CSM News Electronic Edition Volume 4, number 20 June 3, 1995 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 at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== CHARACTERIZATION, SUBCELLULAR LOCALIZATION AND DEVELOPMENTAL REGULATION OF A CYSTEINE PROTEINASE FROM Dictyostelium discoideum. Darshini P. Mehta, James R. Etchison, and Hudson H. Freeze La Jolla Cancer Research Foundation 10901 North Torrey Pines Road, La Jolla, California 92037 Arch. Biochem. Biophys, in press. Summary Previous studies showed that vegetative cells of Dictyostelium discoideum make a cysteine proteinase, called proteinase-1, which contains multiple residues of GlcNAc-1-P linked directly to peptidyl serines. As a prelude to understanding the function of this novel carbohydrate modification, we purified and extensively characterized this proteinase in terms of its enzymatic activity, subcellular localization and developmental regulation. The purified enzyme has an apparent molecular weight of 38 kDa in heat denatured, reducing SDS/PAGE, and 55 kDa under non-reducing conditions. Native gel electrophoresis and isoelectric focusing revealed two protein bands with equal activity and having pI's of 2.5 and 2.6. Even more complex patterns are found in non- heat denatured SDS/PAGE gels. However, partial amino acid sequencing of the purified protein gave predominantly a single sequence. The enzyme is inhibited by E-64, TLCK, TPCK and leupeptin, has a pH optimum of 5.0, and co-fractionates with lysosomal enzymes in bacterially grown cells. It appears to comprise about 90% of the total cysteine proteinase activity in cells at a time when the cells have just finished clearing the bacterial lawn. Prior to this point and after the onset of development, its level is 2-20 fold lower. This remarkably fine regulation parallels the developmental regulation of other cysteine proteinases in Dictyostelium. Based on these results it appears that proteinase-1 may be primarily used for specialized proteolysis just before the onset of development rather than for simply digesting the bacteria for food. ----------------------------------------------------------------------- Three Dimensional Localization of Wild-Type and Myosin II Mutant Cells during Morphogenesis of Dictyostelium David A. Knecht and Eric Shelden Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269 Developmental Biology, in press Abstract Dictyostelium amoebae that lack myosin II (mhcA-) are unable to undergo morphogenesis. The cells aggregate slowly to form hemispherical mounds, but the mounds never extend a tip upward. Expression of developmentally regulated genes appears normal in the absence of morphogenesis. When mixed with an excess of wild-type cells, some mutant cells form differentiated spores, however rescue is extremely inefficient (Knecht and Loomis, 1988). In order to assess how morphogenesis is normally accomplished and why mutants lacking myosin II cannot develop, a new method has been developed that allows individual amoebae to be localized and tracked at high resolution within the multicellular organism during development. Amoebae are labeled with a fluorescent dye at the beginning of starvation, mixed with an excess of unlabeled cells and allowed to develop. The 3 dimensional position of labeled cells in the multicellular organism is then determined using a laser scanning confocal microscope (LSCM). Using this methodology, we have shown that labeled wild-type cells are randomly distributed throughout the organism and complete development normally. When labeled mhcA- mutant cells are mixed with a 20 fold excess of wild type cells, they are non-randomly localized even at the earliest stages of development. Mutant cells in aggregation streams are found primarily at the edges of the streams and many cells never become part of the streams, or are left behind as the wild-type cells complete aggregation. Those that are incorporated into the aggregate are found at the edge and base, the backs of slugs and the base of the fruiting bodies. A few mutant cells can be found in the sorus where they presumably become spores. The segregation of mhcA- mutant cells to the outside of wild-type aggregation streams argues that the mutant cells are unable to penetrate a mass of adhered, wild-type cells. We hypothesize that mutant cells lacking cortical integrity are unable to generate sufficient protrusive force to break the adhesion of wild-type cells to each other. This would make the mutants incapable of moving through a mass of cells (either mutant or wild-type) or of changing shape when adhered to other cells. We propose that mutants lacking myosin II are unable to accomplish morphogenesis because they cannot move correctly in a three dimensional mass of adhered cells. --------------------------------------------------------------------- [End CSM-News, volume 4, number 20]