Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis

T Liu, JG Williams and M Clarke

Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73121.

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.

Volume 3, Issue 12, pp. 1403-1413, 12/01/1992
Copyright © 1992 by The American Society for Cell Biology

This article has been cited by other articles:

				W. Zhang and D. N. Robinson Balance of actively generated contractile and resistive forces controls cytokinesis dynamics PNAS, May 17, 2005; 102(20): 7186 - 7191. [Abstract] [Full Text] [PDF]

				A. Nagasaki and T. Q.P. Uyeda DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium Mol. Biol. Cell, February 1, 2004; 15(2): 435 - 446. [Abstract] [Full Text] [PDF]

				L. Aubry, S. Mattei, B. Blot, R. Sadoul, M. Satre, and G. Klein Biochemical Characterization of Two Analogues of the Apoptosis-linked Gene 2 Protein in Dictyostelium discoideum and Interaction with a Physiological Partner in Mammals, Murine Alix J. Biol. Chem., June 7, 2002; 277(24): 21947 - 21954. [Abstract] [Full Text] [PDF]

				J. M. Gorlach, H. C. McDade, J. R. Perfect, and G. M. Cox Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy Microbiology, January 1, 2002; 148(1): 213 - 219. [Abstract] [Full Text] [PDF]

				A. Angers-Loustau, J.-F. Cote, A. Charest, D. Dowbenko, S. Spencer, L. A. Lasky, and M. L. Tremblay Protein Tyrosine Phosphatase-PEST Regulates Focal Adhesion Disassembly, Migration, and Cytokinesis in Fibroblasts J. Cell Biol., March 8, 1999; 144(5): 1019 - 1031. [Abstract] [Full Text] [PDF]

				K. B. Shannon and R. Li The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast Mol. Biol. Cell, February 1, 1999; 10(2): 283 - 296. [Abstract] [Full Text]

				J Moniakis, M. Coukell, and A Janiec Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum J. Cell Sci., January 2, 1999; 112(3): 405 - 414. [Abstract] [PDF]

				D. C. Wienke, M. L.W. Knetsch, E. M. Neuhaus, M. C. Reedy, and D. J. Manstein Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis in Dictyostelium discoideum Mol. Biol. Cell, January 1, 1999; 10(1): 225 - 243. [Abstract] [Full Text]

				M. A. Osman and R. A. Cerione Iqg1p, a Yeast Homologue of the Mammalian IQGAPs, Mediates Cdc42p Effects on the Actin Cytoskeleton J. Cell Biol., July 27, 1998; 142(2): 443 - 455. [Abstract] [Full Text] [PDF]

				H. Adachi, Y. Takahashi, T. Hasebe, M. Shirouzu, S. Yokoyama, and K. Sutoh Dictyostelium IQGAP-related Protein Specifically Involved in the Completion of Cytokinesis J. Cell Biol., May 19, 1997; 137(4): 891 - 898. [Abstract] [Full Text] [PDF]

				G. Buczynski, B. Grove, A. Nomura, M. Kleve, J. Bush, R. A. Firtel, and J. Cardelli Inactivation of Two Dictyostelium discoideum Genes, DdPIK1 and DdPIK2, Encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, Results in Defects in Endocytosis, Lysosome to Postlysosome Transport, and Actin Cytoskeleton Organization J. Cell Biol., March 24, 1997; 136(6): 1271 - 1286. [Abstract] [Full Text] [PDF]

				Y Xie, M. Coukell, and Z Gombos Antisense RNA inhibition of the putative vacuolar H(+)-ATPase proteolipid of Dictyostelium reduces intracellular Ca2+ transport and cell viability J. Cell Sci., January 2, 1996; 109(2): 489 - 497. [Abstract] [PDF]

				C. Fulton, E. Y. Lai, and S. P. Remillard A Flagellar Calmodulin Gene of Naegleria, Coexpressed during Differentiation with Flagellar Tubulin Genes, Shares DNA, RNA, and Encoded Protein Sequence Elements J. Biol. Chem., March 17, 1995; 270(11): 5839 - 5848. [Abstract] [Full Text] [PDF]

				Q Zhu, T Liu, and M Clarke Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum J. Cell Sci., January 4, 1993; 104(4): 1119 - 1127. [Abstract] [PDF]