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Spatial control of actin polymerization during neutrophil chemotaxis

Abstract

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

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Figure 1: Polarization of a neutrophil in response to a gradient of chemoattractant.
Figure 2: Spatial distribution of incorporation of TMR–actin in a chemoattractant-stimulated permeabilized neutrophil.
Figure 3: Spatial distribution of TMR–actin incorporation in a neutrophil with two pseudopodia.
Figure 4: Response of neutrophils expressing Arp3–GFP to a stationary or moving chemotactic micropipette.
Figure 5: Immunofluorescence localization of endogenous Arp2/3 complex and actin in human neutrophils and the relationship of Arp2/3 localization to sites of actin polymerization.
Figure 6: Model of actin polymerization in response to a chemotactic signal.

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References

  1. Cano, M. L., Lauffenburger, D. A. & Zigmond, S. H. Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution. J. Cell Biol. 115, 677–687 (1991).

    Article  CAS  PubMed  Google Scholar 

  2. Zigmond, S. H. Mechanisms of sensing chemical gradients by polymorphonuclear leukocytes. Nature 249, 450–452 ( 1974).

    Article  CAS  PubMed  Google Scholar 

  3. Zigmond, S. H. & Hirsch, J. G. Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp. Cell Res. 73, 383–393 ( 1972).

    Article  CAS  PubMed  Google Scholar 

  4. Watts, R. G., Crispens, M. A. & Howard, T. H. A quantitative study of the role of F-actin in producing neutrophil shape. Cell. Motil. Cytoskeleton 19, 159–168 (1991).

    Article  CAS  PubMed  Google Scholar 

  5. Mullins, R. D., Heuser, J. A. & Pollard, T. D. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl Acad. Sci. USA 95, 6181–6186 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Welch, M. D., Rosenblatt, J., Skoble, J., Portnoy, D. A. & Mitchison, T. J. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science. 281, 105– 108 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Ma, L., Rohatgi, R. & Kirschner, M. W. The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42. Proc. Natl Acad. Sci. USA 95, 15362–15367 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. McCollum, D., Feoktistova, A., Morphew, M., Balasubramanian, M. & Gould, K. L. The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin. EMBO J. 15 , 6438–6446 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Moreau, V., Madania, A., Martin, R. P. & Winson, B. The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton. J. Cell Biol. 134, 117–132 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Winter, D., Podtelejnikov, A. V., Mann, M. & Li, R. The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches. Curr. Biol. 7, 519–529 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Okabe, S. & Hirokawa, N. Actin dynamics in growth cones . J. Neurosci. 11, 1918– 1929 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Symons, M. H. & Mitchison, T.J. Control of actin polymerization in live and permeabilized fibroblasts. J. Cell Biol. 114, 503–513 (1991).

    Article  CAS  PubMed  Google Scholar 

  13. Chan, A. Y. et al. EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells . J .Cell Sci. 111, 199– 211 (1998).

    CAS  PubMed  Google Scholar 

  14. Redmond, T. & Zigmond, S. H. Distribution of F-actin elongation sites in lysed polymorphonuclear leukocytes parallels the distribution of endogenous F-actin. Cell. Motil. Cytoskeleton 26, 7–18 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Welch, M. D., DePace, A. H., Verma, S., Iwamatsu, A. & Mitchison, T. J. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. J. Cell Biol. 138, 375– 384 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sanger, J. M., Sanger, J. W. & Southwick, F. S. Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes. Infect. Immun. 60, 3609 –3619 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Theriot, J. A., Mitchison, T. J., Tilney, L. G. & Portnoy, D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 357, 257–260 (1992).

    Article  CAS  PubMed  Google Scholar 

  18. Amrein, P. C. & Stossel, T. P. Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood 56, 442–447 ( 1980).

    CAS  PubMed  Google Scholar 

  19. Machesky, L. M. et al. Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins . Biochem. J. 328, 105– 112 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Machesky, L. M., Atkinson, S. J., Ampe, C., Vandekerckhove, J. & Pollard, T. D. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J. Cell Biol. 127 , 107–115 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Kelleher, J. F., Atkinson, S. J. & Pollard, T.D. Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. J. Cell Biol. 131, 385–397 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Mullins, R. D., Kelleher, J. F., Xu, J. & Pollard, T. D. Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments. Mol. Biol.Cell. 9, 841–852 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schafer, D. A. et al. Visualization and molecular analysis of actin assembly in living cells. J. Cell Biol. 143, 1919– 1930 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zigmond, S. H., Joyce, M., Borleis, J., Bokoch, G. M., & Devreotes, P. N. Regulation of actin polymerization in cell-free systems by GTP-γS and Cdc42. J. Cell Biol. 138, 363–374 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ma, L., Cantley, L. C., Janmey, P. A., & Kirschner, M. W. Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts. J.Cell Biol. 140, 1125–1136 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mullins, R. D. & Pollard, T. D. Rho-family G-proteins act through Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts. Curr. Biol. (in the press).

  27. Dabiri, G. A., Sanger, J. M., Portnoy, D. A. & Southwick, F. S. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc. Natl Acad. Sci. USA 87, 6068–6072 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Tilney, L. G. & Tilney, M. S. The wily ways of a parasite: induction of actin assembly by Listeria. Trends Microbiol. 1, 25–31 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Sechi, A. S., Wehland, J. & Small, J. V. The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random. J. Cell Biol. 137, 155–167 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Marchand, J. B. et al. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface. J. Cell Biol. 130, 331–343 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Cassimeris, L., McNeill, H. & Zigmond, S. H. Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities. J. Cell Biol. 110, 1067–1075 (1990).

  32. Gerisch, G. & Keller, H. U. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J. Cell Sci. 52, 1–10 ( 1981).

    CAS  PubMed  Google Scholar 

  33. Pardee, J. D. & Spudich, J. A. Purification of muscle actin . Methods Enzymol. 85B, 164– 181 (1982).

    Article  Google Scholar 

  34. Kellogg, D. R., Mitchison, T. J. & Alberts, B. M. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103, 675–686 (1988).

    CAS  PubMed  Google Scholar 

  35. Small, J. V. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks . J. Cell Biol. 91, 695– 705 (1981).

    Article  CAS  PubMed  Google Scholar 

  36. Tucker, K. A., Lilly, M. B., Heck, L. Jr & Rado, T. A. Characterization of a new human diploid myeloid leukemia cell line (PLB- 985) with granulocytic and monocytic differentiating capacity. Blood 70, 372–378 (1987).

    CAS  PubMed  Google Scholar 

  37. Miller, A. D. & Rosman, G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–982, 984–986, 989–990 ( 1989).

    Google Scholar 

  38. Servant, G., Weiner, O. D., Neptune, E., Sedat, J. W. & Bourne, H. R. Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis. Mol. Biol. Cell. 10, 1163–1178 ( 1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hiraoka, Y., Swedlow, J. R., Paddy, M. R., Agard, D. A. & Sedat, J. W. Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena . Semin. Cell Biol. 2, 153– 165 (1991).

    CAS  PubMed  Google Scholar 

  40. Agard, D. A., Hiraoka, Y., Shaw, P. & Sedat, J. W. Fluorescence microscopy in three dimensions. Methods Cell Biol. 30, 353–377 (1989).

    Article  CAS  PubMed  Google Scholar 

  41. Swedlow, J. R., Sedat, J. W. & Agard, D. A. in in Deconvolution of Images and Spectra (ed. Jansson, P. A.) 284–307 (Academic, San Diego, 1997).

    Google Scholar 

  42. Chen, H., Hughes, D. D., Chan, T. A., Sedat, J. W. & Agard, D. A. IVE (Image Visualization Environment): a software platform for all three-dimensional microscopy applications. J. Struct. Biol. 116, 56–60 (1996).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Abo, D. Agard, C. Bargmann, D. Drubin, Z. Kam, C. Kenyon, R. Mullins, J. Taunton, J. Weissman, S. Zigmond and members of the Bourne and Sedat laboratories for discussions; A. Abo for the PLB-985 promyelocytic cell line; and C. Bargmann, C. Kenyon, J.V. Small, and S. Zigmond for critical reading of the manuscript. This work was supported in part by grants from the NIH (to H.R.B., J.W.S. and T.J.M.). M.D.W. is a Leukemia Society of America Special Fellow; G.S. is a Medical Research Council of Canada Postdoctoral Fellow; and O.D.W. is an HHMI Predoctoral Fellow.

Correspondence and requests for materials should be addressed to H.R.B.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://cellbio.nature.com).

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Weiner, O., Servant, G., Welch, M. et al. Spatial control of actin polymerization during neutrophil chemotaxis . Nat Cell Biol 1, 75–81 (1999). https://doi.org/10.1038/10042

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