Complementary cytoskeletal feedback loops control signal transduction excitability and cell polarity

Abstract

To navigate complex environments, cells integrate chemical and mechanical cues through dynamic feedback between signaling networks and the cytoskeleton. Using synthetic tools to manipulate cytoskeletal components in Dictyostelium and human neutrophils, we uncover feedback mechanisms that regulate Ras/PI3K signaling and control front- and back-states of the cell. Increased branched actin and actin polymerization enhance Ras/PI3K activity. Similarly, decreased myosin II assembly also elevates signaling and chemotactic sensitivity. Conversely, inhibiting branched actin increases cortical actin and blocks Ras/PI3K activation-an effect lessened by decreasing filamentous actin or in myosin II-null cells. Activating RacE to increase actin crosslinking suppresses Ras activity without triggering branched actin nucleators, yet promotes spreading and protrusion. These results informed a computational model incorporating positive cytoskeletal feedback loops, which predicts shifts in polarity and migration with cytoskeletal changes. We propose that such feedback locally tunes signal network excitability, enabling cells to navigate tissues, extracellular matrix, and fluid environments.

Fig. 1. Cytoskeletal feedback loops at the cell front and back control Ras activity.

a Spinning disk confocal imaging of Ras activation (RBD-EGFP/RBD-mCherry) in wild-type (AX3) and actobindin ABC triple knockout (abnABC⁻) Dictyostelium cells. Arpin-OE cells are AX3 cells expressing Arp2/3-inhibitor Arpin-EGFP. b Membrane kymographs (left) of RBD corresponding to the movies in a. Scale bars = 2 minutes (horizontal) and 5 µm (vertical). Diagram (left) depicts the method of creating a membrane kymograph with 2 Fluorescent Proteins (FPs). c, d Individual (dots) and average (lines) percentages of the membrane periphery with RBD localization significantly above background (C) and average total cell intensity in AX3, abnABC⁻, and Arpin-OE cells. n=cells, *=p < 0.05, ***= p < 0.005, 2-sided Mann-Whitney U test. e Scanning confocal imaging of mCherry-FRB-MHCKC membrane recruitment and cell shape (brightfield) in AX3 cells. Cells are also expressing an unlabeled membrane-localized FKBP domain (cAR1-2xFKBP, see methods). t = 00:00 indicates rapamycin addition. f Examples (left) and diagram (right) of temporal color projections of cell outlines corresponding to the movie in (D). Blue and yellow times indicate the first and last images in the projection, respectively. g Average (line) and SEM (shaded area) of normalized (top) or absolute (bottom) cell area before and after rapamycin addition in cells expressing FRB-MHCKC (cyan) or FRB alone (orange). (dashed line, t = 0). n = 21 and 17 cells, respectively. h Scanning confocal imaging of RBD and MHCKC membrane recruitment in AX3 cells. t = 00:00 indicates rapamycin addition. i Membrane kymograph of Ras activation corresponding to the movie in (g). Dashed line indicates rapamycin addition, scale bars = 2 minutes (horizontal) and 5 µm (vertical). j Average and SEM of normalized percentages of the membrane periphery with RBD localization significantly above background before and after rapamycin addition (dashed line, t = 0) in AX3 cells expressing FRB-MHCKC (cyan) and FRB alone (orange) and in myosin II-null (myoII-) cells expressing FRB-MHCKC (magenta). n = 20,32, and 31, respectively. Time is in min:sec; scale bars = 5 µm unless otherwise noted. Source data are provided as a Source Data file.

Fig. 2. Cortical actin suppresses signaling network activation.

a Scanning confocal imaging of polymerizing actin (LimE_ΔCoil-RFP) and PIP₃ (PH_CRAC-YFP) in wild type (AX3) Dictyostelium cells before and after treatment with the Arp2/3 inhibitor CK666 (CK). t = 00:00 indicates CK666 addition. b Average (line) and SEM (shaded area) of the total normalized intensity and the variance of the peripheral intensity of LimE (magenta) and PH_CRAC (green) patches before and after CK666 addition (dashed line, t = 0). n = 27 cells. c TIRF imaging of activated Ras (RBD-mCherry) in electrofused (“giant”) AX3 cells before and after CK666 and latrunculin addition. Cells are incubated in caffeine to raise basal activity levels. t = 00:00 indicates the addition of CK666 or, in parentheses, latrunculin. d Average and SEM of average RBD membrane intensity in giant AX3 cells before and after CK666 addition (left, dashed line, t = 0) or in CK666 before and after latrunculin addition (right, dashed line, t = 0). n = 22 cells. e Individual traces from d. t = 00:00 indicates CK666 addition; dashed lines correspond to drug addition. f Spinning disk confocal imaging of PIP3 (RFP-PH_AKT) in human neutrophil-like (dHL60) cells before and after CK666 and latrunculin addition. t = 00:00 indicates the addition of CK666 or, in parentheses, latrunculin. g Average and SEM of the normalized PH_AKT membrane-to-cytosol ratio before and after CK666 (left, n = 51 cells) and subsequently latrunculin (right, n = 65 cells) addition in dHL60 cells. h Individual (dots) and average (lines) PH_AKT membrane-to-cytosol ratio normalized to the average level before CK666 (left, n = 51 cells) or latrunculin (right, n = 65 cells) addition. ***=P < 0.005, 2-sided Wilcoxon signed-rank test. i Spinning disk confocal imaging of Ras activation levels (RBD-EGFP) in developed AX3 Dictyostelium cells treated with CK666 (CK), latrunculin (Lat), or buffer (Buff) before and 6 seconds after the addition of 0.1 nM, 0.5 nM, and 10 nM cAMP. j Normalized RBD membrane-to-cytosol ratio before and after cAMP addition (dashed lines) in cells treated with CK666 (purple), latrunculin (yellow) or buffer (cyan). n = 31, 40, and 22 cells. k Individual and average maximum values of normalized RBD membrane-to-cytosol ratio in the 42 seconds after addition of cAMP. ***=p < 0.005, 2-sided Mann-Whitney U test. Time=min:sec; scale bars = 5 µm. Source data are provided as a Source Data file.

Fig. 3. The actomyosin cortex exhibits negative feedback onto signaling networks.

a Scanning and spinning disk confocal images of PIP₃ levels (PH_CRAC-YFP/ PH_CRAC-mScarlet3) in wild type (AX3), myosin II-null (myoII-) Dictyostelium cells, and myosin II-null Dictyostelium cells expressing GFP-MyosinII (myoII- rescue) before and after CK666 addition. t = 00:00 indicates CK666 addition. b Heatmap of the number of PH_CRAC patches in individual AX3, myoII-, and rescue cells. Every row is a single cell and every column is a timepoint. c Average (lines) and individual (dots) mean number of PH_CRAC patches over time before and after CK666 addition in AX3 and myoII⁻, and rescue cells. n = cells, ***=p < 0.005, 2-sided Mann-Whitney U test. d Average and individual percent decrease in the average number of PH_CRAC patches after CK666 addition in AX3 and myoII⁻, and rescue cells. Data are from c, *** = p < 0.005 2-sided Mann-Whitney U test. e Scanning confocal images of Ras activation (RBD-EGFP) before and after CK666 addition and subsequently mCherry-FRB-MHCKC membrane recruitment in AX3 cells. Cells are also expressing an unlabeled membrane-localized FKBP domain (cAR1-2xFKBP). t = 00:00 indicates the addition of CK666 or, in parentheses, rapamycin. f Membrane kymograph of Ras activation corresponding to the movie in c. Dashed lines represent the addition of specified drug, scale bars = 2 minutes (horizontal) and 5 µm (vertical). g Average and individual mean number of RBD patches over time before and after CK666 addition and subsequently MHCKC recruitment by rapamycin addition. n = cells, *** = p < 0.005, 2-sided Mann-Whitney U test. Source data are provided as a Source Data file.

Fig. 4. Increasing the abundance of the actomyosin cortex using RacE leads to signaling inhibition.

a Scanning confocal imaging of RacE-GEF (mCherry-FRB-GXCT_ΔNT) membrane recruitment and cell shape in wild type (AX3) Dictyostelium cells. Cells are also expressing an unlabeled membrane-localized FKBP domain (cAR1-2xFKBP). t = 00:00 indicates rapamycin addition. b Temporal color projections of cell outlines corresponding to the movie in a. Blue and yellow times indicate the first and last images in the projection, respectively. c Average (line) and SEM (shaded area) of cell area in AX3 cells before and after GXCT recruitment (dashed line, t = 0). n = 35 cells. d Traces of cell movement in AX3 cells 200 seconds before and after GXCT recruitment. n = 35 cells. e Scanning Confocal imaging of GXCT recruitment and polymerizing actin (LimE_ΔCoil-EGFP) in AX3 cells. t = 00:00 indicates rapamycin addition. f Membrane kymograph of LimE from the movie in e. Dashed line indicates rapamycin addition, scale bars = 2 minutes (horizontal) and 5 µm (vertical). g Average and SEM membrane-to-cytosol ratio of LimE before and after GXCT recruitment by rapamycin addition (dashed line, t = 0). n = 8 cells. h Scanning Confocal imaging of GXCT recruitment and Ras activation (RBD-EGFP) in AX3 cells. t = 00:00 indicates rapamycin addition. i Membrane kymograph of RBD from the movie in h. Dashed line indicates rapamycin addition, scale bars = 2 minutes (horizontal) and 5 µm (vertical). j Average and SEM membrane-to-cytosol ratio of RBD before and after GXCT recruitment by rapamycin addition (dashed line, t = 0). n = 9 cells. Time is in min:sec. Scale bars = 5 µm unless otherwise noted. Source data are provided as a Source Data file.

Fig. 5. RacE reversibly and locally controls cell signaling and actin polymerization.

a Scanning confocal imaging of RacE-GEF (tagRFP-SSPB-GXCT_ΔNT) optical membrane recruitment in wild type (AX3) Dictyostelium cells. Cells also express a membrane-localized iLID domain (N150-ILID). t = 00:00 indicates blue light exposure or blue light loss (parentheses). b Average (lines) and SEM (shaded area) of cell area before and after GXCT recruitment (left, dashed line) or dissociation (right, dashed line). n = 17 cells (left) and 6 cells (right). c Individual traces from (b). d TIRF imaging of Ras activation (RBD-emiRFP670) before, during, and after GXCT recruitment in electrofused (“giant”) AX3 cells. Cells are treated with 50 µg/ml Biliverdin to activate emiRFP670 fluorescence. t = 00:00 indicates blue light exposure. e Average and SEM of the percent of cell membrane with RBD recruitment before and after GXCT recruitment (left, dashed line) or GXCT dissociation (right, dashed line). n = 7 cells. f Individual traces from e. g Scanning confocal imaging of cell protrusion after recruitment of SSPB-GXCT or SSPB alone. +CK indicates cells treated with CK666. Boxes indicate the blue light area; arrows form a line between the center of the protrusion after stimulation and the center of the cell. t = 00:00 indicates the last timepoint before blue light exposure. h Angular histograms of the angle formed between the protrusion and GXCT recruitment relative to the cell center. n = 26 cells, SSPB-GXCT and SSPB-GXCT + CK666. n = 15 cells, SSPB. i TIRF imaging of RBD localization before and after local GXCT recruitment in giant AX3 cells. The yellow box indicates blue light area and the blue arrow is the location of the linear kymograph. t = 00:00 indicates the last timepoint before blue light exposure. j Linear kymograph of GXCT recruitment and RBD intensity from i. Yellow box indicates blue light area. k Quantification of RBD wave motion away from GXCT recruitment (see methods). This measurement is a composite of RBD translocation and disappearance. Kymograph scale bars = 20 seconds (horizontal) and 2 µm (vertical). n=cells, ***=p = 6.31*10⁻⁵, 2-sided Mann-Whitney U test. Time is in min:sec; scale bars = 5 µm unless otherwise noted. Source data are provided as a Source Data file.

Fig. 6. Incorporating cytoskeletal feedback into a core model of the STEN.

a Schematic showing the signal transduction network involving Ras/ PIP2 and PKB and how they couple to the two types of actin feedback loops. b Kymographs showing activity around the cell perimeter as a function of time. PIP2 and Ras are shown in green and magenta, respectively. The top and bottom correspond to simulations without and with the feedback loops, respectively. c Simulated cell trajectories of 10 cells each with feedback loops turned off (top) and on (bottom). d Kymographs showing wave activity across the cell perimeter for varying strengths of the branched actin feedback. e Total Ras activity around the cell perimeter with respect to the strength of the branched actin feedback. Wildtype corresponds to a strength of 1. The black bar denotes the mean of 10 simulations per strength. Simulations with total Ras activity less than 500 showed no firings. f Kymographs showing wave activity across the cell perimeter for varying strengths of the actomyosin feedback. g Total Ras activity around the cell perimeter with respect to the strength of the actomyosin feedback. Wildtype corresponds to a strength of 0.4. The black bar denotes the mean of 10 simulations per strength. Simulations with total Ras activity less than 200 showed no firings. h Frames from a 2D simulation of the effects of adding CK666 and then Latrunculin to wild-type cells. The three rows represent waves in the wild-type cell, waves after CK666 addition, and subsequent Latrunculin treatment. i The total Ras activity for simulations as in h. The solid line and the shaded area represent the mean ± 1 standard deviation. In all the simulations, the CK666 effect is incorporated at the 200 s and the additional Latrunculin effect is added at 400 s. Scale bars = 10 microns. Source data are provided as a Source Data file.

Fig. 7. The STEN model incorporating cytoskeletal feedback predicts changes to motility from modifying branched actin and actomyosin.

a, b 10 simulated tracks of cell motion before (left) and after (right) increasing the strength of the branched actin loop (a) or decreasing the strength of the actomyosin loop (b). Cells begin with low feedback strength for both branched actin and actomyosin, creating low polarity. 10 simulated tracks of cell motion before (left) and after (right) increasing the strength of the branched actin loop (c) or decreasing the strength of the actomyosin loop (d). Cells begin with high feedback strength for both branched actin and actomyosin, creating high polarity. e Tracks of vegetative AX3 and abnABC⁻ cells migrating for 10 minutes. f Tracks of vegetative AX3 cells expressing mCherry-FRB-MHCKC and an unlabeled membrane-localized FKBP domain (cAR1-2xFKBP) migrating for 10 minutes before rapamycin addition to recruit MHCKC and for 10 minutes after 10 minutes of incubation in rapamycin. g Tracks of developed, polarized AX3 and abnABC⁻ cells migrating for 10 minutes. h Tracks of developed, polarized AX3 cells expressing mCherry-FRB-MHCKC and an unlabeled membrane-localized FKBP domain (cAR1-2xFKBP) migrating for 10 minutes without rapamycin or after 10 minutes of incubation in rapamycin. i Average (lines) and individual (dots) estimated 2D diffusion coefficients of cells in each indicated condition from E-F. All cells are vegetative. j Average and individual estimated 2D diffusion coefficients of cells in each indicated condition from G-H. All cells are developed. *=p = 0.041, **=p = 0.0057, ***=p < 0.005, 2-sided Mann-Whitney U test. Source data are provided as a Source Data file.