Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1976 Feb 1;68(2):375–388. doi: 10.1083/jcb.68.2.375

Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis

PMCID: PMC2109630  PMID: 1107335

Abstract

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.

Full Text

The Full Text of this article is available as a PDF (3.8 MB).

Selected References

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

  1. Behnke O., Kristensen B. I., Nielsen L. E. Electron microscopical observations on actinoid and myosinoid filaments in blood platelets. J Ultrastruct Res. 1971 Nov;37(3):351–369. doi: 10.1016/s0022-5320(71)80129-6. [DOI] [PubMed] [Google Scholar]
  2. Burton P. R., Kirkland W. L. Actin detected in mouse neuroblastoma cells by binding of heavy meromyosin. Nat New Biol. 1972 Oct 25;239(95):244–246. doi: 10.1038/newbio239244a0. [DOI] [PubMed] [Google Scholar]
  3. Chang C. M., Goldman R. D. The localization of actin-like fibers in cultured neuroblastoma cells as revealed by heavy meromyosin binding. J Cell Biol. 1973 Jun;57(3):867–874. doi: 10.1083/jcb.57.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen I., Cohen C. A tropomyosin-like protein from human platelets. J Mol Biol. 1972 Jul 21;68(2):383–387. doi: 10.1016/0022-2836(72)90220-3. [DOI] [PubMed] [Google Scholar]
  5. Farquhar M. G., Palade G. E. Cell junctions in amphibian skin. J Cell Biol. 1965 Jul;26(1):263–291. doi: 10.1083/jcb.26.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fine R. E., Blitz A. L., Hitchcock S. E., Kaminer B. Tropomyosin in brain and growing neurones. Nat New Biol. 1973 Oct 10;245(145):182–186. doi: 10.1038/newbio245182a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. GOA J. A micro biuret method for protein determination; determination of total protein in cerebrospinal fluid. Scand J Clin Lab Invest. 1953;5(3):218–222. doi: 10.3109/00365515309094189. [DOI] [PubMed] [Google Scholar]
  10. Goli D. E., Suzuki A., Temple J., Holmes G. R. Studies on purified -actinin. I. Effect of temperature and tropomyosin on the -actinin-F-actin interaction. J Mol Biol. 1972 Jun 28;67(3):469–488. doi: 10.1016/0022-2836(72)90464-0. [DOI] [PubMed] [Google Scholar]
  11. Gorovsky M. A., Carlson K., Rosenbaum J. L. Simple method for quantitive densitometry of polyacrylamide gels using fast green. Anal Biochem. 1970 Jun;35(2):359–370. doi: 10.1016/0003-2697(70)90196-x. [DOI] [PubMed] [Google Scholar]
  12. Hitchcock S. E. Detection of actin filaments in homogenates of developing muscle using heavy meromyosin. Dev Biol. 1971 Aug;25(4):492–501. doi: 10.1016/0012-1606(71)90002-9. [DOI] [PubMed] [Google Scholar]
  13. Humphrey R. R. Genetic and experimental studies on a mutant gene (c) determining absence of heart action in embryos of the Mexican axolotl (Ambystoma mexicanum). Dev Biol. 1972 Mar;27(3):365–375. doi: 10.1016/0012-1606(72)90175-3. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Jacobson A. G., Duncan J. T. Heart induction in salamanders. J Exp Zool. 1968 Jan;167(1):79–103. doi: 10.1002/jez.1401670106. [DOI] [PubMed] [Google Scholar]
  16. Lemanski L. F. Heart development in the Mexican salamander, Ambystoma Mexicanum. II. Ultrastructure. Am J Anat. 1973 Apr;136(4):487–525. doi: 10.1002/aja.1001360408. [DOI] [PubMed] [Google Scholar]
  17. Lemanski L. F. Heart development in the Mexican salamander, Ambystoma mexicanum. I. Gross anatomy, histology and histochemistry. J Morphol. 1973 Mar;139(3):301–327. doi: 10.1002/jmor.1051390303. [DOI] [PubMed] [Google Scholar]
  18. Nachmias V. T., Huxley H. E. Electron microscope observations on actomyosin and actin preparations from Physarum polycephalum, and on their interaction with heavy meromyosin subfragment I from muscle myosin. J Mol Biol. 1970 May 28;50(1):83–90. doi: 10.1016/0022-2836(70)90105-1. [DOI] [PubMed] [Google Scholar]
  19. Orkin R. W., Pollard T. D., Hay E. D. SDS gel analysis of muscle proteins in embryonic cells. Dev Biol. 1973 Dec;35(2):388–394. doi: 10.1016/0012-1606(73)90035-3. [DOI] [PubMed] [Google Scholar]
  20. Perry M. M., John H. A., Thomas N. S. Actin-like filaments in the cleavage furrow of newt egg. Exp Cell Res. 1971 Mar;65(1):249–253. doi: 10.1016/s0014-4827(71)80075-7. [DOI] [PubMed] [Google Scholar]
  21. Pollard T. D., Shelton E., Weihing R. R., Korn E. D. Ultrastructural characterization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic filaments as F-actin by reaction with rabbit heavy meromyosin. J Mol Biol. 1970 May 28;50(1):91–97. doi: 10.1016/0022-2836(70)90106-3. [DOI] [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. Potter J. D. The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils. Arch Biochem Biophys. 1974 Jun;162(2):436–441. doi: 10.1016/0003-9861(74)90202-1. [DOI] [PubMed] [Google Scholar]
  24. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Richards E. G., Chung C. S., Menzel D. B., Olcott H. S. Chromatography of myosin on diethylaminoethyl-Sephadex A-50. Biochemistry. 1967 Feb;6(2):528–540. doi: 10.1021/bi00854a022. [DOI] [PubMed] [Google Scholar]
  26. Rubinstein N. A., Chi J. C., Holtzer H. Actin and myosin in a variety of myogenic and non-myogenic cells. Biochem Biophys Res Commun. 1974 Mar 25;57(2):438–446. doi: 10.1016/0006-291x(74)90950-4. [DOI] [PubMed] [Google Scholar]
  27. SZENT-GYORGYI A. G. Meromyosins, the subunits of myosin. Arch Biochem Biophys. 1953 Feb;42(2):305–320. doi: 10.1016/0003-9861(53)90360-9. [DOI] [PubMed] [Google Scholar]
  28. Sarkar S., Sreter F. A., Gergely J. Light chains of myosins from white, red, and cardiac muscles. Proc Natl Acad Sci U S A. 1971 May;68(5):946–950. doi: 10.1073/pnas.68.5.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schroeder T. E. Actin in dividing cells: contractile ring filaments bind heavy meromyosin. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1688–1692. doi: 10.1073/pnas.70.6.1688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  31. Spooner B. S., Ash J. F., Wrenn J. T., Frater R. B., Wessells N. K. Heavy meromyosin binding to microfilaments involved in cell and morphogenetic movements. Tissue Cell. 1973;5(1):37–46. doi: 10.1016/s0040-8166(73)80004-7. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Tilney L. G., Mooseker M. Actin in the brush-border of epithelial cells of the chicken intestine. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2611–2615. doi: 10.1073/pnas.68.10.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  36. 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