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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Zigmond, S. H. Mechanisms of sensing chemical gradients by polymorphonuclear leukocytes. Nature 249, 450–452 ( 1974).
Zigmond, S. H. & Hirsch, J. G. Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp. Cell Res. 73, 383–393 ( 1972).
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).
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).
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).
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).
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).
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).
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).
Okabe, S. & Hirokawa, N. Actin dynamics in growth cones . J. Neurosci. 11, 1918– 1929 (1991).
Symons, M. H. & Mitchison, T.J. Control of actin polymerization in live and permeabilized fibroblasts. J. Cell Biol. 114, 503–513 (1991).
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).
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).
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).
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).
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).
Amrein, P. C. & Stossel, T. P. Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate. Blood 56, 442–447 ( 1980).
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).
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).
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).
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).
Schafer, D. A. et al. Visualization and molecular analysis of actin assembly in living cells. J. Cell Biol. 143, 1919– 1930 (1998).
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).
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).
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).
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).
Tilney, L. G. & Tilney, M. S. The wily ways of a parasite: induction of actin assembly by Listeria. Trends Microbiol. 1, 25–31 (1993).
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).
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).
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).
Gerisch, G. & Keller, H. U. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J. Cell Sci. 52, 1–10 ( 1981).
Pardee, J. D. & Spudich, J. A. Purification of muscle actin . Methods Enzymol. 85B, 164– 181 (1982).
Kellogg, D. R., Mitchison, T. J. & Alberts, B. M. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development 103, 675–686 (1988).
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).
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).
Miller, A. D. & Rosman, G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–982, 984–986, 989–990 ( 1989).
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).
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).
Agard, D. A., Hiraoka, Y., Shaw, P. & Sedat, J. W. Fluorescence microscopy in three dimensions. Methods Cell Biol. 30, 353–377 (1989).
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).
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).
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).
About this article
Cite this article
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
The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology
The EMBO Journal (2021)
Cellular Signalling (2021)
The actin comet guides the way: How Listeria actin subversion has impacted cell biology, infection biology and structural biology
Cellular Microbiology (2020)
Scientific Reports (2020)