Calmodulin in the Ciliates Paramecium Tetraljrelia and Tetrahymena Thermophila*

CALMODULIN IN THE CILIATES PARAMECIUM TETRALJRELIA AND TETRAHYMENA T H E R M O P HILA* Nita J . Maihle and Birgit H. Satir Department of Anatomy Abert Einstein College of Medicine Bronx, New York 10461

Calmodulin is a protein of special interest, since it is now known that calcium is involved in the regulation of biological processes such as secretion and ciliary motility.8 A more detailed account of the physiology of these processes and a possible mechanism for the involvement of calmodulin in secretion are described in Satir et 01.' In this brief report, we present evidence for the presence and localization of calmodulin in cilia, cortex, and calcium containing crystals in Paramecium tetraurelia and in cilia and cortex of Tetrahymena thermophila. Preliminary reports of these findings for Paramecium have been presented at the Society of Protozoologists' and at American Society of Cell Biologists.s Indirect immunofluorescent localization was performed using goat anticalmodulin supplied by Drs. 1. Dethnan and A. Means, and rhodamine-conjugated rabbit anti-goat IgG as the secondary label (Cappel Laboratories, Pa.). All cells were harvested during early stationary phase and were prepared for immunofluorescence following the methodology described in Maihle et aL4 For all experiments, the following controls were performed in order to insure the immunospecificity of the results: 1)incubation in 2" Ab alone, 2) incubation in a nonabsorbed fraction from the affinity column used in the purification of anticalmodulin, and 3) preabsorption of the 1" Ab in a 6 M excess of mammalian calmodulin. Cells were observed with a Zeiss Universal light microscope and all control cells displayed only a diffuse low level of fluorescence. In experimental aliquots of cells treated with anticalmodulin and 2' Ab, fluorescence is seen in three distinct regions of Paramecium6 and in two distinct 3a & h Satir et a1.'). In Paramecium, label is regions in Tetrahymena (cf. FIGURES present in calcium containing birefringent crystals which are of uniform diameter. These labeled crystals vary in number within the cell ranging from 0-5/cell. The fluorescent images are typically spherical in shape, measuring approximately 10-15 pm in diameter and are present in both axenic and monoaxenic cultures of Paramecium but absent in Tetrahymena. In cilia, both somatic and oral are consistently fluorescent along the entire length of each cilium. Finally, label is present in a linear punctuate pattern of fluorescent spots in both normal and deciliated cells. Based upon the highly organized nature of the cortex of these cells, we currently interpret this pattern of localization to represent the basal bodies of the cell. Calmodulin is present in both ciliates and has been partially purified from both organisms by heating at 80°C for 6 min and ammonium sulfate precipitation. The protein from both genera exhibits an apparent molecular weight of 17.0 Kd which is lower than that of purified bovine brain calmodulin at 18.5 Kd using SDS 15% PAGE analysis. The partially purified protein exhibits a mobility shift

*Supported U S . Public Health Service Grants GMS 24724 and 27298.

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in the presence of 10 FM calcium in SDS PAGE and both preparations stimulate mammalian phosphodiesterase activity. The ability of Paramecium and Tetrahymena extracts to stimulate mammalian phosphodiesterase activity, along with the presence of an appropriate ca. 17 Kd heat-stable protein whose apparent molecular weight shifts in EGTA versus CaCI,, and the affinity of both a Paramecium and Tetrahymena component for anticalmodulin prepared in response to antigen isolated from mammalian tissue,' clearly supports the conclusion that calmodulin, chemically and antigenically similar to mammalian calmodulin, is present in both ciliates. We are currently pursuing the role of calmodulin in Paramecium and Tetrahymena by determining which proteins in subcellular fractions of these cells interact with calmodulin. We have examined whole cell extracts and ciliary fractions of Tetrahymena using immobilized bovine brain calmodulin. There are approximately 12 soluble proteins present in whole cell Tetrahymena extracts that bind to the immobilized calmodulin in a calcium-dependent manner.6 The most prominent of these proteins within the cell appears to originate from the cilia as determined by using both axonemal and ciliary membrane preparations3 These data will aid in providing insights into the role of calmodulin in calciummediated processes and may yield valuable information regarding the fundamental functional significance of calmodulin in eukaryotic cells.

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DEDMAN. J. R.. M. 1. WELSH & A. R. MEANS. 1978. Ca2+-dependentregulator. Production and characterization of a monospecific antibody. J. Biol. Chem. 253: 7515-7521. MAIHLE, N. 1. & B. H. SATIR. 1979. Indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia. J. Protozool. 26( 18): 10a. MAIHLE, N. J., J. AVOLIO, 1. L. SALISBURY, P. SATIR& B. H. SATIR.1980. Isolation of calmodulin regulated ciliary proteins from Tetrahymena thermophila using affinity chromatography. (In preparation.) MAIHLE, N. I., 1. DEDMAN, A. R. MEANS& B. H. SATIR. 1980. Biochemical characterization and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia. (Submitted for publication.] MAIHLE, N. 1,. R. S. GAROFALO & B. H. SATIR. 1979. Biochemical characterization and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia. 1. Cell Biol. 83(2): 476,22919, MAIHLE, N. J,, 1. L. SALISBURY & B. H. SATIR.1980. Calmodulin in Tetrahymena. Isolation, purification, localization, and affinity chromatography. Second International Congress on Cell Biology. September, 1980. REED, W. & P. SATIR.1980. Calmodulin in mussel gill epithelial cells: role in cilary arrest. [This volume.) SATIR. B. H.. R. S. GAROFALO. D. M. GILLIGAN & N. 1. MAIHLE. 1980. Possible functions of calmodulin in protozoa. [This volume.)