Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Sep 1;101(3):1161–1166. doi: 10.1083/jcb.101.3.1161

Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular Ca++

PMCID: PMC2113714  PMID: 4040917

Abstract

When exposed to the N-formylated chemoattractant peptides, neutrophils undergo a transient ruffling followed by a polarization that involves a redistribution of F-actin (Fechheimer, M., and S. H. Zigmond, 1983, Cell Motil., 3:349-361). The cells also undergo a biphasic right angle light scatter response whose first phase is maximal 10-15 s after exposure to the stimulus, and whose second phase is longer in duration and maximal only after 1 min or more (Yuli, I., and R. Snyderman, 1984, J. Clin. Invest. 73:1408-1417). We now report that the first phase is accompanied by a transient polymerization of actin (monitored by cytometric analysis of phallacidin staining according to the method of Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271) and the second phase is accompanied by a more sustained polymerization of actin. Based on correlated measurements of ligand binding (Sklar, L. A., D. A. Finney, Z. G. Oades, A. J. Jesaitis, R. G. Painter, and C. G. Cochrane, 1984, J. Biol. Chem., 259:5661-5669) and intracellular Ca++ elevation (under conditions where we use the fluorescent Ca++ chelator Quin 2 to modulate intracellular Ca++ levels), we conclude that this first phase requires less than 100 receptors/cell (out of 50,000) and does not require the release of intracellular stores of Ca++. In contrast, the sustained polymerization requires both the occupancy of thousands of receptors (an estimated 10% of the receptors per minute) and may be somewhat sensitive to the availability of intracellular Ca++. When ligand binding is interrupted, F-actin rapidly depolymerizes with a half-time of no greater than approximately 15 s, and the transient light scatter response decays toward its initial value in parallel. Partial disaggregation of the cells follows the recovery of these responses. Based on these observations, we suggest that transient actin polymerization and transient cell ruffling give rise to transient aggregation as long as degranulation is limited.

Full Text

The Full Text of this article is available as a PDF (744.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boyles J., Bainton D. F. Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte adherence. J Cell Biol. 1979 Aug;82(2):347–368. doi: 10.1083/jcb.82.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Davis B. H., Walter R. J., Pearson C. B., Becker E. L., Oliver J. M. Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide. Am J Pathol. 1982 Aug;108(2):206–216. [PMC free article] [PubMed] [Google Scholar]
  4. Di Virgilio F., Lew D. P., Pozzan T. Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels. Nature. 1984 Aug 23;310(5979):691–693. doi: 10.1038/310691a0. [DOI] [PubMed] [Google Scholar]
  5. Fechheimer M., Zigmond S. H. Changes in cytoskeletal proteins of polymorphonuclear leukocytes induced by chemotactic peptides. Cell Motil. 1983;3(4):349–361. doi: 10.1002/cm.970030406. [DOI] [PubMed] [Google Scholar]
  6. Gerisch G., Keller H. U. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J Cell Sci. 1981 Dec;52:1–10. doi: 10.1242/jcs.52.1.1. [DOI] [PubMed] [Google Scholar]
  7. Glenney J. R., Jr, Kaulfus P., Weber K. F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end. Cell. 1981 May;24(2):471–480. doi: 10.1016/0092-8674(81)90338-x. [DOI] [PubMed] [Google Scholar]
  8. Henson P. M., Oades Z. G. Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Secretion of granule constituents and increased oxidation of glucose. J Clin Invest. 1975 Oct;56(4):1053–1061. doi: 10.1172/JCI108152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Howard T. H., Meyer W. H. Chemotactic peptide modulation of actin assembly and locomotion in neutrophils. J Cell Biol. 1984 Apr;98(4):1265–1271. doi: 10.1083/jcb.98.4.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hyslop P. A., Sklar L. A. A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes. Anal Biochem. 1984 Aug 15;141(1):280–286. doi: 10.1016/0003-2697(84)90457-3. [DOI] [PubMed] [Google Scholar]
  11. Korchak H. M., Wilkenfeld C., Rich A. M., Radin A. R., Vienne K., Rutherford L. E. Stimulus response coupling in the human neutrophil. Differential requirements for receptor occupancy in neutrophil responses to a chemoattractant. J Biol Chem. 1984 Jun 25;259(12):7439–7445. [PubMed] [Google Scholar]
  12. Korn E. D. Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol Rev. 1982 Apr;62(2):672–737. doi: 10.1152/physrev.1982.62.2.672. [DOI] [PubMed] [Google Scholar]
  13. Lew P. D., Wollheim C. B., Waldvogel F. A., Pozzan T. Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils. J Cell Biol. 1984 Oct;99(4 Pt 1):1212–1220. doi: 10.1083/jcb.99.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. MacLean-Fletcher S., Pollard T. D. Mechanism of action of cytochalasin B on actin. Cell. 1980 Jun;20(2):329–341. doi: 10.1016/0092-8674(80)90619-4. [DOI] [PubMed] [Google Scholar]
  15. McNeil P. L., Kennedy A. L., Waggoner A. S., Taylor D. L., Murphy R. F. Light-scattering changes during chemotactic stimulation of human neutrophils: kinetics followed by flow cytometry. Cytometry. 1985 Jan;6(1):7–12. doi: 10.1002/cyto.990060103. [DOI] [PubMed] [Google Scholar]
  16. Nath J., Flavin M., Gallin J. I. Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome. J Cell Biol. 1982 Nov;95(2 Pt 1):519–526. doi: 10.1083/jcb.95.2.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Omann G. M., Coppersmith W., Finney D. A., Sklar L. A. A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry. Cytometry. 1985 Jan;6(1):69–73. doi: 10.1002/cyto.990060113. [DOI] [PubMed] [Google Scholar]
  18. Pollard T. D., Mooseker M. S. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. doi: 10.1083/jcb.88.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rao K. M., Varani J. Actin polymerization induced by chemotactic peptide and concanavalin A in rat neutrophils. J Immunol. 1982 Oct;129(4):1605–1607. [PubMed] [Google Scholar]
  20. Sklar L. A., Finney D. A. Analysis of ligand-receptor interactions with the fluorescence activated cell sorter. Cytometry. 1982 Nov;3(3):161–165. doi: 10.1002/cyto.990030304. [DOI] [PubMed] [Google Scholar]
  21. Sklar L. A., Finney D. A., Oades Z. G., Jesaitis A. J., Painter R. G., Cochrane C. G. The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil. J Biol Chem. 1984 May 10;259(9):5661–5669. [PubMed] [Google Scholar]
  22. Sklar L. A., Jesaitis A. J., Painter R. G., Cochrane C. G. The kinetics of neutrophil activation. The response to chemotactic peptides depends upon whether ligand-receptor interaction is rate-limiting. J Biol Chem. 1981 Oct 10;256(19):9909–9914. [PubMed] [Google Scholar]
  23. Sklar L. A., Jesaitis A. J., Painter R. G. The neutrophil N-formyl peptide receptor: dynamics of ligand-receptor interactions and their relationship to cellular responses. Contemp Top Immunobiol. 1984;14:29–82. doi: 10.1007/978-1-4757-4862-8_2. [DOI] [PubMed] [Google Scholar]
  24. Sklar L. A., McNeil V. M., Jesaitis A. J., Painter R. G., Cochrane C. G. A continuous, spectroscopic analysis of the kinetics of elastase secretion by neutrophils. The dependence of secretion upon receptor occupancy. J Biol Chem. 1982 May 25;257(10):5471–5475. [PubMed] [Google Scholar]
  25. Sklar L. A., Oades Z. G., Finney D. A. Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation. J Immunol. 1984 Sep;133(3):1483–1487. [PubMed] [Google Scholar]
  26. Sklar L. A., Oades Z. G., Jesaitis A. J., Painter R. G., Cochrane C. G. Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7540–7544. doi: 10.1073/pnas.78.12.7540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stossel T. P., Hartwig J. H., Yin H. L., Zaner K. S., Stendahl O. I. Actin gelation and structure of cortical cytoplasm. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):569–578. doi: 10.1101/sqb.1982.046.01.053. [DOI] [PubMed] [Google Scholar]
  28. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wallace P. J., Wersto R. P., Packman C. H., Lichtman M. A. Chemotactic peptide-induced changes in neutrophil actin conformation. J Cell Biol. 1984 Sep;99(3):1060–1065. doi: 10.1083/jcb.99.3.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wegner A., Engel J. Kinetics of the cooperative association of actin to actin filaments. Biophys Chem. 1975 Jul;3(3):215–225. doi: 10.1016/0301-4622(75)80013-5. [DOI] [PubMed] [Google Scholar]
  31. White J. R., Naccache P. H., Sha'afi R. I. The synthetic chemotactic peptide formyl-methionyl-leucyl-phenylalanine causes an increase in actin associated with the cytoskeleton in rabbit neutrophils. Biochem Biophys Res Commun. 1982 Oct 15;108(3):1144–1149. doi: 10.1016/0006-291x(82)92120-9. [DOI] [PubMed] [Google Scholar]
  32. Yuli I., Snyderman R. Rapid changes in light scattering from human polymorphonuclear leukocytes exposed to chemoattractants. Discrete responses correlated with chemotactic and secretory functions. J Clin Invest. 1984 May;73(5):1408–1417. doi: 10.1172/JCI111345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zigmond S. H. Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J Cell Biol. 1977 Nov;75(2 Pt 1):606–616. doi: 10.1083/jcb.75.2.606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zigmond S. H., Levitsky H. I., Kreel B. J. Cell polarity: an examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis. J Cell Biol. 1981 Jun;89(3):585–592. doi: 10.1083/jcb.89.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zigmond S. H., Sullivan S. J. Sensory adaptation of leukocytes to chemotactic peptides. J Cell Biol. 1979 Aug;82(2):517–527. doi: 10.1083/jcb.82.2.517. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES