Eukaryotic Chemotaxis: A Network of Signaling Pathways Controls Motility, Directional Sensing, and Polarity

Supplementary Data

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  Supplemental Movie 1. Actin binding proteins form propagating waves on the basal surface of cells. A migrating undifferentiated Dictyostelium cell, expressing MyoB-GFP and LimE_Δcoil-RFP, was observed by two color total internal reflection microscopy (TIRF) microscopy. Imaging reveals the sequential recruitment of the actin binding proteins to the basal surface of the cell in the form of propagated waves. Images were captured at 1 second intervals. Movie is reproduced from (1). Similar waves of actin and other actin binding proteins have been observed in Dictyostelium cells and neutrophils (2, 4-7). Mathematical simulations of the waves have been presented (3, 7).

  Literature Cited

  1. Bretschneider T, Anderson K, Ecke M, Muller-Taubenberger A, Schroth-Diez B, et al. 2009. The three-dimensional dynamics of actin waves, a model of cytoskeletal self-organization. Biophys J 96:2888-900
  2. Bretschneider T, Jonkman J, Kohler J, Medalia O, Barisic K, et al. 2002. Dynamic organization of the actin system in the motile cells of Dictyostelium. J Muscle Res Cell Motil 23:639-49
  3. Doubrovinski K, Kruse K. 2008. Cytoskeletal waves in the absence of molecular motors. EPL (Europhysics Letters) 83:18003
  4. Millius A, Dandekar SN, Houk AR, Weiner OD. 2009. Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion. Curr Biol 19:253-9
  5. Vicker MG. 2000. Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion. Biophys Chem 84:87-98
  6. Vicker MG. 2002. Eukaryotic cell locomotion depends on the propagation of self-organized reactiondiffusion waves and oscillations of actin filament assembly. Exp Cell Res 275:54-66
  7. Weiner OD, Marganski WA, Wu LF, Altschuler SJ, Kirschner MW. 2007. An actin-based wave generator organizes cell motility. PLoS Biol 5:e221

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  Supplemental Movies 2 and 3. Latrunculin A-treated cells retain the ability to selectively accumulate PIP3 at the membrane. Wild-type cells expressing Crac-GFP were treated with 0.5 µM Latrunculin for 20 minutes and imaged with wide-field fluorescence microscopy. Images were captured at 5 second intervals. Response to a uniform stimulus is transient. In Movie 2, cells were exposed to a uniform increase of 1 µM cAMP. Response towards the high side of the chemoattractant gradient is persistent. In Movie 3, a micropipette releasing 1 µM cAMP was located near the lower right corner of the field. The GFP signal decreases slightly over time due to photobleaching. Movie 3 is reproduced from (4). Similar experiments were done in (2, 3). Ras binding domain, which senses the activated form of Ras proteins, behaves similarly to Crac-GFP (5). The responses seen can be explained by modeling (1).

  Literature Cited

  1. Iglesias PA, Devreotes PN. 2008. Navigating through models of chemotaxis. Curr Opin Cell Biol 20:35-40
  2. Janetopoulos C, Ma L, Devreotes PN, Iglesias PA. 2004. Chemoattractant-induced phosphatidylinositol 3,4,5-trisphosphate accumulation is spatially amplified and adapts, independent of the actin cytoskeleton. Proc Natl Acad Sci U S A 101:8951-6
  3. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, Devreotes PN. 1998. G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95:81-91
  4. Parent CA, Devreotes PN. 1999. A cell's sense of direction. Science 284:765-70
  5. Sasaki AT, Chun C, Takeda K, Firtel RA. 2004. Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J Cell Biol 167:505-18

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  Supplemental Movie 4. Loss of FRET signal, representing G-protein dissociation, is persistent. Cells co-expressing G-protein subunits α2-cerulean and β-venus were imaged during persistent stimulation with 1 µM cAMP. FRET signal is reflected by the ratio of fluorescence in the YFP to CFP channels. Images were captured at 2 second intervals. Stimulus was applied by perfusion after 15 frames and held constant for five minutes, then removed. See (1, 2) for similar experiments.

  Literature Cited

  1. Janetopoulos C, Jin T, Devreotes P. 2001. Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science 291:2408-11
  2. Xu X, Meier-Schellersheim M, Jiao X, Nelson LE, Jin T. 2005. Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium. Mol Biol Cell 16:676-88

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  Supplemental Movies 5 and 6. Proteins that are found at the leading or lagging edge of polarized cells transiently redistribute in response to uniform cAMP stimulation. "Leading edge" proteins translocate uniformly to the membrane or cortex within 10 seconds, then return to the cytosol roughly 30 seconds after stimulation. "Lagging edge" proteins, with similar kinetics, transiently fall off of the membrane or cortex and into the cytosol before returning to the cell periphery. Dictyostelium cells in early differentiation were imaged with wide-field fluorescence microscopy and uniformly stimulated with 1 µM cAMP after the second frame. Images were captured at 6 second intervals for 2.5 minutes and played at a rate of 5 frames per second. In Movie 5, cells are expressing Crac-GFP. In Movie 6, cells are expressing PTEN-GFP. See (1-3) for similar experiments; also see Supplemental Table 2 for additional proteins that exhibit this behavior.

  Literature Cited

  1. Iijima M, Devreotes P. 2002. Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109:599-610
  2. Meili R, Ellsworth C, Lee S, Reddy TB, Ma H, Firtel RA. 1999. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. Embo J 18:2092-105
  3. Parent CA, Blacklock BJ, Froehlich WM, Murphy DB, Devreotes PN. 1998. G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95:81-91