CSM News Electronic Edition Volume 3, number 14 October 22, 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. ------------- Corrections ------------- There were two problems with the last edition of the newsletter the first was the omission of journal in which the Chen, Ostrow, Tafuri and Chisholm paper will be published. It is in press in J. Cell Biol. The second was the absence of title and author list for the abstract from Dave Knecht's lab. The entire abstract will all of the data appears below: Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis Eric Shelden and David A. Knecht Department of Molecular and Cell Biology University of Connecticut Storrs, CT 06269 J. Cell Science, 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. =========== Abstracts =========== Identification and characterization of multiple A/T-rich cis-acting elements that control expression from Dictyostelium Actin promoters: The Dictyostelium Actin Upstream Activating Sequence confers growth-phase expression and has enhancer-like properties Roderick Hori and Richard A. Firtel Department of Biology, Center for Molecular Genetics, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0634 Nucleic Acids Research, in press. Summary The promoter elements in the Dictyostelium Actin 15 and Actin 6 genes required for full growth-phase expression were identified by assaying promoter/luciferase reporter constructs. We find that these promoters contain common cis-acting elements, an Actin Upstream Activating Sequence (UAS) and sequences proximal to the transcription startsite that overlap with a poly dT region. The Actin 15 promoter has two additional cis-acting elements not present in the Actin 6 promoter that may account for the higher level of expression from the Actin 15 promoter. All of the identified promoter elements are unusual for Dictyostelium in that they are all A/T-rich. Two cis-acting elements, the Actin UAS and the poly dT domain were studied in greater detail. The Actin UAS was tested on a heterologous promoter from the prespore-specific gene SP60 and shown to have the ability to confer growth-phase expression. The Actin UAS also exhibited the ability to function in a distance- and orientation-independent manner and activate expression synergistically when present in two copies. The poly dT domain of the Actin 15 promoter was studied in greater detail by using a genetic selection scheme to define parameters that effect the strength of this element. This element is comprised of 45 consecutive dT residues immediately upstream of the putative TATA box. We show that the length of the homopolymer dT region correlates with the expression level of the promoter. The poly dT element is also shown to function to promote wild-type levels of expression with small deviations in the sequence, indicating that the element is not required to be homopolymeric to function. ---------------------------------------------------------------------- [End CSM News, vol. 3, number 14]