Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1980 Jul 1;86(1):315–325. doi: 10.1083/jcb.86.1.315

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm

PMCID: PMC2110665  PMID: 6893453

Abstract

A biochemical assay employing DNase-I affinity chromatography, two- dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two- dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.

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. Bennett V., Branton D. Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin. J Biol Chem. 1977 Apr 25;252(8):2753–2763. [PubMed] [Google Scholar]
  2. Blikstad I., Markey F., Carlsson L., Persson T., Lindberg U. Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. Cell. 1978 Nov;15(3):935–943. doi: 10.1016/0092-8674(78)90277-5. [DOI] [PubMed] [Google Scholar]
  3. Bray D., Thomas C. Unpolymerized actin in fibroblasts and brain. J Mol Biol. 1976 Aug 25;105(4):527–544. doi: 10.1016/0022-2836(76)90233-3. [DOI] [PubMed] [Google Scholar]
  4. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  5. Forer A., Behnke O. An actin-like component in sperm tails of a crane fly (Nephrotoma suturalis Loew). J Cell Sci. 1972 Sep;11(2):491–519. doi: 10.1242/jcs.11.2.491. [DOI] [PubMed] [Google Scholar]
  6. Forer A., Behnke O. An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew). II. The cell cortex. Chromosoma. 1972;39(2):175–190. doi: 10.1007/BF00319841. [DOI] [PubMed] [Google Scholar]
  7. Gordon D. J., Boyer J. L., Korn E. D. Comparative biochemistry of non-muscle actins. J Biol Chem. 1977 Nov 25;252(22):8300–8309. [PubMed] [Google Scholar]
  8. Gordon D. J., Yang Y. Z., Korn E. D. Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin. J Biol Chem. 1976 Dec 10;251(23):7474–7479. [PubMed] [Google Scholar]
  9. Hainfeld J. F., Steck T. L. The sub-membrane reticulum of the human erythrocyte: a scanning electron microscope study. J Supramol Struct. 1977;6(3):301–311. doi: 10.1002/jss.400060303. [DOI] [PubMed] [Google Scholar]
  10. Hellewell S. B., Taylor D. L. The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis. J Cell Biol. 1979 Dec;83(3):633–648. doi: 10.1083/jcb.83.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hitchcock S. E., Carisson L., Lindberg U. Depolymerization of F-actin by deoxyribonuclease I. Cell. 1976 Apr;7(4):531–542. doi: 10.1016/0092-8674(76)90203-8. [DOI] [PubMed] [Google Scholar]
  12. Ishikawa H., Bischoff R., Holtzer H. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J Cell Biol. 1969 Nov;43(2):312–328. [PMC free article] [PubMed] [Google Scholar]
  13. KASAI M., NAKANO E., OOSAWA F. POLYMERIZATION OF ACTIN FREE FROM NUCLEOTIDES AND DIVALENT CATIONS. Biochim Biophys Acta. 1965 Mar 29;94:494–503. doi: 10.1016/0926-6585(65)90058-0. [DOI] [PubMed] [Google Scholar]
  14. Kahn R., Rubin R. W. Quantitation of submicrogram amounts of protein using coomassie brilliant blue R on sodium dodecyl sulfate-polyacrylamide slab-gels. Anal Biochem. 1975 Jul;67(1):347–352. doi: 10.1016/0003-2697(75)90305-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LaFountain J. R., Jr What moves chromosomes, microtubules or microfilaments? Biosystems. 1975 Nov;7(3-4):363–369. doi: 10.1016/0303-2647(75)90017-9. [DOI] [PubMed] [Google Scholar]
  16. LaFountain J. R., Jr, Zobel C. R., Thomas H. R., Galbreath C. Fixation and staining of F-actin and microfilaments using tannic acid. J Ultrastruct Res. 1977 Jan;58(1):78–86. doi: 10.1016/s0022-5320(77)80009-9. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luna E. J., Kidd G. H., Branton D. Identification by peptide analysis of the spectrin-binding protein in human erythrocytes. J Biol Chem. 1979 Apr 10;254(7):2526–2532. [PubMed] [Google Scholar]
  20. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  21. Nakashima K., Beutler E. Comparison of structure and function of human erythrocyte and human muscle actin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):935–938. doi: 10.1073/pnas.76.2.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  23. Rohr G., Mannherz H. G. The activation of actin:DNase I complex with rat liver plasma membranes. The possible role of 5'-nucleotidase. FEBS Lett. 1979 Mar 15;99(2):351–356. doi: 10.1016/0014-5793(79)80990-4. [DOI] [PubMed] [Google Scholar]
  24. Schroeder T. E. Dynamics of the contractile ring. Soc Gen Physiol Ser. 1975;30:305–334. [PubMed] [Google Scholar]
  25. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  26. Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
  27. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  28. Taylor D. L., Condeelis J. S. Cytoplasmic structure and contractility in amoeboid cells. Int Rev Cytol. 1979;56:57–144. doi: 10.1016/s0074-7696(08)61821-5. [DOI] [PubMed] [Google Scholar]
  29. Tilney L. G., Detmers P. Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ. J Cell Biol. 1975 Sep;66(3):508–520. doi: 10.1083/jcb.66.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tilney L. G., Hatano S., Ishikawa H., Mooseker M. S. The polymerization of actin: its role in the generation of the acrosomal process of certain echinoderm sperm. J Cell Biol. 1973 Oct;59(1):109–126. doi: 10.1083/jcb.59.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Uyemura D. G., Brown S. S., Spudich J. A. Biochemical and structural characterization of actin from Dictyostelium discoideum. J Biol Chem. 1978 Dec 25;253(24):9088–9096. [PubMed] [Google Scholar]
  32. Weisenberg R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972 Sep 22;177(4054):1104–1105. doi: 10.1126/science.177.4054.1104. [DOI] [PubMed] [Google Scholar]
  33. YAGI K., MASE R., SAKAKIBARA I., ASAI H. FUNCTION OF HEAVY MEROMYOSIN IN THE ACCELERATION OF ACTIN POLYMERIZATION. J Biol Chem. 1965 Jun;240:2448–2454. [PubMed] [Google Scholar]

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

RESOURCES