CSM News Electronic Edition Volume 3, number 13 October 15, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development. Pengxin Chen, Bruce D. Ostrow, Sherrie R. Tafuri and Rex L. Chisholm Dept. of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 Abstract Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment, et al. 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay. Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC coding region, leading to cells devoid of both the RMLC transcript and the 18 Kd RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to that from myosin heavy chain mutants, suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR- cells are able to cap cell surface receptors following con A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild type myosin, but the filaments then exhibited abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function. ---------------------------------------------------------------------- Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells. Bruce D. Ostrow, Pengxin Chen and Rex L. Chisholm Dept. of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 J. Cell Biol. in press Abstract In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate non-muscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium, phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed from an actin promoter on an integrating vector introduced into RMLC null Dictyostelium (mlcR-) cells. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wildtype RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions. ---------------------------------------------------------------------- Calcium uptake and gp80 mRNA destabilization follows cAMP receptor down regulation in Dictyostelium discoideum Glaucia Mendes Souza1, Claudette Klein2, Jose Carlos da Costa Maia1, and Aline Maria da Silva1, 1Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Sao Paulo, Brasil; 2Edward A. Doisy Department of Biochemistry and Molecular Biology, St.Louis University School of Medicine, St.Louis, Missouri Cellular Signalling, in press. Abstract The mechanism by which high concentrations of cAMP selectively destabilize the gp80 mRNA in Dictyostelium discoideum was investigated. This treatment which leads to down-regulation of the cAMP receptor was also found to cause an increase in calcium uptake, as measured using 45Ca2+. Given this observation, we sought a role for calcium as a second messenger in the degradation of the gp80 mRNA. Changes in the mRNA levels were examined by Northern blot after treating cells with compounds known to alter their intracellular Ca2+ concentrations. This included the use of A23187, Ca2+, 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate HCl (TMB-8), LiCl and 8-p- chlorophenylthioadenosine 3',5'-cyclic monophosphate (ClPhS- Ado-3':5'-P). The sum of the data suggests that it is the cAMP- induced influx of Ca2+ across the plasma membrane, as apposed to a cAMP-mediated release of Ca2+ from intracellular stores, that initiates gp80 mRNA degradation. Treatment of cells with Concanavalin A (ConA) to induce cAMP receptor down- regulation, also causes a reduction in gp80 mRNA levels and an increase in calcium uptake. ---------------------------------------------------------------------- Title Not Provided Knecht lab Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269 J. Cell Sci., in press ABSTRACT We have used fluorescent labeling, confocal microscopy and computer assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 um/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 um/min, twice as fast as reported previously for isolated myosin II mutant cells, and failed to display regular periodic movement. When removed from aggregation streams myosin II mutant cells moved at only 2.5 +/- 2.0 um/min, while wild-type cells under these conditions moved at 5.9 +/- 4.5 um/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams demonstrates both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. The distortion of myosin II mutant cells within wild-type aggregation streams further argues that myosin is not required for the formation of cell-cell contacts, although it remains possible that such attachments are not completely normal. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium. ---------------------------------------------------------------------- Nucleoside Diphosphate Kinase: Investigation of the Active Site and of the Conformational Stability by Site-Directed Mutagenesis Annemiek D. Tepper, Heike Dammann, Anthony A. Bominaar and Michel Veron Unite de Biochimie Cellulaire, CNRS-URA 1129, Institut Pasteur, 75724 Paris Cedex 15, France J. Biol. Chem., in press. Abstract Nucleoside diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of eight amino-acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124 and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis or substrate binding or both, previously proposed on the basis of the X-ray structure (Morera et al., 1994, Biochemistry, 33, 459-467). Furthermore, residues Lys16, Arg109 and Asn119 were identified to play an important role in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure, important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual role of Lys16, Arg109 and Asn119 in both substrate binding and subunit assembly provides further evidence for a functional coupling between catalytic activity and quaternary structure in NDP kinase. ----------------------------------------------------------------- AUTOPHOSPHORYLATION OF NUCLEOSIDE DIPHOSPHATE KINASE ON NON-HISTIDINE RESIDUES Anthony A. Bominaar, Annemiek D. Tepper and Michel Veron1. Unite de Biochimie Cellulaire, CNRS-URA 1129, Institut Pasteur, 75724 Paris cedex 15, France. FEBS Letters, in press Abstract Recently, several reports appeared which described auto-phosphorylation of NDP kinase on residues different from the active-site histidine. Based on these findings conclusions were drawn with respect to a regulation of enzyme activity and to a possible role as a metastasis suppressor. In this paper we show that although non-histidine autophosphorylation occurs on NDP kinases from mammals, lower eukaryotes and bacteria, less than 0.2% of the subunits are phosphorylated. Using site-directed mutagenesis, we show that the active site histidine is essential for non-histidine autophosphorylation. The low stoichiometry of phosphate incorporation excludes a role of autophosphorylation in regulating overall enzyme activity. -------------------------------------------------------------------- [End CSM News, volume 3, number 13]