Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration

Abstract

Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P₂]. In addition, depletion of PI(3,4)P₂ by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P₂, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P₂ levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P₂ down, causing the PI(3,4)P₂-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P₂ as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P₂ mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Keywords: chemotaxis; excitability; phosphoinositides; positive feedback loop; signal transduction.

Fig. 1.

Back-to-front gradient and transient chemoattractant-induced depletion of PI(3,4)P₂. (A) Growth-stage wild-type Ax3 cell coexpressing tPH_CynA-KikGR (green) and LimE-RFP (red). (Scale bar: 5 μm.) (B) Kymographs of tPH_CynA and LimE intensity on the perimeter of cell in A undergoing random migration. Confocal images collected at 5-s intervals. (C) Binding of supernatants from cells expressing full-length CynA-GFP, tPH_CynA-GFP, or cPH_TAPP1-GFP to PIP strips. (D) “Pull-down” assay showing TIRF images of tPH_CynA-GFP binding to tethered vesicles containing PI(3,4,5)P₃, PI(4,5)P₂, or PI(3,4)P₂ (Top row). DiD staining of vesicles (Bottom row). (Scale bar: 5 μm.) (E) Quantification of tPH_CynA-GFP binding in pull-down assays. Background fluorescence spots in the GFP channel obtained by adding lysates to vesicles lacking PIs were subtracted from all samples. Error bars are SEM. (F) Growth-stage wild-type Ax2 cell coexpressing ttPH_CynA-GFP (green) and LimE-RFP (red). (Scale bar: 5 μm.) (G) Four-hour–stage wild-type Ax3 cells expressing PH_CRAC-RFP and tPH_CynA-GFP were treated with 5 μM latrunculin A for 20 min and then stimulated with cAMP. Time-lapse confocal representative images showing redistributions of PH_CRAC-RFP (red) and tPH_CynA-GFP (green) in the same cell. Images were collected at −10, 0, and 10 s. (Scale bar: 5 μm.) (H) Confocal images of tPH_CynA-GFP (Top) in independent experiment similar to that in A at representative times. Corresponding kymograph of cortical tPH_CynA intensity (Bottom). (Scale bar: 5 μm.) (I) Field of 4-h–stage wild-type Ax3 cells expressing tPH_CynA-KikGR 60 s before and 360 s after exposure to a micropipette containing 1 µM cAMP. (Scale bar: 10 μm.) (J) Time-lapse images of an individual cell in the micropipette assay. Kymograph of the cortical tPH_CynA-KikGR intensity is shown at Bottom. (Scale bar: 5 μm.) (K) Fluctuations in the angle of the tPH_CynA-GFP crescents. To determine the positions of the crescents, the angle was defined by measuring the angle formed by two lines: the line drawn between the centroid of the cell and the center of the crescent, and the line drawn between the centroid of the cell and the tip of the micropipette. Crescent fluctuations of four cells from experiments in I are shown (n = 6).

Fig. 2.

Deletion of OCRL homolog Dd5P4 leads to lowered PI(3,4)P₂ and elevated Ras activity. (A) Representative confocal images of tPH_CynA-KikGR in growth-stage, wild-type Ax3 and Dd5P4− cells treated with 5 μM latrunculin A. (Scale bar: 10 μm.) (B) Ratio of membrane to cytosol intensity of tPH_CynA-KikGR in wild-type Ax3 and Dd5P4− cells; mean ± SEM (n = 18). (C) Representative confocal images of RBD-GFP in migrating growth-stage, wild-type Ax3 and Dd5P4− cells. (Scale bar: 10 μm.) (D) Kymographs of cortical RBD-GFP intensities in representative individual cells from C. (E and F) Basal surface area (E) and fraction of cell perimeter covered by RBD-GFP patches (F) in cells from C. *P < 0.05 versus Ax3 group; mean ± SEM (n = 18). (G) Elevated RalGDS activity in Dd5P4− cells. Representative confocal images of Ax3 and Dd5P4− cells expressing RalGDS-GFP are shown. (Scale bar: 5 μm.) (H) Kymographs of movies of cells in G. (I) Quantification of RalGDS patch activity in Ax3 and Dd5P4− cells. Fraction of the perimeter occupied by RalGDS patches was quantified (Materials and Methods); n = 35. (J) Time-lapse confocal images of individual cells from independent experiment similar to that in C highlighting oscillatory Dd5P4− cell. (Scale bar: 10 μm.) (K) Color-coded tracing of cell outlines at 4-min intervals of several cells from independent experiment similar to that in C. (Scale bar: 10 μm.)

Fig. 3.

Lowering of PI(3,4)P₂ by exogenous INPP4B leads to hyperactive cell behavior. (A) Growth-stage, wild-type Ax3 cells expressing mCherry-FRB-INPP4B_510–924 (red), N150-tFKBP and tPH_CynA-GFP (green) were treated with 5 μM latrunculin A for 20 min. The time-lapse confocal images of the same cell were obtained every 20 s for 1 h. Representative images before (Top) and 30 min after rapamycin (Rapa) treatment (Bottom) are shown. (Scale bar: 10 μm.) (B) Fractional changes of ratio of membrane to cytosol intensity of tPH_CynA-GFP in experiment in A (n = 5). (C) Kymograph of cortical tPH_CynA-GFP membrane intensity of representative cell from A. (D) Randomly migrating growth-stage cells were imaged every 20 s. Confocal images of three individual cells showing the transition of the cell migratory modes before and after rapamycin treatment. Wild-type Ax3 cells expressing mCherry-FRB-INPP4B_510–924 (red) and N150-tFKBP in the second and third rows. Control cells in the first row are expressing mCherry-FRB (red) instead of mCherry-FRB-INPP4B_510–924. (Scale bars: 10 μm.) (E) Cell areas and cell perimeters were quantified before and after addition of rapamycin for control (red) and experimental cells (blue) in an independent experiment similar to that in D (n = 12). (F and G) Normalized areas of six control (F) and six experimental cells (G) at 1-min intervals. Rapamycin was added at t = 0. Cells were segmented into amoeboid or oscillatory migratory modes, black and red, respectively, using MATLAB. (H) Centroid tracks showing random movement of cells from D before and after rapamycin addition. Each track lasts 10 min and was repositioned to the same origin. Quantification of the cell speed is on the Right (n = 18). ***P < 0.001 versus −Rapa group. (I) Time-lapse confocal images of Dd5P4− cells expressing mCherry-FRB-INPP4B_510–924 and N150-tFKBP before (Left) and 30 min after (Right) rapamycin treatment. (Scale bar: 10 μm.) (J) Basal surface area covered by tPH_CynA-GFP in cells from I. ***P < 0.001 versus −Rapa group; mean ± SEM (n = 10).

Fig. 4.

The contribution of PI(3,4,5)P₃ to Ras activity. (A) Confocal representative images of vegetative, wild-type Ax3 (Top row) and Dd5P4− cells (Bottom row) expressing RBD-GFP treated with 50 μM LY294002 for −1 (Left), 5 (Middle), and 51 min (Right). (Scale bar: 10 μm.) (B) Quantification of fraction of cell perimeter covered by RBD-GFP patches in A. (C) Representative confocal images of tPH_CynA-GFP in growth-stage, wild-type Ax3, Pten−, and Dd5P4− cells treated with 5 μM latrunculin A. (Scale bar: 10 μm.) (D) Ratio of membrane to cytosol intensity of tPH_CynA. Mean ± SEM (n = 18). ***P < 0.001 versus Ax3 group. (E) RBD patch dynamics in latrunculin A-treated cells. Representative images from time-lapse movies of latrunculin A-treated cells expressing RBD-GFP are shown above 180° rotated views of t stacks generated from 4-min time lapses. (Scale bar: 4 μm.) (F) Quantification of the fraction of the perimeter from cells in E, occupied by RBD patches (n = 18). ***P < 0.005. (G) Quantification of the number of RBD patches generated during 4-min time-lapse movies of cells in E. Error bars indicate SD (n = 18). ***P < 0.005.

Fig. 5.

RasGAP2 and RapGAP3 bind to and are regulated by PI(3,4)P₂. (A) Unrooted phylogenetic trees of Dictyostelium genes with consensus RasGAP and RapGAP domains. Uniprot IDs are listed for uncharacterized genes. (B) Domain organization of RasGAP2 (RG2), accession number DDB_G0278483, and RapGAP3 (RG3), accession number DDB_G0271806. (C and D) Single confocal sections of cells coexpressing RFP-tagged RG2 (C) or RG3 (D) with LimE-GFP. The arrows point to the localization of GAP proteins to the base of cup-shaped macropinosome crowns. (Scale bars: 5 μm.) (E) Selected frames from time-lapse movies of RG2-RFP– and GFP-LimE–coexpressing cells. The arrows point to the accumulation of RG2 at the base of macropinocytotic crowns and nascent macropinosomes. (Scale bar: 7 μm.) (F) Analysis of RG3 localization as in E. (G) Selected frames from time-lapse movies of RG3-RFP– and GFP-LimE–coexpressing cells. The arrows point to the accumulation of RG3 at the base of macropinocytotic crowns and nascent macropinosomes. (Scale bar: 7 μm.) (H) Analysis of RG3 localization as in G. (I) Four-hour–stage Ax2 cells expressing RG3-RFP were treated with 5 µM latrunculin A for 10 min before beginning the time course. cAMP was added at time 0. (Scale bar: 7 μm.) (J) Binding of RG2-GFP and RG3-GFP to the indicated lipids immobilized on PIP strips; see Materials and Methods. (K) Confocal representative images of Ax3 and Dd5P4-null cells coexpressing LimE and one of RG2-GFP, or RG3-GFP. (Scale bar: 5 μm.)

Fig. 6.

Deletion of RasGAP2 and RapGAP3 leads to Ras and Rap activation and the hyperactive phenotype. (A) Ras patch dynamics in wild-type and RG2− cells. Selected frames from time-lapse movies of Raf1-RBD-GFP–expressing wild-type and RG2− cells. (Scale bar: 5 μm.) (B) Similar experiments as in A showing Rap1 (of RalGDS-GFP) patch dynamics in RG3− cells. (Scale bar: 10 μm.) (C) Comparison of Ras patch dynamics in latrunculin A-treated cells. Frames from time-lapse movies were stacked vertically to create time-stacked kymographs, which are shown in two 180° rotated views. (Scale bar: 5 μm.) (D) Quantification of the portion of the cell perimeter from C encompassed by RBD patches. ***P < 0.001. (E) Similar experiments as in C illustrating Rap1 patch dynamics using RalGDS-GFP biosensor. (Scale bar: 5 μm.) (F) Experiments in E were quantified as in D. ***P < 0.001. (G and H) Rose plots of cell-tracking data from time-lapse movies of RG2− and RG3− cells. Quantification of the cell speed is on the Right, respectively.

Fig. 7.

Simulation of cell behavior based on mutually inhibitory positive-feedback loop. (A) Three state model of excitability. Front (F) in red, back (B) in green, and refractory (R) in blue are connected by positive-feedback (arrows) and delayed negative-feedback (bars) loops. (B) Typical responses of the F, B, and R states when the system is triggered. The arrows emphasize the undershoot and overshoot in F and B, respectively. (C) Simulated kymographs generated using a one-dimensional discretized domain resembling the cell perimeter. (Left) shadow wave activity of B, with the dark regions denoting the lowest levels and bright green the overshoot; (Middle), F and B activity; (Right) all three states. The horizontal dashed white line corresponds to the time-course shown in B. (D) Level set simulations modeling the protrusion forces corresponding to the dashed line in C, Middle. (E) Close up of the the time course, left to right, of a single protrusion from D. (F, Left) Ras regulation of PI(3,4,5)P₃ production showing positive feedback. (Right) molecular architecture of the mutually inhibitory Ras-RasGAPs-PI(3,4)P₂ feedback loop. (G) Schematic representation of the opposing spatial and temporal patterns of Ras activity and PI(3,4)P₂ in migrating cells.