Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1979 Aug 1;82(2):369–379. doi: 10.1083/jcb.82.2.369

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

PMCID: PMC2110455  PMID: 479306

Abstract

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Becker E. L., Showell H. J., Henson P. M., Hsu L. S. The ability of chemotactic factors to induce lysosomal enzyme release. I. The characteristics of the release, the importance of surfaces and the relation of enzyme release to chemotactic responsiveness. J Immunol. 1974 Jun;112(6):2047–2054. [PubMed] [Google Scholar]
  2. Becker E. L., Showell H. J. The effect of Ca2+ and Mg2+ on the chemotactic responsiveness and spontaneous motility of rabbit polymorphonuclear leukocytes. Z Immunitatsforsch Exp Klin Immunol. 1972 Jun;143(5):466–476. [PubMed] [Google Scholar]
  3. Becker E. L. Some interrelations of neutrophil chemotaxis, lysosomal enzyme secretion, and phagocytosis as revealed by synthetic peptides. Am J Pathol. 1976 Nov;85(2):385–394. [PMC free article] [PubMed] [Google Scholar]
  4. Boucek M. M., Snyderman R. Calcium influx requirement for human neutrophil chemotaxis: inhibition by lanthanum chloride. Science. 1976 Sep 3;193(4256):905–907. doi: 10.1126/science.948752. [DOI] [PubMed] [Google Scholar]
  5. Durham A. C. A unified theory of the control of actin and myosin in nonmuscle movements. Cell. 1974 Jul;2(3):123–135. doi: 10.1016/0092-8674(74)90087-7. [DOI] [PubMed] [Google Scholar]
  6. Gallin J. I., Clark R. A., Frank M. M. Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways. Clin Immunol Immunopathol. 1975 Jan;3(3):334–346. doi: 10.1016/0090-1229(75)90020-3. [DOI] [PubMed] [Google Scholar]
  7. Gallin J. I., Clark R. A., Kimball H. R. Granulocyte chemotaxis: an improved in vitro assay employing 51 Cr-labeled granulocytes. J Immunol. 1973 Jan;110(1):233–240. [PubMed] [Google Scholar]
  8. Gallin J. I., Durocher J. R., Kaplan A. P. Interaction of leukocyte chemotactic factors with the cell surface. I. Chemotactic factor-induced changes in human granulocyte surface charge. J Clin Invest. 1975 May;55(5):967–974. doi: 10.1172/JCI108026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gallin J. I., Rosenthal A. S. The regulatory role of divalent cations in human granulocyte chemotaxis. Evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol. 1974 Sep;62(3):594–609. doi: 10.1083/jcb.62.3.594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Malech H. L., Root R. K., Gallin J. I. Structural analysis of human neutrophil migration. Centriole, microtubule, and microfilament orientation and function during chemotaxis. J Cell Biol. 1977 Dec;75(3):666–693. doi: 10.1083/jcb.75.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Manery J. F. Effects of Ca ions on membranes. Fed Proc. 1966 Nov-Dec;25(6):1804–1810. [PubMed] [Google Scholar]
  12. Mathieson A. R., Olayemi J. Y. The interaction of calcium and magnesium ions with deoxyribonucleic acid. Arch Biochem Biophys. 1975 Jul;169(1):237–243. doi: 10.1016/0003-9861(75)90337-9. [DOI] [PubMed] [Google Scholar]
  13. Mogensen C. E. The glomerular permeability determined by dextran clearance using Sephadex gel filtration. Scand J Clin Lab Invest. 1968;21(1):77–82. doi: 10.3109/00365516809076979. [DOI] [PubMed] [Google Scholar]
  14. Naccache P. H., Showell H. J., Becker E. L., Sha'afi R. I. Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. Effect of chemotactic factor. J Cell Biol. 1977 May;73(2):428–444. doi: 10.1083/jcb.73.2.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Robinson K. R., Jaffe L. F. Polarizing fucoid eggs drive a calcium current through themselves. Science. 1975 Jan 10;187(4171):70–72. doi: 10.1126/science.1167318. [DOI] [PubMed] [Google Scholar]
  16. SMOLELIS A. N., HARTSELL S. E. The determination of lysozyme. J Bacteriol. 1949 Dec;58(6):731–736. doi: 10.1128/jb.58.6.731-736.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Seimiya T., Ohki S. Ionic structure of phospholipid membranes, and binding of calcium ions. Biochim Biophys Acta. 1973 Mar 29;298(3):546–561. doi: 10.1016/0005-2736(73)90073-4. [DOI] [PubMed] [Google Scholar]
  18. Showell H. J., Freer R. J., Zigmond S. H., Schiffmann E., Aswanikumar S., Corcoran B., Becker E. L. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils. J Exp Med. 1976 May 1;143(5):1154–1169. doi: 10.1084/jem.143.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Simson J. A., Spicer S. S. Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique. J Histochem Cytochem. 1975 Aug;23(8):575–598. doi: 10.1177/23.8.51037. [DOI] [PubMed] [Google Scholar]
  20. Spicer S. S., Hardin J. H., Greene W. B. Nuclear precipitates in pyroantimonate-osmium tetroxide-fixed tissues. J Cell Biol. 1968 Oct;39(1):216–221. doi: 10.1083/jcb.39.1.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Williams L. T., Snyderman R., Pike M. C., Lefkowitz R. J. Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1204–1208. doi: 10.1073/pnas.74.3.1204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wright D. G., Malawista S. E. The mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis. J Cell Biol. 1972 Jun;53(3):788–797. doi: 10.1083/jcb.53.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Zigmond S. H., Hirsch J. G. Leukocyte locomotion and chemotaxis. New methods for evaluation, and demonstration of a cell-derived chemotactic factor. J Exp Med. 1973 Feb 1;137(2):387–410. doi: 10.1084/jem.137.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES