) Representative normalized intensity curve of active Rho and F-actin after extract addition on a control SLB. Dotted lines indicate best fit sigmoidal curve. F) Quantification of the t1/2 and the temporal phase shift between active Rho and F-actin on control, sphingomyelin, and cholesterol SLBs. Mean ± SD is shown. For all, n = 3 videos, 3 extract preps, 2 experimental days. *p ≤ 0.05.

	FIGURE 1: PE SLBs alter active Rho and F-actin dynamics. A) Traveling waves on SLBs with 0% or 20% PE. Arrowheads mark merging wave fronts. Dashed lines mark regions used for kymographs. Yellow dashed boxes mark regions used for intensity traces. Time is shown in minutes:seconds. B) Kymographs of traveling waves. Arrowheads indicate consecutive wave fronts. C) Intensity over time traces of traveling waves. D) Traveling wave velocity, mean ± 95% confidence interval (CI) is shown. Control (0% PE), n = 20, 4, 3, 3, 20% PE, n = 26, 5, 4, 4 for wave fronts, videos, extract preps, experimental days, respectively, **p ≤ 0.001. E) Quantification of active Rho dynamics in traveling waves. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± standard deviation (SD). Control, n = 3083, 4, 3, 3, 20% PE, n = 5288, 5, 5, 4 for boxes, videos, extract preps, experimental days, respectively, p ≤ 0.05. F) Standing oscillations on SLBs with 0% or 20% PE. Time is shown in seconds. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Yellow dashed boxes mark regions used for intensity traces. G) Kymographs of standing oscillations. H) Intensity over time traces of standing oscillations. I) Quantification of the active Rho and F-actin dynamics in standing oscillations. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. Control, n = 7415, 6, 5, 4, 20% PE, n = 9754, 6, 4, 2, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, *p ≤ 0.01.
	Still from Movie S1: Traveling waves and standing oscillations on SLBs containing 0 or 20% PE. Related to Figure 1. Time is shown in minutes:seconds. Acquisition interval is 10 seconds. Playback is 10 frames per second (fps).
	FIGURE 2: Active Rho and F-actin self-organize in the artificial cortex independent of intracellular organelles. A) Schematic of low-speed supernatant (LSS) or membrane-depleted extract (MDE) preparation. 15 min centrifugation to generate LSS, and subsequent 90 and 30 min centrifugation steps to generate MDE. B) Normalized whole-field intensity of active Rho and F-actin over time in LSS (black) and MDE (blue). Mean (solid line) ± SD (shading) is shown. For LSS, n = 7, 4, 3, for MDE, n = 13, 9, 6, for videos, extract preps, experimental days, respectively. C) Standing oscillations in the artificial cortex in LSS or MDE. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Time is indicated in seconds after the start of oscillatory dynamics. D) Kymographs of standing oscillations in LSS or MDE. E) Quantification of oscillatory dynamics in LSS (black) or MDE (blue). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For LSS, n = 6352, 6 videos, 4, 3, for MDE, n = 4753, 6, 4, 3 for boxes, videos, extract preps, experimental days, respectively.
	Still from Movie S2: Standing oscillations in low-speed supernatant (LSS) and membrane-depleted extract (MDE). Related to Figure 2. Time is shown in minutes:seconds. Acquisition interval is 10 seconds for LSS and 5 seconds for MDE. Playback is 10 fps.
	FIGURE 3: PIP2 supports F-actin assembly on SLBs. A) Quantification F-actin assembly PIP2 SLBs after 10 min. SLBs are comprised of 0% PIP2 (60% PC, 30% PS, 10% PI), or 0.1% PIP2 (60% PC, 30% PS, 9.9% PI, 0.1% PIP2) or 1% PIP2 (60% PC, 30% PS, 9% PI, 1% PIP2) or 5% PIP2 (60% PC, 30% PS, 5% PI, 5% PIP2). For 0% n = 12, 11, 6, For 0.1% n = 12, 11, 6, for 1% n = 11, 10, 5, for 5% n = 10, 10, 5 for videos, extract preps, experimental days, respectively. B) Fluorescence intensity of PIP2 biosensor (GFP-C1-PLCδ-PH) over time 0% (left) or 0.1% (right) PIP2 SLBs in the absence (buffer) or presence of MDE. Mean (solid line) ± standard error of the mean (shading) is shown. C) Relative change in fluorescence intensity of a PIP2 biosensor after 20 min of incubation with buffer (−) or MDE (+). Mean ± SD is shown. For buffer, n = 3 experimental replicates on one experimental day, for MDE n = 5 videos, 5 extract preps, 2 experimental days p ≤ 0.05, p ≤ 0.005. D) Normalized active Rho (left) or F-actin (right) intensity over time after treatment with DMSO or inhibitors of PIP2 synthesis (30 µM PI4KIIIɑ inhibitor [GSK-A1] + 20 µM PIP5K1α inhibitor [ISA-2011B]). Extract was incubated with PIP2 synthesis inhibitors for 10 min at room temperature before adding the extract to the SLB. Time = 0 indicates time of extract addition to the SLB. Mean (solid line) ± SD (shading) is shown. For both n = 3 videos, 3 extract preps, 2 experimental days. E) Standing oscillations in MDE on PIP2 SLBs at time = 0 s. Dashed lines mark regions used for kymographs. F) Kymographs of standing oscillations on PIP2 SLBs. G) Oscillatory dynamics in MDE on PIP2 SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PIP2, n = 4554, 4, 4, 3, for 0.1% PIP2, n = 6773, 6 videos, 4, 3, for 1% PIP2, n = 9190, 8, 6, 4, for 5% PIP2, n = 9209, 8, 5, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	Still from Movie S3: Standing oscillations in MDE on SLBs containing 0 or 0.1% PIP2 and on SLBs containing 0, 10, 20, or 30% PE. Related to Figures 3 and 4. Time is shown in minutes:seconds. Acquisition interval is 10 seconds. Playback is 10 fps.
	FIGURE 4: Cortical oscillation amplitude increases in MDE on PE SLBs. A) Standing oscillations in MDE on PE SLBs. SLBs are comprised of 0% PE (60% PC, 30% PS, 10% PI), or 10% PE (50% PC, 10% PE, 30% PS, 10% PI) or 20% PE (40% PC, 20% PE, 30% PS, 10% PI) or 30% PE (30% PC, 30% PE, 30% PS, 10% PI). Time is shown in seconds (s). Dashed lines marks regions used for kymographs. B) Kymographs of standing oscillations in MDE on PE SLBs. C) Quantification of oscillatory dynamics in MDE on PE SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 3872, 4, 3, 3, for 10% PE, n = 5099, 5, 3, 3, for 20% PE, n = 3354, 3, 3, 2, for 30% PE, n = 8222, 4, 4, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Quantification of oscillatory dynamics in MDE on PE SLBs with PIP2. SLBs are comprised of 0% PE (60% PC, 30% PS, 9.9% PI, 0.1% PIP2), or 10% PE (50% PC, 10% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 20% PE (40% PC, 20% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 30% PE (30% PC, 30% PE, 30% PS, 9.9% PI, 0.1% PIP2). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 5338, 4, 3, 3, for 10% PE, n = 4832, 5, 4, 4, for 20% PE, n = 4671, 5, 5, 4, for 30% PE, n = 5552, 5, 4, 4, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 5: Standing oscillations are altered on sphingomyelin and cholesterol SLBs. A) Standing oscillations on control, sphingomyelin, and cholesterol SLBs. White dashed lines indicate regions used for kymographs. Right: kymographs of standing oscillations on control, sphingomyelin, and cholesterol SLBs. B) Quantification of oscillatory dynamics in MDE on control, sphingomyelin, and cholesterol SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For control, n = 5623, 6, 3, 4, for sphingomyelin, n = 6606, 6, 3, 4, for cholesterol, n = 6647, 8, 6, 3 for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. C) Left: active Rho on control, cholesterol and sphingomyelin SLBs and masks used to determine spatial Rho activity. Right: percent area of increasing active Rho at each time point and average active Rho cluster size. Violin plot = all time points, dots = mean of each experiment, bars = total mean ± SD. For control, n = 1000, 6, 3, 4, for cholesterol, n = 991, 8, 6, 3, for sphingomyelin, n = 868, 6, 3, 4 time points, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Kymographs of Rho activation and F-actin association (arrowheads) with control, sphingomyelin, and cholesterol SLBs. E) Representative normalized intensity curve of active Rho and F-actin after extract addition on a control SLB. Dotted lines indicate best fit sigmoidal curve. F) Quantification of the t1/2 and the temporal phase shift between active Rho and F-actin on control, sphingomyelin, and cholesterol SLBs. Mean ± SD is shown. For all, n = 3 videos, 3 extract preps, 2 experimental days. *p ≤ 0.05.
	Still from Movie S4: Standing oscillations and cortical self-assembly in MDE on SLBs containing cholesterol or sphingomyelin. Related to Figure 5. Time is shown in seconds. For oscillations, acquisition interval is 10 seconds. For cortical assembly, acquisition interval is 2 seconds. Playback is 10 fps.
	FIGURE 1:. PE SLBs alter active Rho and F-actin dynamics. A) Traveling waves on SLBs with 0% or 20% PE. Arrowheads mark merging wave fronts. Dashed lines mark regions used for kymographs. Yellow dashed boxes mark regions used for intensity traces. Time is shown in minutes:seconds. B) Kymographs of traveling waves. Arrowheads indicate consecutive wave fronts. C) Intensity over time traces of traveling waves. D) Traveling wave velocity, mean ± 95% confidence interval (CI) is shown. Control (0% PE), n = 20, 4, 3, 3, 20% PE, n = 26, 5, 4, 4 for wave fronts, videos, extract preps, experimental days, respectively, **p ≤ 0.001. E) Quantification of active Rho dynamics in traveling waves. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± standard deviation (SD). Control, n = 3083, 4, 3, 3, 20% PE, n = 5288, 5, 5, 4 for boxes, videos, extract preps, experimental days, respectively, p ≤ 0.05. F) Standing oscillations on SLBs with 0% or 20% PE. Time is shown in seconds. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Yellow dashed boxes mark regions used for intensity traces. G) Kymographs of standing oscillations. H) Intensity over time traces of standing oscillations. I) Quantification of the active Rho and F-actin dynamics in standing oscillations. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. Control, n = 7415, 6, 5, 4, 20% PE, n = 9754, 6, 4, 2, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, *p ≤ 0.01.
	FIGURE 2:. Active Rho and F-actin self-organize in the artificial cortex independent of intracellular organelles. A) Schematic of low-speed supernatant (LSS) or membrane-depleted extract (MDE) preparation. 15 min centrifugation to generate LSS, and subsequent 90 and 30 min centrifugation steps to generate MDE. B) Normalized whole-field intensity of active Rho and F-actin over time in LSS (black) and MDE (blue). Mean (solid line) ± SD (shading) is shown. For LSS, n = 7, 4, 3, for MDE, n = 13, 9, 6, for videos, extract preps, experimental days, respectively. C) Standing oscillations in the artificial cortex in LSS or MDE. Left: micrograph of the whole field of view at time = 0 s. Dashed lines mark regions used for kymographs. Yellow boxes mark regions for enlarged micrographs. Right: Enlarged micrographs of standing oscillations. Time is indicated in seconds after the start of oscillatory dynamics. D) Kymographs of standing oscillations in LSS or MDE. E) Quantification of oscillatory dynamics in LSS (black) or MDE (blue). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For LSS, n = 6352, 6 videos, 4, 3, for MDE, n = 4753, 6, 4, 3 for boxes, videos, extract preps, experimental days, respectively.
	FIGURE 3:. PIP2 supports F-actin assembly on SLBs. A) Quantification F-actin assembly PIP2 SLBs after 10 min. SLBs are comprised of 0% PIP2 (60% PC, 30% PS, 10% PI), or 0.1% PIP2 (60% PC, 30% PS, 9.9% PI, 0.1% PIP2) or 1% PIP2 (60% PC, 30% PS, 9% PI, 1% PIP2) or 5% PIP2 (60% PC, 30% PS, 5% PI, 5% PIP2). For 0% n = 12, 11, 6, For 0.1% n = 12, 11, 6, for 1% n = 11, 10, 5, for 5% n = 10, 10, 5 for videos, extract preps, experimental days, respectively. B) Fluorescence intensity of PIP2 biosensor (GFP-C1-PLCδ-PH) over time 0% (left) or 0.1% (right) PIP2 SLBs in the absence (buffer) or presence of MDE. Mean (solid line) ± standard error of the mean (shading) is shown. C) Relative change in fluorescence intensity of a PIP2 biosensor after 20 min of incubation with buffer (−) or MDE (+). Mean ± SD is shown. For buffer, n = 3 experimental replicates on one experimental day, for MDE n = 5 videos, 5 extract preps, 2 experimental days p ≤ 0.05, p ≤ 0.005. D) Normalized active Rho (left) or F-actin (right) intensity over time after treatment with DMSO or inhibitors of PIP2 synthesis (30 µM PI4KIIIɑ inhibitor [GSK-A1] + 20 µM PIP5K1α inhibitor [ISA-2011B]). Extract was incubated with PIP2 synthesis inhibitors for 10 min at room temperature before adding the extract to the SLB. Time = 0 indicates time of extract addition to the SLB. Mean (solid line) ± SD (shading) is shown. For both n = 3 videos, 3 extract preps, 2 experimental days. E) Standing oscillations in MDE on PIP2 SLBs at time = 0 s. Dashed lines mark regions used for kymographs. F) Kymographs of standing oscillations on PIP2 SLBs. G) Oscillatory dynamics in MDE on PIP2 SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PIP2, n = 4554, 4, 4, 3, for 0.1% PIP2, n = 6773, 6 videos, 4, 3, for 1% PIP2, n = 9190, 8, 6, 4, for 5% PIP2, n = 9209, 8, 5, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 4:. Cortical oscillation amplitude increases in MDE on PE SLBs. A) Standing oscillations in MDE on PE SLBs. SLBs are comprised of 0% PE (60% PC, 30% PS, 10% PI), or 10% PE (50% PC, 10% PE, 30% PS, 10% PI) or 20% PE (40% PC, 20% PE, 30% PS, 10% PI) or 30% PE (30% PC, 30% PE, 30% PS, 10% PI). Time is shown in seconds (s). Dashed lines marks regions used for kymographs. B) Kymographs of standing oscillations in MDE on PE SLBs. C) Quantification of oscillatory dynamics in MDE on PE SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 3872, 4, 3, 3, for 10% PE, n = 5099, 5, 3, 3, for 20% PE, n = 3354, 3, 3, 2, for 30% PE, n = 8222, 4, 4, 3, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Quantification of oscillatory dynamics in MDE on PE SLBs with PIP2. SLBs are comprised of 0% PE (60% PC, 30% PS, 9.9% PI, 0.1% PIP2), or 10% PE (50% PC, 10% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 20% PE (40% PC, 20% PE, 30% PS, 9.9% PI, 0.1% PIP2) or 30% PE (30% PC, 30% PE, 30% PS, 9.9% PI, 0.1% PIP2). Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For 0% PE, n = 5338, 4, 3, 3, for 10% PE, n = 4832, 5, 4, 4, for 20% PE, n = 4671, 5, 5, 4, for 30% PE, n = 5552, 5, 4, 4, for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05, **p ≤ 0.005.
	FIGURE 5:. Standing oscillations are altered on sphingomyelin and cholesterol SLBs. A) Standing oscillations on control, sphingomyelin, and cholesterol SLBs. White dashed lines indicate regions used for kymographs. Right: kymographs of standing oscillations on control, sphingomyelin, and cholesterol SLBs. B) Quantification of oscillatory dynamics in MDE on control, sphingomyelin, and cholesterol SLBs. Violin plot = data points from all experiments, dots = mean of each experiment, bars = total mean ± SD. For control, n = 5623, 6, 3, 4, for sphingomyelin, n = 6606, 6, 3, 4, for cholesterol, n = 6647, 8, 6, 3 for boxes, videos, extract preps, experimental days, respectively. p ≤ 0.05. C) Left: active Rho on control, cholesterol and sphingomyelin SLBs and masks used to determine spatial Rho activity. Right: percent area of increasing active Rho at each time point and average active Rho cluster size. Violin plot = all time points, dots = mean of each experiment, bars = total mean ± SD. For control, n = 1000, 6, 3, 4, for cholesterol, n = 991, 8, 6, 3, for sphingomyelin, n = 868, 6, 3, 4 time points, videos, extract preps, experimental days, respectively. p ≤ 0.05. D) Kymographs of Rho activation and F-actin association (arrowheads) with control, sphingomyelin, and cholesterol SLBs. E) Representative normalized intensity curve of active Rho and F-actin after extract addition on a control SLB. Dotted lines indicate best fit sigmoidal curve. F) Quantification of the t1/2 and the temporal phase shift between active Rho and F-actin on control, sphingomyelin, and cholesterol SLBs. Mean ± SD is shown. For all, n = 3 videos, 3 extract preps, 2 experimental days. *p ≤ 0.05.