CSM News Electronic Edition Volume 9, number 3 July 19, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============================================ Dicty Researcher Database Now Back On-Line ============================================ The searchable Dicty research community database is now back on line after a software upgrade. Give it a try--check out your entry. If you have not updated your entry, just follow this link: http://apps.basic.northwestern.edu/dicty/ =============== Job Available =============== INDIAN INSTITUTE OF SCIENCE BANGALORE 560 012 Applications are invited from Indian nationals preferably below the age of 35 years for faculty positions at the level of Assistant Professor in the Division of Biological Sciences. Those with interest in the following areas may apply: (a) Genetics and developmental biology of higher organisms including humans. (b) Primate reproductive biology, reproductive immunology, endocrinology, molecular reproduction; cardiovascular diseases, neuroendocrinology using non-human primates. (c) Macromolecular structure determination by NMR and other biophysical methods, biological mass spectrometry and related areas. (d) Computer modeling of biomolecules and related areas of biomolecular structure and interactions. The selected candiates are expected to develop and maintain independent research in the concerned area as well as collaborate with other faculty and contribute to the teaching programme. Small start up funds could be provided. The candidates should have Ph.D. degreee with about 3 years post- doctoral experience. The total emoluments at the minimum of the scale of Rs.3700-125-4950-150-5700 (likely to be revised) are around Rs.1,32,000/- per annum. Interested persons should send their curriculum vitae, list of publications, important reprints, a brief description of the research programme and names of at least three referees with their full address including fax and e- mail address to Prof. M. Vijayan, Chairman, Division of Biological Sciences, Indian Institute of Science, Bangalore 560 012, India, within 45 days of the appearance of this advertisement. The post in which the candidate is interested should be indicated in the covering letter. The referees may be requested to send their assessment directly to Prof Vijayan (E.mail: dcbio@admin.iisc.ernet.in OR Fax: 91-80-3340416) The following persons may be contacted for further details. Posts (a) & (b) : Prof.A.J.Rao, Chairman, CRBME/DBGL E-mail: ajrme@crbme.iisc.ernet.in Posts (c) & (d) : Prof.P. Balaram, Chairman, Molecular Biophysics Unit E-mail: pb@mbu.iisc.ernet.in =========== Abstracts =========== Cell shape, motility and distribution of F-actin in amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea. A comparison with Dictyostelium discoideum Christian Zuppinger and Urs-Peter Roos Institut für Pflanzenbiologie, Universität Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland European J. Protistology, in press. SUMMARY We investigated, by a computer-assisted video analysis, by F-actin cytochemistry, and by scanning electron microscopy (SEM), the dynamic morphology, the locomotory behavior, and the actin cytoskeleton of trophic amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea, and compared them with amoebae of the cellular slime mold Dictyostelium discoideum. The three species represent protist taxa marked by different reproductive systems and by differences of amoeboid motility in the non-reproductive phase of the life cycle. Live observations and the numerical analysis of video recordings revealed characteristic traits of cell shape changes and motility in each species. The leading edge of Protostelium amoebae is dominated by a large lamellipodium that is studded with pseudodigits. We computed a mean cell size (outline area) of 498 µm2 and a mean velocity of 26.5 µm/min (centroid displacement) for this species (N=30, as for the other organisms). Most amoebae followed a sinuous path during the observation period, which resulted in a mean standard deviation of vector direction (SDV) of 0.64, but some hardly moved at all. Amoebae of Acrasis are essentially monopodial, with lobose, smooth pseudopodia. Their mean cell size was 759 µm2 and their mean velocity 71.6 µm/min. They are thus among the fastest ‘crawling' eucaryotic cells, which may be related to the fact that they contain no cytoplasmic microtubules. Most amoebae followed a straight path, but some made one or two abrupt turns during the observation period (SDV = 0.58). The corresponding values for Dictyostelium amoebae with filose or lobose pseudopodia were 220 µm2, 10.3 µm/min, and SDV = 0.80. In amoebae of all the three species F-actin was localized in the cell cortex, especially in hyaline pseudopodia. As seen by SEM, Protostelium amoebae had the largest area of contact with the substratum, whereas Acrasis amoebae adhered with a foot-like structure, the leading edge and the uropod being detached from the support. --------------------------------------------------------------------- 14-3-3 Inhibits the Dictyostelium Myosin II Heavy Chain Specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14-3-3 Binding Domain Meirav Matto-Yelin#, Alastair Aitken+ and Shoshana Ravid#* #Department of Biochemistry, Hadassah Medical School, The Hebrew University Jerusalem 91120, Israel and +Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, U. K. Mol. Biol. Cell, in press Summary Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP [Abu-Elneel et al (1996) J. Biol. Chem. 271:977-984]. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show here that one of these proteins is a homologue of the 14-3-3 protein (Dd14-3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14-3-3 z isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14-3-3 in the cytosol in a cAMP-dependent manner while the membrane-bound active MHC-PKC was not found in a complex with Dd14-3-3. This suggests that Dd14-3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14-3-3 as well as 14-3-3z through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14-3-3 family, and demonstrate that MHC-PKC interacts directly with Dd14-3-3 and 14-3-3z through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase. --------------------------------------------------------------------- A Novel Role for Clathrin in Cytokinesis Maria L. Niswonger and Theresa J. O˜Halloran Department of Cell Biology, Duke University Medical Center, Durham, NC PNAS, in press Abstract Using clathrin-minus Dictyostelium cells, we identified a novel requirement for clathrin during cytokinesis. In suspension culture, clathrin-minus cells failed to divide and became large and multinucleate. This cytokinesis deficiency was not attributable to a pleiotropic effect on the actomyosin cytoskeleton, since other cellular events driven by myosin II (e.g., cortical contraction and capping of concanavalin A receptors) remained intact in clathrin-minus cells. Examination of cells expressing myosin II tagged with green fluorescent protein demonstrated that clathrin-minus cells failed to assemble myosin II into a functional contractile ring. This inability to localize myosin II to a particular location was specific for cytokinesis, as clathrin-minus cells moving across a substrate localized myosin II properly to their posterior cortexes. These results demonstrate that clathrin is essential for construction of a functional contractile ring during cell division. --------------------------------------------------------------------- Characterization of the Dictyostelium discoideum cellulose-binding protein CelB and regulation of the gene's expression Ramachandran Ramalingam@ and Herbert L. Ennis§ Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110 Present addresses: @Pulmonary Research Laboratory, Cornell University Medical College, 520 East 70th Street, New York, NY 10021; §Department of Anatomy and Cell Biology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, NY 10032 SUMMARY Similar to other stages of Dictyostelium development, spore germination is a particularly suitable model for studying regulation of gene expression. The transition from spore to amoeba is accompanied by developmentally regulated changes in both protein and mRNA synthesis. A number of spore germination-specific cDNAs have been isolated previously. Among these are 2 members of the 270 gene family, a group of four genes defined by the presence of a common tetrapeptide repeat of threonine-glutamic acid-threonine-proline. celA (formerly called 270-6) and celB (formerly 270-11) are expressed solely and coordinately during spore germination, the levels of the respective mRNAs being low in dormant spores, rising during germination to a maximum level at about 2 h, and then rapidly declining as amoebae are released from spores. The mRNAs are not found in growing cells or during multicellular development. The rapidity with which these transcripts accumulate and then disappear during germination implies that the respective products may be important for the process. We reported previously that the CelA protein is a cellulase [endo-(1,4)-ß-glucanase (EC 3.2.1.4)]. In the present investigation properties of the CelB protein, a glycosylated protein of 532 amino acids, 36% of which are serine or threonine, were examined; and the upstream sequences involved in the developmental regulation of the expression of the gene have been determined. The CelB protein does not demonstrate cellulase activity, but it has a cellulose binding domain. Its role, if any, in degradation of the cellulose-containing spore wall is unknown. To identify cis-acting elements in the celB promoter, unidirectional 5' deletions of the celB upstream non-coding region were constructed and used to transform amoebae. Analysis of promoter activity during different stages of development shows that a short, very A/T-rich sequence of approximately 81 bp1 is sufficient for spore-specific celB transcription.the ained in this sequence is 4 Contained in this sequence is the Myb oncogene protein binding site, TAACTG, which was shown previously to be a negative regulator of celA transcription. Dictyostelium and mouse Myb proteins bind to this region of the promoter, suggesting that Myb might regulate celB gene expression negatively as it does in celA. --------------------------------------------------------------------- A ubiquitin conjugating enzyme is essential for developmental transitions in Dictyostelium Alexandra Clark, Anson Nomura, Sudhasri Mohanty, and Richard A. Firtel Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634 Mol. Biol. Cell, in press. ABSTRACT We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB null cells. ubcB null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ~10 h prior to forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared to that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells (ALCs), a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB null cells being present as a mass of cells located in the very posterior of the developing organism. The amino acid sequence analysis of the UbcB ORF and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation. --------------------------------------------------------------------- Activation of the MAP kinase ERK2 by the chemoattractant folic acid in Dictyostelium Mineko Maeda1,2 and Richard A. Firtel2,3 1Department of Biology, Graduate School of Science, Osaka University Machikaneyama-cho 1-16, Toyonaka Osaka 560 JAPAN 2Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634, USA J. Biological Chemistry, in press. ABSTRACT The Dictyostelium MAP kinase ERK2 is activated by extracellular cAMP in aggregation-competent cells and is required for receptor activation of adenylyl cyclase (1-3) . This cAMP-dependent activation of ERK2 is mediated by the serpentine, G protein-coupled cAMP receptors (cARs). However, ERK2 activation by cAMP is at least partially heterotrimeric G protein-independent, with a level of activation in cells lacking the sole Gb subunit or the cAR-coupled Ga2 subunit that is ~50% that of wild-type cells (1,2) . Folic acid, a chemoattractant in the vegetative cells that enables amoebae to find bacteria in the wild, also triggers the activation of adenylyl cyclase, which is impaired in the vegetative cells lacking the Ga protein subunit Ga4 (4) . In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity. Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled Ga subunit Ga4 are maximally expressed. During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE. Interestingly, this activation is not elicited in the absence of Gb subunits, in contrast to the response to cAMP. This response also requires the Ga4 subunit known to be required for other folic acid-mediated responses (4) . Furthermore, we show that the activation of ERK2 by cAMP is independent of the Ga4 subunit, while the activation of ERK2 by folate is independent of Ga2. Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G protein-dependent and -independent pathways. --------------------------------------------------------------------- [End CSM News, volume 9, number 3]