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Altering the threshold of an excitable signal transduction network changes cell migratory modes

Abstract

The diverse migratory modes displayed by different cell types are generally believed to be idiosyncratic. Here we show that the migratory behaviour of Dictyostelium was switched from amoeboid to keratocyte-like and oscillatory modes by synthetically decreasing phosphatidylinositol-4,5-bisphosphate levels or increasing Ras/Rap-related activities. The perturbations at these key nodes of an excitable signal transduction network initiated a causal chain of events: the threshold for network activation was lowered, the speed and range of propagating waves of signal transduction activity increased, actin-driven cellular protrusions expanded and, consequently, the cell migratory mode transitions ensued. Conversely, innately keratocyte-like and oscillatory cells were promptly converted to amoeboid by inhibition of Ras effectors with restoration of directed migration. We use computational analysis to explain how thresholds control cell migration and discuss the architecture of the signal transduction network that gives rise to excitability.

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Figure 1: Acute clamping of PtdIns(4,5)P2 at lowered levels triggers cell migratory mode transitions.
Figure 2: Actin profiles show distinct spatiotemporal patterns in amoeboid, fan-shaped and oscillatory cells.
Figure 3: A signal transduction network mediates the transitions of cell migratory modes.
Figure 4: Properties of the STEN waves are altered independent of cytoskeleton.
Figure 5: The threshold for STEN activation is lowered.
Figure 6: Simulations of the excitable network with varying thresholds successfully capture various cell migratory modes.
Figure 7: Innately fan-shaped or oscillatory cells can be converted to amoeboid mode.
Figure 8: Propagating waves of STEN activity control cell migratory modes.

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Acknowledgements

The authors would like to thank C. Janetopoulos (University of the Sciences) for discussions on the roles of PtdIns(4,5)P2. We thank all members of the Devreotes, Iglesias and Inoue laboratories as well as members of the D. Robinson and M. Iijima laboratories (Johns Hopkins University) for helpful suggestions. We thank the R. Kay laboratory (MRC Laboratory of Molecular Biology, UK) for providing pikI cells, and the M. Ueda laboratory (Osaka University, Japan) for - cells. We thank the DictyBase stock centre for providing rasC/rasG and amiB cells. We thank the V. Bankaitis laboratory (Texas A&M Health Sciences Center) for providing constructs of nodulin. This work was supported by NIH grant R35 GM118177 (to P.N.D.), AFOSR MURI FA95501610052, DARPA HR0011-16-C-0139, and NIH Grant S10 OD016374 (to S. Kuo of the JHU Microscope Facility).

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Authors and Affiliations

Authors

Contributions

Y.M. performed the majority of experiments, S.B. conducted computational simulations, and M.E. performed chemotaxis assays and experiments regarding amiB cells. All authors analysed the data and wrote the manuscript. P.N.D. supervised the study.

Corresponding author

Correspondence to Peter N. Devreotes.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Multiple acute perturbations altered cell migratory profile.

(a) Ratios of membrane to cytosol intensity of PIP2 biosensors following rapamycin treatment at time 0 (mean ± s.d., n = 21 cells for each biosensor). “Nodulin” refers to the Nlj6-like nodulin domain of the Arabidopsis Sec14-nodulin protein AtSfh141, which has been shown to specifically bind PIP2. NodulinQ534P is a mutant with defects in self-oligomerization and thus diminished affinity for PIP2. (bf) Centroid tracks of random cell migration before and after rapamycin addition. Cells expressed myr-FKBP-FKBP as well as mCherry-FRB-Inp54pD281A (b), mCherry-FRB-Inp54p (c), mCherry-FRB-RasCQ62L(ΔCAAX) (d), mCherry-FRB-GbpD(GEF) (e), and mCherry-FRB-Rap1G12V (ΔCAAX) (f). Rapamycin was added at time 0. Blue box, −10 to 0 min time window; Red box, 10 to 20 min time window. Tracks are reset to the same origin and n = 50 cells for each, mean ± s.d.

Supplementary Figure 2 Rapidly increasing RasC activity led to cell migratory modes changes.

(a,b) Still images of cells expressing mCherry-FRB-RasCQ62L(ΔCAAX) and myr-FKBP-FKBP before (a) and after (b) rapamycin addition in the same field. Left, confocal image of mCherry-FRB-RasCQ62L(ΔCAAX). Right, bright-field image. Red arrows point to fans or the spreading phase of oscillators. (c) Percentage (mean ± s.d., n = 3 experiments, >500 cells in each experiment, P = 0.015, two-tailed t-test) of fan-shaped plus oscillatory cells before and after rapamycin addition (recruiting RasCQ62L(ΔCAAX)). Blue, 10 to 0 min before rapamycin addition; Red, 10 to 20 min after rapamycin addition. (d) Time-lapse phase-contrast images showing oscillations in induced RasCQ62L expressing cells. Scale bar represents 10 μm. (e) Percentage (mean ± s.d., n = 4 experiments, >100 cells in each experiment) of fan-shaped plus oscillatory cells before (blue) and after (red) induction of RasCQ62L. Doxycycline was added 12 hours prior, and cells were scored over a one-hour time window before and after induction.

Supplementary Figure 3 Rapidly increasing Rap1-related activity led to cell migratory modes changes.

(a,b) Still Phase-contrast images of cells expressing mCherry-FRB-GbpD(GEF) (a) or mCherry-FRB-Rap1G12V (ΔCAAX) (b), in addition to myr-FKBP-FKBP, before and after rapamycin addition in the same field. Red arrows point to fans or the spreading phase of oscillators, which appeared phase-dark under Phase-contrast microscopy. (c,d) Percentage (mean ± s.d., n = 3 experiments, >300 cells in each experiments, P = 0.019 in c and 0.04 in d, two-tailed t-test) of fan-shaped plus oscillatory cells before and after rapamycin addition, recruiting GbpD(GEF) in c and Rap1G12V (ΔCAAX) in d. Blue, 10 to 0 min before rapamycin addition; Red, 10 to 20 min after rapamycin addition.

Supplementary Figure 4 Automatic classification of migratory modes.

(a) Algorithm for classification of cells begins with segmentation (Step 1) which is used to determine how cell area changes over time. Cells whose coefficient of variation (COV) of area is higher than a threshold (COV th = 18) are deemed to be oscillatory (Step 2). Otherwise, they are classified into fan-shaped or amoeboid based on the direction of motion relative to cell polarity angle (Step 3). (b) Histograms of coefficient of variation of area (COV) for WT amoeboid cells (blue) and the population mixture after rapamycin addition (red). Data was fit by two Gaussians of varying means (8.7, 89% and 16.1, 11% for amoeba; 10.9, 27% and 16.1, 73% for rapamycin-treated cells). The COVth threshold used in Step 2 was chosen based on this data. The fitted curves are shown by the dotted, shaded regions. (c) Histograms representing the mean value of O (from Step 3 in a) in cells with low variation (less than 30°) of the angle. The rapamycin-treated cells have a shift in the peak from around 130° to 100° depicting the presence of fan-shaped cells in the population. Dotted, shaded regions represent Gaussian fits (126° and 103°, respectively). (d) Comparison of the classification of cells into different migratory modes using automatic (right) versus manual inspection (left). Blue, green and red correspond to amoeboid, fan-shaped and oscillatory cells, respectively.

Supplementary Figure 5 Profiles of recruited mCherry-FRB-Inp54p as membrane control.

(a,c) Overlays color-coded at 2 sec intervals, corresponding to Fig. 2d, f, respectively. Scale bars represent 10 μm. (b,dg) Kymographs of membrane mCherry intensity, corresponding to Figs 2e, g, 3a, b, and c, respectively.

Supplementary Figure 6 PKBs negatively regulate Ras and PI3K activities.

(a) Confocal images of RBD-GFP in DMSO control cells (left) and cells treated with LY294002 plus PP242 for 60 min (right). Scale bars represent 20 μm. (be), Distributions of different sizes of RBD-GFP activity in cells treated with DMSO (blue) and both inhibitors (red) for 30, 60, 90, and 120 min, respectively (>4500 cells from 3 independent experiments). (f) Fractions of cells with large RBD size (>10% of cell perimeter) after treatment of DMSO (blue) and both inhibitors (red) for different amount of time (mean ± s.d., n = 3 experiments, >1500 cells in each experiment, P = 0.028, 0.0061, P = 0.00068, 0.0002, two-tailed t-test). 5 μM LatrunculinA, 50 μM LY294002, and 20 μM PP242 were used. (g) Illustration of recruiting PKBA by the inducible dimerization system. (h) Kymographs of cortical PHcrac-YFP in LatrunculinA-treated cells before and after recruiting PKBA. The black dashed lines indicate rapamycin addition. Solid vertical lines on the left of each kymograph represent 20 μm.

Supplementary Figure 7 Threshold for STEN activation was lowered by clamping activated RasC activities.

Mean normalized responses of PHcrac-YFP to doses of cAMP before (blue) and after (red) recruiting RasCQ62L(ΔCAAX). n = 23 cells for each condition and error bars indicate standard error, P = 0.0017, two-tailed t-test.

Supplementary Figure 8 Model of the excitable signal transduction network.

(a) The excitable network with the polarization module. (b) Phase-plane plot of the excitable system, with the activator (F, green) and inhibitor (R, red). These plots assume that B is in quasi-steady, state and that P is constant. The black star denotes the equilibrium state of the system, while the black arrows describe the system evolution at different points in the phase plane. (c) Maximum (dashed line) and average (solid line) activities for six sample simulated cells with randomly generated thresholds. The grey line at 50 time units depicts the simulated addition of rapamycin, after which the threshold is gradually lowered. Based on the changes in the activity plots, the response was categorized as amoeboid (blue), fan-shaped (green) or oscillatory (red).

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Time-lapse confocal videos of mCherry in cells before and after recruiting Inp54pD281A (left) and Inp54p (right), corresponding to Fig. 1.

Cells on the left expressed myr-FKBP-FKBP and mCherry-FRB-Inp54pD281A, and cells on the right expressed myr-FKBP-FKBP and mCherry-FRB-Inp54p. Rapamycin was added at time 10 min. Scale bars represent 20 μm. Images were acquired every 12 sec and the videos are shown at 25 frame/sec. (MOV 7452 kb)

Time-lapse phase video of cells before and after recruiting Inp54p, corresponding to Fig. 1c.

Cells expressed myr-FKBP-FKBP and mCherry-FRB-Inp54p. Rapamycin was added at time 6 min. Examples of amoeboid-to-fan-shaped and amoeboid-to-oscillatory transitions were outlined, with blue, green, and red representing amoeboid, fan-shaped, and oscillatory, respectively. Scale bar represents 20 μm. Images were acquired every 12 sec and the video is shown at 20 frame/sec. (MOV 20038 kb)

Time-lapse confocal videos of LimE-YFP in cells before and after recruiting Inp54pD281A (left) and Inp54p (right), corresponding to Fig. 2a.

The cells were aligned to the same centroid. Three-dimensional kymographs are shown below each video. Rapamycin was added at time 10 min. Scale bars represent 5 μm. Images were acquired every 12 sec and the videos are shown at 15 frame/sec. (MOV 1521 kb)

Time-lapse confocal videos of LimE-YFP in an amoeboid (top left), fan-shaped (right), and oscillatory (bottom left) cell, corresponding to Fig. 2b–h. Scale bars represent 5 μm.

Images were acquired every 2 sec and the videos are shown at 15 frame/sec. (MOV 1184 kb)

Time-lapse confocal videos of LimE-YFP in cells before and after recruiting RasCQ62L(ΔCAAX) (left), GbpD(GEF) (middle), and Rap1G12V (ΔCAAX) (right).

Rapamycin was added at time 5 min. Scale bars represent 20 μm. Images were acquired every 15 sec and the videos are shown at 15 frame/sec. (MOV 6117 kb)

Time-lapse confocal videos of RBD-YFP (left), PHcrac-YFP (middle), and PTEN-GFP (right) in oscillatory cells, corresponding to Fig. 3a–c.

Scale bars represent 10 μm. Images were acquired every 5 sec and the videos are shown at 15 frame/sec. (MOV 1008 kb)

Time-lapse confocal videos of LimE-YFP in cells coexpressing myr-FKBP-FKBP and mCherry-FRB-Inp54p, where 5 μM rapamycin and 1mM cycloheximide were added at time 4:45 min.

Scale bars represent 20 μm. Images were acquired every 15 sec and the videos are shown at 20 frame/sec. (MOV 1587 kb)

Time-lapse TIRF videos of PHcrac-YFP in an amoeboid (left), fan-shaped (middle), and oscillatory (right) cell. These videos correspond to Fig. 4a.

Cell outlines were imposed based on videos of mCherry-FRB-Inp54p concurrently. Scale bar represents 5 μm. Images were acquired every 15 sec and the videos are shown at 6 frame/sec. (MOV 679 kb)

Time-lapse TIRF videos of LimE-YFP in an amoeboid (top left), fan-shaped (right), and oscillatory (bottom left) cell.

These videos correspond to Fig. 4b. Cell outlines were imposed based on videos of mCherry-FRB-Inp54p concurrently. Scale bar represents 5 μm. Images were acquired every 10 sec and the videos are shown at 8 frame/sec. (MOV 1142 kb)

Time-lapse confocal videos, focused on the bottom plane, of LimE-RFP (left) and PHcrac-YFP (right) in an oscillatory cell.

These videos correspond to Fig. 4c. Scale bar represents 5 μm. Images were acquired every 2 sec and the videos are shown at 16 frame/sec. (MOV 3135 kb)

Time-lapse confocal videos of mCherry-FRB-Inp54p (left) and PHcrac-YFP (right) in cells treated with 5 μM LatrunculinA before and after addition of rapamycin at time 10 min, corresponding to Fig. 4e, f.

Scale bars represent 20 μm. Images were acquired every 15 sec and the videos are shown at 20 frame/sec. (MOV 3202 kb)

Two-dimensional simulations on a periodic grid showing waves of activity propagating outward and collapsing corresponding to Fig. 6a–c, with the green and red denoting the activator and inhibitor, respectively.

The bottom panel shows how the slope of the R-nullcline is gradually lowered thereby decreasing the threshold for activation. (MOV 8139 kb)

Level set simulations of the three different migratory modes corresponding to Fig. 6d–f.

The video also shows the trajectory of the equilibrium state on the phase plane, for the three different thresholds, where the particular state shown reflects the behavior of one of the points on the cell membrane, indicated by the moving white bar on the kymograph. Scale bars represent 10 μm. (MOV 8319 kb)

Level set simulations of the transitions between the amoeboid to fan-shaped and the fan-shaped to oscillatory states corresponding to Fig. 6g.

The transitions are simulated with a constant threshold up to the black line, after which the threshold is gradually lowered, with the red line indicating the progress in time. The transition from amoeboid to fan-shaped is demarcated by the shift in activity from a fluctuating to a constant maxima, while the transition from fan-shaped to oscillatory occurs when the maxima shifts back to a periodic cycle. Scale bars represent 10 μm. (MOV 8142 kb)

Time-lapse phase-contrast video of pikI cells before (left) and after (right) 40 μM LY294002 treatment, corresponding to Fig. 7a, c.

Scale bar represents 10 μm. Images were acquired every 12 sec and the video is shown at 7 frame/sec. (MOV 3630 kb)

Time-lapse phase-contrast video of amiB cells in the same field before and after 30 μM LY294002 treatment, corresponding to Fig. 7d–f.

Scale bar represents 50 μm. Images were acquired every minute and the video is shown at 7 frame/sec. (MOV 5177 kb)

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Miao, Y., Bhattacharya, S., Edwards, M. et al. Altering the threshold of an excitable signal transduction network changes cell migratory modes. Nat Cell Biol 19, 329–340 (2017). https://doi.org/10.1038/ncb3495

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