Abstract
Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10) was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical MTs of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these MTs, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized MTs were lysed in the presence of various concentrations of calcium and examined for the association of CaM with cortical MTs. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical MTs, whereas at 3.6 [mu]M calcium, this association was completely abolished. Control experiments were performed to eliminate alternate explanations including differential antibody binding in the presence of calcium and/or taxol, detergent-induced redistribution of antigen, and epitope masking. The results are discussed in terms of a model in which CaM associates with MTs via two types of interactions, one that occurs in the presence of calcium and another that occurs only in its absence.
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- Alexander K. A., Wakim B. T., Doyle G. S., Walsh K. A., Storm D. R. Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein. J Biol Chem. 1988 Jun 5;263(16):7544–7549. [PubMed] [Google Scholar]
- 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]
- Gilroy S., Read N. D., Trewavas A. J. Elevation of cytoplasmic calcium by caged calcium or caged inositol triphosphate initiates stomatal closure. Nature. 1990 Aug 23;346(6286):769–771. doi: 10.1038/346769a0. [DOI] [PubMed] [Google Scholar]
- Greenlee D. V., Andreasen T. J., Storm D. R. Calcium-independent stimulation of Bordetella pertussis adenylate cyclase by calmodulin. Biochemistry. 1982 May 25;21(11):2759–2764. doi: 10.1021/bi00540a028. [DOI] [PubMed] [Google Scholar]
- Harper J. F. Antigenic structure of calmodulin: production and characterization of antisera specific for plant calmodulins or Ca2+-replete vs. Ca2+-free calmodulins. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(1):3–17. [PubMed] [Google Scholar]
- Hulen D., Baron A., Salisbury J., Clarke M. Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum. Cell Motil Cytoskeleton. 1991;18(2):113–122. doi: 10.1002/cm.970180206. [DOI] [PubMed] [Google Scholar]
- 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]
- Kumagai H., Nishida E. The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer. J Biochem. 1979 May;85(5):1267–1274. [PubMed] [Google Scholar]
- 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]
- Margolis R. L., Rauch C. T., Job D. Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior. Proc Natl Acad Sci U S A. 1986 Feb;83(3):639–643. doi: 10.1073/pnas.83.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marusich M. F. Efficient hybridoma production using previously frozen splenocytes. J Immunol Methods. 1988 Nov 10;114(1-2):155–159. doi: 10.1016/0022-1759(88)90167-6. [DOI] [PubMed] [Google Scholar]
- Melan M. A., Sluder G. Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy. J Cell Sci. 1992 Apr;101(Pt 4):731–743. doi: 10.1242/jcs.101.4.731. [DOI] [PubMed] [Google Scholar]
- Morejohn L. C., Fosket D. E. Higher plant tubulin identified by self-assembly into microtubules in vitro. Nature. 1982 Jun 3;297(5865):426–428. doi: 10.1038/297426a0. [DOI] [PubMed] [Google Scholar]
- Morejohn L. C., Fosket D. E. Inhibition of Plant Microtubule Polymerization in vitro by the Phosphoric Amide Herbicide Amiprophos-Methyl. Science. 1984 May 25;224(4651):874–876. doi: 10.1126/science.224.4651.874. [DOI] [PubMed] [Google Scholar]
- Ranjeva R., Refeno G., Boudet A. M., Marmé D. Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5222–5224. doi: 10.1073/pnas.80.17.5222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts D. M., Zielinski R. E., Schleicher M., Watterson D. M. Analysis of suborganellar fractions from spinach and pea chloroplasts for calmodulin-binding proteins. J Cell Biol. 1983 Nov;97(5 Pt 1):1644–1647. doi: 10.1083/jcb.97.5.1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van Eldik L. J., Burgess W. H. Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virus-transformed fibroblasts. J Biol Chem. 1983 Apr 10;258(7):4539–4547. [PubMed] [Google Scholar]
- Van Eldik L. J., Wolchok S. R. Conditions for reproducible detection of calmodulin and S100 beta in immunoblots. Biochem Biophys Res Commun. 1984 Nov 14;124(3):752–759. doi: 10.1016/0006-291x(84)91022-2. [DOI] [PubMed] [Google Scholar]
- Vantard M., Lambert A. M., De Mey J., Picquot P., Van Eldik L. J. Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus. J Cell Biol. 1985 Aug;101(2):488–499. doi: 10.1083/jcb.101.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]