A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration

Abstract

Ca2+ signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca2+-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca2+ pulses, local depletion of Ca2+ in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca2+ influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca2+ pump rates in the front relative to the back of the plasma membrane enable effective local Ca2+ signalling by locally decreasing basal Ca2+. Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca2+ control system with polarized Ca2+ signalling proteins and second messengers to synergistically promote directed cell migration.

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Figure 1: RTK signalling is restricted to the front of migrating leader cells.
Figure 2: SOC influx controls cell migration by regulating cell–matrix adhesion in the front of migrating cells.
Figure 3: SOC increases migration speed when cell–matrix adhesion is weak, but slows down migration when adhesion is strong.
Figure 4: STIM1 is enriched in the front of migrating cells.
Figure 5: STIM1 is locally activated in the front of migrating cells.
Figure 6: Polarized PM Ca2+ pump activity keeps cytosolic Ca2+ low in the front.
Figure 7: Higher Ca2+ pump activity in the front when compared with the back generates a gradient of basal [Ca2+] in migrating cells.
Figure 8: A PLC-induced gradient of DAG controls cell motility and directionality.

References

  1. 1

    Vorotnikov, A. V. Chemotaxis: movement, direction, control. Biochem. Mosc. 76, 1528–1555 (2011).

    Article  CAS  Google Scholar 

  2. 2

    Li, S., Huang, N. F. & Hsu, S. Mechanotransduction in endothelial cell migration. J. Cell. Biochem. 96, 1110–1126 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. 3

    Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445–457 (2009).

    Article  CAS  PubMed  Google Scholar 

  4. 4

    Vitorino, P., Hammer, M., Kim, J. & Meyer, T. A steering model of endothelial sheet migration recapitulates monolayer integrity and directed collective migration. Mol. Cell. Biol. 31, 342–350 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. 5

    Giannone, G. et al. Periodic lamellipodial contractions correlate with rearward actin waves. Cell 116, 431–443 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. 6

    Giannone, G. et al. Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561–575 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Burnette, D. T. et al. A role for actin arcs in the leading-edge advance of migrating cells. Nat. Cell Biol. 13, 371–381 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Machacek, M. et al. Coordination of Rho GTPase activities during cell protrusion. Nature 461, 99–103 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Tkachenko, E. et al. Protein kinase A governs a RhoA-RhoGDI protrusion-retraction pacemaker in migrating cells. Nat. Cell. Biol. 13, 660–667 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Evans, J. H. & Falke, J. J. Ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages. Proc. Natl Acad. Sci. USA 104, 16176–16181 (2007).

    Article  PubMed  Google Scholar 

  11. 11

    Wei, C. et al. Calcium flickers steer cell migration. Nature 457, 901–905 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. 12

    Tsai, F-C. & Meyer, T. Ca2+ pulses control local cycles of lamellipodia retraction and adhesion along the front of migrating cells. Curr. Biol. 22, 837–842 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Franco, S. J. et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat. Cell Biol. 6, 977–983 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. 14

    Brundage, R. A., Fogarty, K. E., Tuft, R. A. & Fay, F. S. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science 254, 703–706 (1991).

    Article  CAS  PubMed  Google Scholar 

  15. 15

    Gilbert, S. H., Perry, K. & Fay, F. S. Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils. J. Cell. Biol. 127, 489–503 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. 16

    Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. 17

    Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Brandman, O., Liou, J., Park, W. S. & Meyer, T. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels. Cell 131, 1327–1339 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Vitorino, P. & Meyer, T. Modular control of endothelial sheet migration. Genes Dev. 22, 3268–3281 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Gozgit, J. M. et al. Ponatinib (AP24534), a multitargeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models. Mol. Cancer Ther. 11, 690–699 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. 21

    Lamalice, L., Boeuf, F. L. & Huot, J. Endothelial cell migration during angiogenesis. Circ. Res. 100, 782–794 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. 22

    Chen, T-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Zitt, C. et al. Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J. Biol. Chem. 279, 12427–12437 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. 24

    Kasturi, R., Vasulka, C. & Johnson, J. D. Ca2+, caldesmon, and myosin light chain kinase exchange with calmodulin. J. Biol. Chem. 268, 7958–7964 (1993).

    CAS  PubMed  Google Scholar 

  25. 25

    Chen, Y-F. et al. Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Proc. Natl Acad. Sci. 108, 15225–15230 (2011).

    Article  PubMed  Google Scholar 

  26. 26

    Yang, S., Zhang, J. J. & Huang, X-Y. Orai1 and STIM1 are critical for breast tumor cell migration and metastasis. Cancer Cell 15, 124–134 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. 27

    Potier, M. et al. Evidence for STIM1- and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: role in proliferation and migration. FASEB J. 23, 2425–2437 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Bisaillon, J. M. et al. Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration. Am. J. Physiol., Cell Physiol. 298, C993–C1005 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Schafer, C., Rymarczyk, G., Ding, L., Kirber, M. T. & Bolotina, V. M. Role of molecular determinants of store-operated Ca2+ entry (Orai1, phospholipase A2 group 6 and STIM1) in focal adhesion formation and cell migration. J. Biol. Chem. 287, 40745–40757 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Ridley, A. J. et al. Cell migration: integrating signals from front to back. Science 302, 1704–1709 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. 31

    Grigoriev, I. et al. STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER. Curr. Biol. 18, 177–182 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Honnappa, S. et al. An EB1-binding motif acts as a microtubule tip localization signal. Cell 138, 366–376 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. 33

    Abell, E., Ahrends, R., Bandara, S., Park, B. O. & Teruel, M. N. Parallel adaptive feedback enhances reliability of the Ca2+ signaling system. Proc. Natl Acad. Sci. USA 108, 14485–14490 (2011).

    Article  PubMed  Google Scholar 

  34. 34

    Bandara, S., Malmersjö, S. & Meyer, T. Regulators of calcium homeostasis identified by inference of kinetic model parameters from live single cells perturbed by siRNA. Sci. Signal. 6, ra56 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Thastrup, O., Cullen, P. J., Drøbak, B. K., Hanley, M. R. & Dawson, A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc. Natl Acad. Sci. USA 87, 2466–2470 (1990).

    Article  CAS  PubMed  Google Scholar 

  36. 36

    Smallwood, J. I., Gügi, B. & Rasmussen, H. Regulation of erythrocyte Ca2+ pump activity by protein kinase C. J. Biol. Chem. 263, 2195–2202 (1988).

    CAS  PubMed  Google Scholar 

  37. 37

    Pérez-Gordones, M. C., Lugo, M. R., Winkler, M., Cervino, V. & Benaim, G. Diacylglycerol regulates the plasma membrane calcium pump from human erythrocytes by direct interaction. Arch. Biochem. Biophys. 489, 55–61 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. 38

    James, P. et al. Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain. J. Biol. Chem. 264, 8289–8296 (1989).

    CAS  PubMed  Google Scholar 

  39. 39

    Chicka, M. C. & Strehler, E. E. Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting. J. Biol. Chem. 278, 18464–18470 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. 40

    De Chaffoy de Courcelles, D. C., Roevens, P. & Van Belle, H. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J. Biol. Chem. 260, 15762–15770 (1985).

    CAS  PubMed  Google Scholar 

  41. 41

    Larsson, C. Protein kinase C and the regulation of the actin cytoskeleton. Cell. Signal. 18, 276–284 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. 42

    Smith, G. & MacQuaide, N. Cytoplasmic versus Intra-SR: the Battle of the Ca2+ diffusion coefficients in cardiac muscle. Biophys. J. 95, 1005–1006 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Turner, N. & Grose, R. Fibroblast growth factor signalling: from development to cancer. Nat. Rev. Cancer 10, 116–129 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. 44

    Arisz, S. A., Testerink, C. & Munnik, T. Plant PA signaling via diacylglycerol kinase. Biochim. Biophys. Acta 1791, 869–875 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. 45

    Almena, M. & Mérida, I. Shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling. Trends Biochem. Sci. 36, 593–603 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. 46

    Carrasco, S. & Merida, I. Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes. Mol. Biol. Cell 15, 2932–2942 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Spitaler, M., Emslie, E., Wood, C. D. & Cantrell, D. Diacylglycerol and protein kinase D localization during T lymphocyte activation. Immunity 24, 535–546 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. 48

    Quann, E. J., Merino, E., Furuta, T. & Huse, M. Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells. Nat. Immunol. 10, 627–635 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. 49

    May, R. C. & Machesky, L. M. Phagocytosis and the actin cytoskeleton. J. Cell. Sci. 114, 1061–1077 (2001).

    CAS  PubMed  Google Scholar 

  50. 50

    Stephens, L., Ellson, C. & Hawkins, P. Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr. Opin. Cell Biol. 14, 203–213 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. 51

    Scott, C. C. et al. Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J. Cell. Biol. 169, 139–149 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Botelho, R. J. et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell. Biol. 151, 1353–1368 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    De Jong, A. P. H. & Verhage, M. Presynaptic signal transduction pathways that modulate synaptic transmission. Curr. Opin. Neurobiol. 19, 245–253 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. 54

    Kim, K., Yang, J. & Kim, E. Diacylglycerol kinases in the regulation of dendritic spines. J. Neurochem. 112, 577–587 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. 55

    Carrasco, S. & Mérida, I. Diacylglycerol, when simplicity becomes complex. Trends Biochem. Sci. 32, 27–36 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. 56

    Rosse, C. et al. PKC and the control of localized signal dynamics. Nat. Rev. Mol. Cell Biol. 11, 103–112 (2010).

    Article  CAS  PubMed  Google Scholar 

  57. 57

    Ludowyke, R. I. et al. Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C η II and coincides with the onset of stimulated degranulation of RBL-2H3 mast cells. J. Immunol. 177, 1492–1499 (2006).

    Article  CAS  PubMed  Google Scholar 

  58. 58

    Woods, A. & Couchman, J. R. Protein kinase C involvement in focal adhesion formation. J. Cell. Sci. 101, 277–290 (1992).

    CAS  PubMed  Google Scholar 

  59. 59

    Hillman, R. T. et al. Neuropilins are positive regulators of Hedgehog signal transduction. Genes Dev. 25, 2333–2346 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Stauffer, T. P., Ahn, S. & Meyer, T. Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr. Biol. 8, 343–346 (1998).

    Article  CAS  PubMed  Google Scholar 

  61. 61

    Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J. & Meyer, T. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat. Methods 2, 415–418 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Liou, J., Fivaz, M., Inoue, T. & Meyer, T. Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion. Proc. Natl Acad. Sci. USA 104, 9301–9306 (2007).

    Article  CAS  PubMed  Google Scholar 

  63. 63

    Groden, D. L., Guan, Z. & Stokes, B. T. Determination of Fura-2 dissociation constants following adjustment of the apparent Ca-EGTA association constant for temperature and ionic strength. Cell Calcium 12, 279–287 (1991).

    Article  CAS  PubMed  Google Scholar 

  64. 64

    Petr, M. J. & Wurster, R. D. Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG. Cell Calcium 21, 233–240 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. 65

    Fluorescent Calcium Indicators - 154.pdf. http://probes.invitrogen.com/media/publications/154.pdf

  66. 66

    Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  PubMed  Google Scholar 

  67. 67

    Huang, A. J. et al. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J. Cell. Biol. 120, 1371–1380 (1993).

    Article  CAS  PubMed  Google Scholar 

  68. 68

    Ikeda, M. et al. Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells. J. Cell. Biochem. 63, 23–36 (1996).

    Article  CAS  PubMed  Google Scholar 

  69. 69

    Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using μ Manager. Curr. Protoc. Mol. Biol. 92 (2010) 14.20.1–14.20.17

  70. 70

    Otsu, N. A Threshold Selection Method from Gray-Level Histograms. IEEE Trans. Syst., Man Cybernet. 9, 62–66 (1979).

    Article  Google Scholar 

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Acknowledgements

We thank S. R. Collins, M. Galic and R. Wollman for technical support and discussions, S. Bandara for the modified T1ER construct, N. Borghi for the paxillin construct, X. Ge for the CD4 construct, C. J. Lin for H1299 cells, E. E. Strehler for the PMCA constructs and A. Winans for critical reading of the manuscript. The research was supported by a Stanford Graduate Fellowship (F-C.T.) and the NIGMS (T.M.)

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Contributions

F-C.T. conceived, designed and carried out experiments, analysed the data and wrote a draft of the manuscript. A.S. developed the DAG sensor and helped with the DAG-related experiments and western blotting. H.W.Y. repeated and validated experiments with the lipid sensors, and compared gradients in leader and follower cells. A.H. helped generate the paxillin constructs, prepared cells stably expressing reference membrane markers and helped write the manuscript. S.C. developed the ER–PM and ER-membrane markers. S.M. developed the membrane-targeted version of GCaMP6s and helped with ruboxistaurin experiments. T.M. conceived the project together with F-C.T., and helped interpret the data and write the manuscript.

Corresponding authors

Correspondence to Feng-Chiao Tsai or Tobias Meyer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Local Ca2+ pulses in the front of migrating cells are generated via polarized receptor tyrosine kinase (RTK) signaling.

(a) Quantification of the differential signal intensities of phosphotyrosine between the front and the back (front back) of migrating cells (n = 107, 105, 115, 110 and 107 cells for SFM, follower cells, and Ponatinib 0 nM, 25 nM and 100 nM, respectively). SFM: The migration assay was performed in serum-free medium. FGF: The migration assay was performed in the presence of fibroblast growth factor 20 ng/mL. Bars denote mean ± SEM. (b) Negative control for Fig. 1c–f. The distribution of mCD4-YFP did not show a front-back gradient when normalized by mCD4-CFP both in leader and follower cells. (n = 64 and 212 cells for leader and follower cells, respectively). (c) The PI(4,5)P2 sensorPLCδ-PH-YFP did not show a significant front-back gradient in migrating cells (n = 25 and 81 cells for leader and follower cells, respectively). (d) Localization gradients quantified as ratio between front and back localization. Bars denote mean ± SEM (for leader and follower cells, n = 64 and 212 cells for CD4, 24 and 42 cells for Akt–PH, 25 and 81 cells for PLCδ-PH and 28 and 62 cells for PKC-C1). (e,f) [Ca2+] levels and pulse activities increased after adding 10% fetal bovine serum and decreased after after addition of the pan-RTK inhibitor Ponatinib 100 nM. Individual frames of a time-lapse sequence are shown. (g) Calibration of cytosolic [Ca2+] using Fura-2, as described in Methods. FR denotes (F – Fmin)/(Fmax-F), where F is the Fura 340/380 ratio, Fmin is the ratio at zero [Ca2+]and Fmax is the ratio at saturating [Ca2+](39 μM). (h) Similar experiment as shown in Fig. 1h and Supplementary Figure 1e,f, but showing the effect of Ponatinib inhibition on local Ca2+ pulses in the front of FGF rather than of serum stimulated cells (middle). Ponatinib (100 nM) reversed the effect of FGF stimulation (n = 25 cells). FGF could not trigger local Ca2+ pulses when cells were pretreated with Ponatinib (100 nM for 6 minutes) (right) (n = 31 cells). The amplitude of Ca2+ fluctuation was normalized using average cell wide basal cytosolic levels (0.3 R.U. means the fluctuation was 30% of the average cytosolic [Ca2+] level). Bars denote mean ± SEM. Student t test was used for Supplementary Figure 1a,h.

Supplementary Figure 2 Store-operated Ca2+ (SOC) influx controls cell migration by regulating cell–matrix adhesion in the front of migrating cells.

(a) Knockdown efficiency of siPMCA and siSTIM1 shown by Western blot. (b) siSTIM1, siSTIM2, siORAIl1 and siORAI2 all accelerated sheet migration of HUVEC. Bars of siSTIM1, siSTIM2 and control siRNA are also shown in Fig. 3a. Student t test was used to compare control siRNA with siSTIM1, siSTIM2, siORAI1 or siORAI2. (p = 0.0199, 0.1587, 0.0506 and 0.0610, respectively.) (c) Over-expression of YFP–STIM1 rescued the effect of STIM1 knockdown on migration speed in HUVEC cells. (d) The SOC inhibitor BTP2 rescued the inhibitory effect of STIM1 overexpression on cell migration. (e)Ca2+ buffering by Fura-2 increased the speed HUVEC migration. The control compound Mag Fura-2 had no significant effect. Bars in (b) through (e) are mean ± SEM (n = 4 experiments for each group). (f) Example showing that BTP2 alters local Ca2+ signals in the front of migrating cells. The right pseudocolor graph shows time-lapse changes (x-axis) of local Ca2+ signals along the leading edge (y-axis) of the cell shown in the left panel. Both basal Ca2+ levels and the amplitudes of Ca2+ pulses decreased after BTP2 treatment. [Ca2+] was normalized using average cytosolic levels before BTP2 treatment. (g) Quantification of the amplitudes of Ca2+ signals relative to the basal level before BTP2 treatment. The amplitude of Ca2+ fluctuation was normalized using average cell-wide basal cytosolic levels as a reference. Bars are mean ± SEM. (n = 20 cells) (h) Examples of migrating HUVEC expressing GFP–paxillin after Blebbistatin treatment. Control and BTP2-treated cells (same as Fig. 2h,i) were included for comparison. Notice the immediate decrease of the GFP–paxillin after Blebbistatin addition and the slightly delayed response after BTP2 addition. (i) Time course of loss in GFP–paxillin puncta in front following addition of the SOC inhibitor BTP2, the myosin inhibitor Blebbistatin or control (medium). Note that Blebbistatin decreased paxillin signals initially faster than BTP2. Bars are mean ± SEM (n = 14 cells for control cells, n = 36 cells for BTP2 treated cells, n = 20 cells for Blebbistatin treated cells).. One-way ANOVA was used for Supplementary Figure 2e to compare groups treated with different concentrations of (Calcium) Fura-2 or Mag Fura-2.

Supplementary Figure 3 Fibronectin and SOC regulate focal adhesions.

H1299 cells were plated on different concentrations of fibronectin and treated with BTP2 or transfected with mCherry-STIM1 before fixation and staining with anti-paxillin antibody to label endogenous nascent focal adhesion complexes. (a) A separate experiment from that shown in Fig. 3d also shows more punctate paxillin signals in cells on high compared with low fibronectin. Bars are mean ± SEM (n = 47 cells on high fibronectin and 51 cells on low fibronectin group). (b) BTP2 decreases punctate paxillin signals in cells grown on high or low fibronectin. Data are analyzed from the same experiment as shown in Fig. 3d. Averaged punctate paxillin signals were compared between different cell treatments. Bars are mean ± SEM (n = 115, 121, 104 and 133 cells from left to right for each group). (c-f) H1299 cells with overexpressing mCherry-STIM1 have more punctate paxillin signals, compared with control cells expressing tdimer2-CAAX. Data were analyzed from the same experiment shown in Fig. 3f. (c,d) Correlation between the expression level of overexpressed proteins and punctate paxillin signals. Solid lines denote mean while dotted lines denote SEM; n = 89 (red) & 62 (blue) cells in (c), and 108 (red) & 70 (blue) cells in (d). (e,f) Integrated punctate paxillin signals from each cell were binned and averaged according to the level of STIM1 overexpression. N: logeRFP < 1.5. H: logeRFP > 4. Bars are mean ± SEM (n =15, 17, 45 & 16 cells from left to right for each group in (e), and 17, 18, 42 & 23 cells from left to right for each group in (f)). (g) Full Western blot scans of Supplementary Figure 2a, with molecular weight (MW) size markers. Student t test was used for Supplementary Figure 3a,b,e,f.

Supplementary Figure 4 STIM1 is enriched at the front of migrating cells to generate polarized SOC influx.

(a) The ratio image of STIM1/ER in the HUVEC cell shown in Fig. 4a indicates an enrichment of STIM1 in the front. (b) Assay to test whether mutant STIM1 constructs regulate SOC equally well as wild-type STIM1. (c) The STIM1 mutant S1NN deficient in binding microtubule plus ends induced store-operated Ca2+ (SOC) influx similarly as the wild-type STIM1 protein. Bars are mean ± SEM (n 10,000 for each of the wild-type or NN mutant group). (d) Quantification of the signal ratios for STIM1/ER-PM puncta of the cell as shown in Fig. 5a. (e,f) Images of the example shown in Fig. 5e. (e) Timelapse images of a cell expressing mCherry-CAAX as amembrane marker and the ER Ca2+ probe T1ER, migrating in the direction indicated by the arrow. (f) Original images of FRET and CFP signals. Dotted lines reveal the boundary of the cell based on the plasma membrane marker. Notice that the density of ER was very low within about 5 μm of the cell border, consistent with our previous data12, and the notion that local Ca2+ pulses 6.6 μm behind the cell front induce lamellipodia retraction and to enhance focal adhesion. (g-i) Treatment of cells with Ponatinib reduced the activation of STIM1 in the front of migrating cells. (g) Another example showing the increase in STIM1 punctate relative to ER-PM markers in the front compared with the back of a migrating cell. The white arrow indicates the direction of cell migration. (h) Quantification of STIM1/ER-PM ratios along the axis front-back in the cell shown in (g), before and after the addition of Ponatinib (100 nM). The white arrow indicates the reduction of relative STIM1 activation in the front. (i) The boxplot shows the median reduction of STIM1/ER-PM gradient after addition of Ponatinib. Each box with whiskers shows, from bottom to top, minima, 25th percentile, median, 75th percentile and maxima values. Paired t test was used to generate the p value. (n = 3 cells).

Supplementary Figure 5 Higher Ca2+ pump activity in the front generates a gradient of basal cytosolic [Ca2+] in migrating HUVEC.

(a,b) The amplitude of local lamellipodia protrusion and retraction cycles only correlated with Ca2+ pulses when front cytosolic Ca2+ levels were low. (a) Addition of the SERCA inhibitor thapsigargin to migrating HUVEC showed that the correlation between local Ca2+ and front retraction was decreased when cytosolic Ca2+ was elevated. HUVEC were loaded with Fura-2/AM as described in Methods. [Ca2+] was normalized to average cytosolic levels before thapsigargin treatment. (b) Average changes of the front membrane speed with local Ca2+ levels before and after thapsigargin addition. Bars are mean ± SEM (n = 6 cells). (c,d) The SERCA inhibitor thapsigargin increased the Ca2+ gradient in migrating cells, indicating that SERCA does not contribute to generate the Ca2+ gradient. (c) Cells were plated and loaded with Fura-2/AM and imaged for 3 minutes to record baseline Ca2+ levels before addition of thapsigargin (2 μM). Cells were then imaged for 10 more minutes. (d) Average change in cytosolic Ca2+ levels in front versus back. Notice the differential decrease of cytosolic Ca2+ levels in the front (left) and the back (middle) of migrating cells. The Ca2+ gradient (back – front, right) was increased. Bars are mean ± SEM (n = 32 cells). (e,f) The PMCA inhibitors Caloxin 2A1 and La3+ both suppressd the Ca2+ gradient and the differential Ca2+ pump activities in migrating cells. Bars are mean ± SEM. (e) Both PMCA inhibitors increased cytosolic Ca2+ levels in the front and in the back (n = 47 cells for each group). At the same time, the Ca2+ gradient([Ca2+]back–[Ca2+]front) was decreased (Fig. 6c). (f) Cells pre-incubated with PMCA inhibitors were treated with thapsigargin, EGTA, and BTP2 as in Fig. 6d. This resulted in a decreased Ca2+ pump activity in the front (left) but not in the back (right). Notice that the effect of La3+ on the front pumping activity was weaker than that of Caloxin 2A1, likely because the Ca2+ chelator EGTA also partially chelated La3+ and weakened the La3+ inhibitory effect on PMCA. Bars are mean ± SEM (n = 47 cells for each group.) Student t test was used for Supplementary Figure 5b,e,f.

Supplementary Figure 6 Higher Ca2+ pump activity in the front compared with the back generates a gradient of basal Ca2+ in migrating cells.

(a,b)10 μM of 2,4-DCB or 3,4-DCB slightly decreases Ca2+ pump activities in the front and in the back of migrating HUVECs. However, the differential pump activities were not affected (Fig. 7f). La3+ was used as a positive control. Bars are mean ± SEM (n = 41, 40, 35 & 42 cells in DMSO, 2,4-DCB, 3,4-DCB and LaCl3 group, respectively). (c) The PMCA inhibitor La3+ increased cytosolic Ca2+ and reduced HUVEC sheet migration, whereas EGTA (1mM) decreased cytosolic Ca2+ and enhanced sheet migration. Bars are mean ± SEM (n = 4 wells for each group). (d) PMCA is enriched in the front of migrating cells. Leader cells co-expressing GFP–PMCA4b and lyn-tdimer2 were analyzed using the sheet migration assay. Images of GFP–PMCA4b (left) and lyn-tdimer2 (the rest) of a migrating cell are shown. The pseudo-color image shows how the cell migrated in the direction indicated by the arrow. The corresponding ratio image GFP–PMCA4b/lyn-tdimer2 is shown in Fig. 7g. (e) H293T cells transfected with GFP–PMCA4b showed localization in the plasma membrane Student t test was used for Supplementary Figure 6a,b. One-way ANOVA was used in Supplementary Figure 6c for comparison of dose-dependent effect of La3+ on sheet migration speed.

Supplementary Figure 7 Polarized phospholipase C (PLC) activitiy in the front of migrating cells controls migration through DAG production.

(a) The PLC inhibitor U73122 slowed down HUVEC sheet migration in addition to single cell speed as shown in Fig. 8b of HUVEC. U73122 was added to the cell sheets prior to time-lapse imaging. (n = 2 experiments for each group). (b) Inhibition of DAG kinase caused a small but significant increase in single cell migration speed in control cells. DAGKI II had a stronger effect when cell density was low than when the density was high. 1X density 31,250 cells/cm2. Bars are mean ± SEM (n = 3 experiments for each group). (c) Addition of the PKCβ inhibitor Ruboxistaurin (0, 5 and 10 μM) suppressed migration in a dose-dependent manner (the black, red and blue Bars at the leftmost column of the graph), while increasing doses of a DAG kinase inhibitor (0 to 4 μM) increased directed cell migration when PKCβ activity was partially inhibited. Note that the effect of DAG kinase inhibition was clearly apparent when PKC was partially inhibited. Bars are mean ± SEM (n = 4 experiments for each group). (d) Histogram of the length distribution of leader cells (mean ± s.d. = 55.23 ± 16.82 μm, n = 115 cells). One-way ANOVA was used for Supplementary Figure 7a,b.

Supplementary Figure 8 Schematic representation of the polarized Ca2+ signaling system components that orchestrate directed cell migration.

(a) Receptor tyrosine kinase signaling is polarized in the front of migrating cells, resulting front-restricted generation of PIP3 The local RTK activation also generates local InsP3-mediatedCa2+ pulses that regulate retraction and adhesion via activation of MLCK. The co-generated gradient in DAG controls speed and directionality by polarized activation of conventional PKCs. (b) STIM1 is enriched in the cell front and, in addition, selectively activated by lower luminal Ca2+ levels in the front of the ER. The resulting polarized store-operated Ca2+ influx in the front triggers local Ca2+ signals of its own and restores luminal Ca2+, enabling local InsP3-gatedCa2+ release pulses to be continuously triggered. (c)Ca2+ extrusion by plasma membrane Ca2+-ATP pumps is higher in the cell front, causing lower basal Ca2+ levels in the front, enabling local Ca2+ signals to regulate MLCK.

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The frequency of local Ca2+ pulses is higher in the front than in the middle or the back of migrating leader cells during collective migration.

The movie shows local Ca2+ activities in leader cells. GCaMP6 tethered to the PM using a CAAX sequence was used to monitor temporal and spatial [Ca2+] changes. Three of the nine traces are also shown as a static image in Fig. 1g. (AVI 10133 kb)

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Tsai, F., Seki, A., Yang, H. et al. A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration. Nat Cell Biol 16, 133–144 (2014). https://doi.org/10.1038/ncb2906

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