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Locally excitable Cdc42 signals steer cells during chemotaxis

Nature Cell Biology volume 18, pages 191–201 (2016)Cite this article

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Abstract

Neutrophils and other amoeboid cells chemotax by steering their front ends towards chemoattractant. Although Ras, Rac, Cdc42 and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, pre-existing local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement on uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, whereas Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.

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Figure 1: Neutrophil chemotaxis controlled by automated photorelease of chemoattractant.
Figure 2: Cdc42 and opposing RhoA activities are steeply polarized at the front.
Figure 3: Endogenous local Cdc42 signal differences between the left- and right-hand sides of the cell front direct turning towards chemoattractant.
Figure 4: Basal Cdc42 activity fluctuations in morphologically unpolarized cells predict the future direction of cell polarization.
Figure 5: Locally pulsatile Cdc42 activity in the absence of actin polymerization argues for the existence of an excitable network.
Figure 6: Cdc42 and RhoA activities polarize before Rac and Ras activities during de novo polarization and induced chemokinetic movement.
Figure 7: Cdc42 and opposing RhoA activities correlate spatiotemporally with local protrusion during cell migration.
Figure 8: Cdc42 antagonizes RhoA activity.

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Acknowledgements

We thank K. Aoki and M. Matsuda for GTPase sensors; A. Hayer, D. Garbett and A. Winans for critical reading of the manuscript and helpful discussions; and the Stanford Shared FACS Facility for cell sorting and the National Institute of General Medical Sciences for funding.

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Author notes

  1. Sean R. Collins

    Present address: Present address: Department of Microbiology and Molecular Genetics, University of California, Davis, California 95616, USA.,

  2. Hee Won Yang and Sean R. Collins: These authors contributed equally to this work.

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  1. Department of Chemical & Systems Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    Hee Won Yang, Sean R. Collins & Tobias Meyer

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Contributions

H.W.Y., S.R.C. and T.M. designed the experiments. H.W.Y. and S.R.C. carried out the experiments and analysed the data. H.W.Y., S.R.C. and T.M. interpreted the data. H.W.Y., S.R.C. and T.M. wrote the paper.

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Correspondence to Hee Won Yang, Sean R. Collins or Tobias Meyer.

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Integrated supplementary information

Supplementary Figure 1 Systematic analysis of cell motility and chemotaxis for control PLB-985 cells and PLB-985 cells stably expressing the indicated GTPase biosensors.

All cells expressed an H2B-mCherry marker which was used for cell tracking. Chemoattractant gradients were generated at time zero by UV uncaging, cells were tracked from frame-to-frame, and statistics of cell movement were calculated37. (a) Scheme for quantifying speed and direction of movement. For b and c: n = 160 (Control), n = 318 (Rac), n = 309 (Cdc42), n = 136 (Ras), and n = 123 (RhoA) cells. (b) Measurement of mean cell speed as function of time relative to chemoattractant gradient generation. (c) Histograms of instantaneous cell direction relative to the chemoattractant gradient for moving cells (an angle of zero indicates perfect directionality). For the number of cells indicated above, the data includes: n = 19743 (Control), n = 42954 (Rac), n = 39045 (Cdc42), n = 13901 (Ras), and n = 15307 (RhoA) frame-to-frame movement steps. (d) Histograms of instantaneous cell direction relative to the chemoattractant gradient from the steeper gradient condition used for all high resolution imaging in this study. The data includes: n = 2056 (Rac), n = 1307 (Cdc42), n = 1801 (Ras), and n = 2798 (RhoA) frame-to-frame movement steps for 59, 49, 80, and 103 independent cells, respectively. (e) Intensity of the Fluo-3 dye inside PLB-985 cells after chemoattractant photorelease. Scale bar is 50 μm. Color bar indicates relative fluorescence unit. (f) Quantitative analysis of timecourse of fluorescence intensity of Fluo-3 as a function of time relative to Nv-fMLF photorelease. Green dotted line marks the time of chemoattractant release. Data were normalized by the initial fluorescence intensity for each cell. Data represent the mean ± s.e.m. of n = 22 cells.

Supplementary Figure 2 Measurement of relative activities of GTPases in migrating cells as a function of distance backwards from the leading edge.

(a) A protrusion/retraction map computed by overlaying and subtracting cell masks from sequential images (left), a color coded map of the distance from the leading edge with associated color bar (middle), and Cdc42 activity (right). Color bar (right) indicates dynamic range of Cdc42 activity. The direction of the fMLF gradient is indicated with a ∗. Scale bar is 10 μm. (b) Computed intracellular gradients of FRET ratio as a function of distance from the leading edge are shown for multiple time points for the individual cells shown in Fig. 2a–d. Each single timepoint curve is shown in gray. The averaged curve over all time points is shown in red. (c) The time-averaged activity curves for each individual cell used in our analysis for Fig. 2e–h (Control (Gradient)) are shown in gray. The curves for the cells depicted in the images in Fig. 2a–d are shown in red. Each curve is normalized by the levels at the front edge (mean of 5 front pixels).

Supplementary Figure 3 Spatial gradients of GTPase activities in the absence of PI3K activation.

(a) Inhibition of the polarization of the PHAkt domain fused to YFP (a biosensor for PIP3) by LY29 (50 μM). Time relative to stimulation is indicated and the direction of the chemoattractant gradient is indicated with a ∗. Scale bar is 10 μm. (b) Relative intensity of PHAkt as a function of distance from the leading edge. Values are normalized to the levels at the front edge. Error bars indicate ± s.e.m. of n = 15 (control) and n = 20 (LY29) averaged traces from timecourses of independent cells. (c) Spatial gradients of GTPase activity in the presence of LY29 (50 μM). Time relative to stimulation is indicated, and the direction of the chemoattractant gradient is indicated with a ∗. Color bars indicate the range of biosensor FRET ratios. Scale bar is 10 μm.

Supplementary Figure 4 Spatial GTPases activity at the front during cell turning and alternate method to assess correlations between asymmetric GTPase signaling and cell turning towards chemoattractant.

(a–c) Rac (a), Ras (b), and RhoA (c) activities in cells responding to a changing gradient generated by photorelease of Nv-fMLF. White line connects the centroid of the cell to the center of the cell front. Yellow arrow shows GTPase activity bias at the cell front. Time relative to stimulation is indicated and the direction of the chemoattractant gradient is indicated with a ∗. Color bars indicate the range of biosensor FRET ratios. Scale bar is 10 μm. (d) Schematic of the correlation analysis between a ‘signaling angle’ and a cell turning angle. Cell centroid positions are used to compute cell movement vectors, and a cell turning angle for each frame. A ‘signaling vector’ is computed as a weighted sum of vectors pointing from the cell centroid to parametrized regions on the cell periphery with greater than average FRET ratio. The signaling angle is computed as the angle between the previous cell movement vector and the signaling vector. (e) Comparison of the temporal cross-correlation analysis between the signaling angle and the turning angle for each GTPase sensor. Negative time offsets indicate that signaling asymmetry precedes turning. Error bars indicate ±s.e.m. of n = 41 (Rac), n = 47 (Cdc42), n = 42 (Ras), and n = 47 (RhoA) cells.

Supplementary Figure 5 Local fluctuations or enrichments of GTPases activity in unpolarized cells.

(a) Schematic of the assay to generate a rapid spatially uniform increase of the chemoattractant fMLF to induce a polarization and chemokinesis migration response. (b) Histogram of the maximum or minimum FRET ratio over the periphery of individual cells. Ratios were computed after smoothing to minimize the effects of imaging noise. Values were normalized by the mean FRET ratio for each cell. The red curves indicate histograms for the observed maximum (left) or minimum (right) FRET ratios. The blue curves show control values computed for the same cells in which the pixel positions in the cell periphery were randomly permuted before smoothing and detection of the maximum and minimum signals. The control curves are intended to simulate the distributions expected for uniform signaling activity with similar levels of imaging noise. n = 146 (Rac), n = 137 (Cdc42), n = 168 (Ras), n = 162 (RhoA) cells.

Supplementary Figure 6 Cdc42 activity in cells having two competitive protrusions and prediction of future direction.

(a) Rose plots showing distributions of the angle between the minimum local GTPase activity before stimulation and the subsequent migration direction. ∗ indicates p-value is less than 0.001. P-values were calculated using the sign test applied to the cosine of the angles. n = 68 (Rac), n = 69 (Cdc42), n = 63 (Ras), and n = 65 (RhoA) cells. (b) Two examples of cells which initially generated two active protrusions after chemoattractant stimulation are shown. Cdc42 activity is indicated by the color scale. White arrows indicate the sites of pre-existing Cdc42 activity at cell periphery. Pink arrows mark the presence of two protruding fronts in the same cell. Scale bar is 10 μm.

Supplementary Figure 7 Wave like behavior Cdc42 activity in the absence of PI3K activation.

(a) Examples of autonomous waves of Cdc42 activity in the presence of LY29. Cells were treated with LatA (1 μM) and LY (50 μM). White dots mark the location of maximum Cdc42 activity. Cdc42 activity is indicated by the color scale. The images were taken every 2 s for 120 s. Scale bar is 10 μm. (b) Kymograph of Cdc42 activity for a cell 1 treated with both LatA (1 μM) and LY29 (50 μM). The Cdc42 activity was averaged over the horizontal (x-axis) direction to get a one dimensional profile for each timepoint. For this analysis, images were taken at 2 s intervals. Cdc42 activity is indicated by the color scale. (c) Quantitative measurements of local Cdc42 activity in LatA (1 μM) and LY29 (50 μM)-treated cells. Shown are temporal traces for selected 5 μm square regions within individual cells. For this analysis, images were taken every 5 s for 420 s.

Supplementary Figure 8 Timecourses of directed speed during de novo polarization, effect of ZCL278 on GTPase activities, efficiency and specificity of Cdc42 and RhoA knock down, and effect of ZCL278 on pMLC.

(a) Directed speed was measured as the rate of movement of the cell centroid in the direction of eventual cell polarization. Green dotted line marks the time of chemoattractant release. Error bars indicate ±s.e.m. of n = 113 cells. (b) Mean activity of GTPases in PLB-985 cells treated with different doses of ZCL278. Values were normalized by the control condition. Error bars indicate ±s.e.m. of n = 163 (Ras 0 μM), n = 170 (Ras 10 μM), n = 160 (Ras 50 μM), n = 209 (Ras 100 μM), n = 136 (Rac 0 μM), n = 159 (Rac 10 μM), n = 173 (Rac 50 μM), n = 130 (Rac 100 μM), n = 141 (Cdc42 0 μM), n = 179 (Cdc42 10 μM), n = 144 (Cdc42 50 μMl), and n = 162 (Cdc42 100 μM).∗ indicates p-value is less than 0.01. P-values were calculated using the rank sum test. (c) Western blots of whole cell lysates were performed to assess siRNA-mediated gene knockdown efficiency of Cdc42 and RhoA. Blotting for GAPDH is also shown to demonstrate equal loading. (d) Histogram of pMLC intensities measured in individual cells by immunofluorescence for cells treated with ZCL278 (50 μM) for 30 min. n = 50892 (DMSO), and n = 57584 (ZCL278) cells.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1412 kb)

Rac activity during neutrophil-like PLB-985 cell chemotaxis.

These movie files contains 360 s imaging sequences with 5 s intervals between frames, corresponding to the examples shown in Fig. 2a. Time relative to stimulation is indicated and direction of the chemoattractant gradient and sequential UV photorelease are indicated with UV. (AVI 2172 kb)

Cdc42 activity during neutrophil-like PLB-985 cell chemotaxis.

These movie files contains 360 s imaging sequences with 5 s intervals between frames, corresponding to the examples shown in Fig. 2b. Time relative to stimulation is indicated and direction of the chemoattractant gradient and sequential UV photorelease are indicated with UV. (AVI 4686 kb)

Ras activity during neutrophil-like PLB-985 cell chemotaxis.

These movie files contains 360 s imaging sequences with 5 s intervals between frames, corresponding to the examples shown in Fig. 2c. Time relative to stimulation is indicated and direction of the chemoattractant gradient and sequential UV photorelease are indicated with UV. (AVI 2190 kb)

RhoA activity during neutrophil-like PLB-985 cell chemotaxis.

These movie files contains 360 s imaging sequences with 5 s intervals between frames, corresponding to the examples shown in Fig. 2d. Time relative to stimulation is indicated and direction of the chemoattractant gradient and sequential UV photorelease are indicated with UV. (AVI 2346 kb)

Cdc42 activity bias at the cell front predicts direction of cell turning.

This movie file contains a 380 s imaging sequence with 5 s intervals between frames, corresponding to the example shown in Fig. 3b. Time relative to stimulation is indicated and direction of the chemoattractant gradient and sequential UV photorelease are indicated with UV. (AVI 2356 kb)

Prepolarized Cdc42 predicts cell direction after uniform fMLF stimulation.

This movie file contains a 50 s imaging sequence with 2 s intervals between frames, corresponding to the example shown in Fig. 4a. Time relative to stimulation is indicated. (AVI 1632 kb)

Locally pulsatile activation and propagation of Cdc42 in the absence of actin polymerization.

The original video was incorrect; a new file was uploaded on 5 January 2016. This movie file contains a 120 s imaging sequence with 2 s intervals between frames, corresponding to the example shown in Fig. 5a. (AVI 4198 kb)

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Yang, H., Collins, S. & Meyer, T. Locally excitable Cdc42 signals steer cells during chemotaxis. Nat Cell Biol 18, 191–201 (2016). https://doi.org/10.1038/ncb3292

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