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. 1985 Aug 1;101(2):488–499. doi: 10.1083/jcb.101.2.488

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus

PMCID: PMC2113678  PMID: 2410433

Abstract

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

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Selected References

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  1. Andersen B., Osborn M., Weber K. Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge. Cytobiologie. 1978 Aug;17(2):354–364. [PubMed] [Google Scholar]
  2. Berkowitz S. A., Wolff J. Intrinsic calcium sensitivity of tubulin polymerization. The contributions of temperature, tubulin concentration, and associated proteins. J Biol Chem. 1981 Nov 10;256(21):11216–11223. [PubMed] [Google Scholar]
  3. Blinks J. R., Wier W. G., Hess P., Prendergast F. G. Measurement of Ca2+ concentrations in living cells. Prog Biophys Mol Biol. 1982;40(1-2):1–114. doi: 10.1016/0079-6107(82)90011-6. [DOI] [PubMed] [Google Scholar]
  4. Burgess W. H. Characterization of calmodulin and calmodulin isotypes from sea urchin gametes. J Biol Chem. 1982 Feb 25;257(4):1800–1804. [PubMed] [Google Scholar]
  5. Caswell A. H. Methods of measuring intracellular calcium. Int Rev Cytol. 1979;56:145–181. doi: 10.1016/s0074-7696(08)61822-7. [DOI] [PubMed] [Google Scholar]
  6. De Mey J., Lambert A. M., Bajer A. S., Moeremans M., De Brabander M. Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immuno-gold staining method. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1898–1902. doi: 10.1073/pnas.79.6.1898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Mey J., Moeremans M., Geuens G., Nuydens R., De Brabander M. High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol Int Rep. 1981 Sep;5(9):889–899. doi: 10.1016/0309-1651(81)90204-6. [DOI] [PubMed] [Google Scholar]
  8. Deery W. J., Means A. R., Brinkley B. R. Calmodulin-microtubule association in cultured mammalian cells. J Cell Biol. 1984 Mar;98(3):904–910. doi: 10.1083/jcb.98.3.904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fuller G. M., Brinkley B. R. Structure and control of assembly of cytoplasmic microtubules in normal and transformed cells. J Supramol Struct. 1976;5(4):497(349)–514(366). doi: 10.1002/jss.400050407. [DOI] [PubMed] [Google Scholar]
  10. Izant J. G. The role of calcium ions during mitosis. Calcium participates in the anaphase trigger. Chromosoma. 1983;88(1):1–10. doi: 10.1007/BF00329497. [DOI] [PubMed] [Google Scholar]
  11. Job D., Fischer E. H., Margolis R. L. Rapid disassembly of cold-stable microtubules by calmodulin. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4679–4682. doi: 10.1073/pnas.78.8.4679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Job D., Rauch C. T., Fischer E. H., Margolis R. L. Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks. Biochemistry. 1982 Feb 2;21(3):509–515. doi: 10.1021/bi00532a015. [DOI] [PubMed] [Google Scholar]
  13. Kakiuchi S., Sobue K., Yamazaki R., Nagao S., Umeki S., Nozawa Y., Yazawa M., Yagi K. Ca2+-dependent modulator proteins from Tetrahymena pyriformis, sea anemone, and scallop and guanylate cyclase activation. J Biol Chem. 1981 Jan 10;256(1):19–22. [PubMed] [Google Scholar]
  14. Keith C., DiPaola M., Maxfield F. R., Shelanski M. L. Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules. J Cell Biol. 1983 Dec;97(6):1918–1924. doi: 10.1083/jcb.97.6.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keller T. C., 3rd, Jemiolo D. K., Burgess W. H., Rebhun L. I. Strongylocentrotus purpuratus spindle tubulin. II. Characteristics of its sensitivity to Ca++ and the effects of calmodulin isolated from bovine brain and S. purpuratus eggs. J Cell Biol. 1982 Jun;93(3):797–803. doi: 10.1083/jcb.93.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kiehart D. P. Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J Cell Biol. 1981 Mar;88(3):604–617. doi: 10.1083/jcb.88.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klee C. B., Vanaman T. C. Calmodulin. Adv Protein Chem. 1982;35:213–321. doi: 10.1016/s0065-3233(08)60470-2. [DOI] [PubMed] [Google Scholar]
  18. Klumpp S., Kleefeld G., Schultz J. E. Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase. J Biol Chem. 1983 Oct 25;258(20):12455–12459. [PubMed] [Google Scholar]
  19. Lee Y. C., Wolff J. Two opposing effects of calmodulin on microtubule assembly depend on the presence of microtubule-associated proteins. J Biol Chem. 1982 Jun 10;257(11):6306–6310. [PubMed] [Google Scholar]
  20. Lukas T. J., Iverson D. B., Schleicher M., Watterson D. M. Structural characterization of a higher plant calmodulin : spinacia oleracea. Plant Physiol. 1984 Jul;75(3):788–795. doi: 10.1104/pp.75.3.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marcum J. M., Dedman J. R., Brinkley B. R., Means A. R. Control of microtubule assembly-disassembly by calcium-dependent regulator protein. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3771–3775. doi: 10.1073/pnas.75.8.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marshak D. R., Clarke M., Roberts D. M., Watterson D. M. Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum. Biochemistry. 1984 Jun 19;23(13):2891–2899. doi: 10.1021/bi00308a007. [DOI] [PubMed] [Google Scholar]
  23. Nishida E., Kumagai H. Calcium sensitivity of sea urchin tubulin in in vitro assembly and the effects of calcium-dependent regulator (CDR) proteins isolated from sea urchin eggs and porcine brains. J Biochem. 1980 Jan;87(1):143–151. doi: 10.1093/oxfordjournals.jbchem.a132719. [DOI] [PubMed] [Google Scholar]
  24. Nishida E., Kumagai H., Ohtsuki I., Sakai H. The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of microtubule assembly. J Biochem. 1979 May;85(5):1257–1266. [PubMed] [Google Scholar]
  25. Olmsted J. B., Borisy G. G. Ionic and nucleotide requirements for microtubule polymerization in vitro. Biochemistry. 1975 Jul;14(13):2996–3005. doi: 10.1021/bi00684a032. [DOI] [PubMed] [Google Scholar]
  26. Roberts D. M., Burgess W. H., Watterson D. M. Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins. Plant Physiol. 1984 Jul;75(3):796–798. doi: 10.1104/pp.75.3.796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rosenfeld A. C., Zackroff R. V., Weisenberg R. C. Magnesium stimulation of calcium binding to tubulin and calcium induced depolymerization of microtubules. FEBS Lett. 1976 Jun 1;65(2):144–147. doi: 10.1016/0014-5793(76)80466-8. [DOI] [PubMed] [Google Scholar]
  28. Salmon E. D., Segall R. R. Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus). J Cell Biol. 1980 Aug;86(2):355–365. doi: 10.1083/jcb.86.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schleicher M., Lukas T. J., Watterson D. M. Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare). Plant Physiol. 1983 Nov;73(3):666–670. doi: 10.1104/pp.73.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schliwa M., Euteneuer U., Bulinski J. C., Izant J. G. Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1037–1041. doi: 10.1073/pnas.78.2.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Eldik L. J., Piperno G., Watterson D. M. Similarities and dissimilarities between calmodulin and a Chlamydomonas flagellar protein. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4779–4783. doi: 10.1073/pnas.77.8.4779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Van Eldik L. J., Zendegui J. G., Marshak D. R., Watterson D. M. Calcium-binding proteins and the molecular basis of calcium action. Int Rev Cytol. 1982;77:1–61. doi: 10.1016/s0074-7696(08)62463-8. [DOI] [PubMed] [Google Scholar]
  33. Wallace A. D., Shapira R., Fritz R. B. Isolation and characterization of rabbit antibodies to bovine myelin basic protein. Immunochemistry. 1978 Jan;15(1):47–54. doi: 10.1016/0161-5890(78)90025-1. [DOI] [PubMed] [Google Scholar]
  34. Watterson D. M., Iverson D. B., Van Eldik L. J. Spinach calmodulin: isolation, characterization, and comparison with vertebrate calmodulins. Biochemistry. 1980 Dec 9;19(25):5762–5768. doi: 10.1021/bi00566a015. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1867–1871. doi: 10.1073/pnas.75.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Tubulin and calmodulin. Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J Cell Biol. 1979 Jun;81(3):624–634. doi: 10.1083/jcb.81.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Willingham M. C., Wehland J., Klee C. B., Richert N. D., Rutherford A. V., Pastan I. H. Ultrastructural immunocytochemical localization of calmodulin in cultured cells. J Histochem Cytochem. 1983 Apr;31(4):445–461. doi: 10.1177/31.4.6338107. [DOI] [PubMed] [Google Scholar]
  39. Wolniak S. M., Hepler P. K., Jackson W. T. Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline. J Cell Biol. 1980 Oct;87(1):23–32. doi: 10.1083/jcb.87.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wolniak S. M., Hepler P. K., Jackson W. T. Ionic changes in the mitotic apparatus at the metaphase/anaphase transition. J Cell Biol. 1983 Mar;96(3):598–605. doi: 10.1083/jcb.96.3.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zavortink M., Welsh M. J., McIntosh J. R. The distribution of calmodulin in living mitotic cells. Exp Cell Res. 1983 Dec;149(2):375–385. doi: 10.1016/0014-4827(83)90350-6. [DOI] [PubMed] [Google Scholar]

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