Dicty News Electronic Edition Volume 19, number 12 November 30, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= An RGS-Containing Kinase is Important for Chemotaxis and Multicellular Development in Dictyostelium Binggang Sun and Richard A. Firtel in press: Molecular Biology of the Cell ABSTRACT We have identified a gene encoding RCK1, a novel RGS domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ~50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ~40%. Upon cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ~10 sec, similar to the kinetics of membrane localization of the PH domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g. Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Ga2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium. submitted by: Rick Firtel [rafirtel@ucsd.edu] ---------------------------------------------------------------------------- - THE ROLE OF BEACH PROTEINS IN DICTYOSTELIUM. Arturo De Lozanne Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. TRAFFIC, in press. ABSTRACT The BEACH family of proteins is a novel group of proteins with diverse roles in eukaryotic cells. The identifying feature of these proteins is the BEACH domain named after the founding members of this family, the mouse beige and the human Chediak-Higashi Syndrome (CHS) proteins. Although all BEACH proteins share a similar structural organization they appear to have very distinct cellular roles ranging from lysosomal traffic, to apoptosis and cytokinesis. Very little is currently known about the function of most of these proteins, few binding-partner proteins have been identified and no molecular mechanism for any of these proteins has been discovered. Thus, it is important to establish good model systems for the study of these novel proteins. Dictyostelium contains six BEACH proteins that can be classified into four subclasses. Two of them, LvsA and LvsB, have clearly distinct roles in the cell. LvsA is localized on the contractile vacuole membrane and is essential for cytokinesis and osmoregulation. LvsB is most similar in sequence to the mammalian beige/CHS proteins and shares with them a common function in lysosomal trafficking. Structural and functional analysis of these proteins in Dictyostelium will help elucidate the function of this enigmatic novel family of proteins. submitted by: (a.delozanne@mail.utexas.edu) ---------------------------------------------------------------------------- - The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus Masashi Fukuzawa+, Tomoaki Abe+ and Jeffrey G. Williams*, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK + These two authors contributed equally to this work DEVELOPMENT IN PRESS Summary Dd-STATc becomes tyrosine phosphorylated, dimerises and accumulates in the nuclei of Dictyostelium cells exposed to DIF, the chlorinated hexaphenone that directs prestalk cell differentiation. By performing cytoplasmic photobleaching of living cells, we show that DIF inhibits the nuclear export of Dd-STATc. Within Dd-STATc there is a 50 amino acid region containing several consensus CRM1 (exportin 1)-dependent nuclear export signals (NESs). Deletion of this region causes Dd-STATc to accumulate in the nucleus constitutively and, when coupled to GFP, the same region directs nuclear export. We show that the N terminal-proximal 46 amino acids are necessary for nuclear accumulation of Dd-STATc and sufficient to direct constitutive nuclear accumulation when fused to GFP. Combining the photobleaching and molecular analyses, we suggest that DIF-induced dimerisation of Dd-STATc functionally masks the NES-containing region and that this leads to nett nuclear accumulation, directed by the N terminal-proximal import signals. These results show that the regulated nuclear accumulation of a STAT protein can be controlled at the level of nuclear export and they also provide a better understanding of the mechanism whereby DIF directs cell type divergence. submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] ---------------------------------------------------------------------------- - Mitochondrial membrane dynamics are altered in cluA- mutants of Dictyostelium. Fields, S.D., Arana, Q., Heuser, J., and Clarke, M. Musc. Res. Cell Motil. (special Dictyostelium issue), in press. In cluA- mutants of Dictyostelium, mitochondria are clustered near the cell center rather than being dispersed throughout the cytoplasm. We have examined two possible mechanisms that could account for this phenotype. First, we sought evidence that the cytoskeleton or a presumptive mitochondrion- cytoskeleton linkage was altered in mutant cells. We found that cytoskeletal structures in cluA- cells appeared normal by immunostaining, and that the distribution of peroxisomes in mutant cells was indistinguishable from that in wild type cells. Treatment of wild type cells with drugs that disrupted microtubules or actin filaments did not mimic the cluA- phenotype. Thus, cytoskeletal defects seemed unlikely to account for the mitochondrial clustering in cluA- cells. Observation of the movement of GFP-tagged mitochondria in wild type cells suggested that mitochondria are transported along microtubules, as in mammalian cells, rather than along actin filaments, as in budding yeast. Therefore, the similar phenotypes of cluA- Dictyostelium cells and clu1D yeast cells argued against CluA/Clu1p acting as a mitochondrion-cytoskeleton linker. We next examined the ultrastructure of mitochondria in freeze-substituted, thin-sectioned cells. We found that the clustered mitochondria in cluA-cells are interconnected. Often, adjacent mitochondria are linked by narrow membranous strands, although sometimes the mitochondria are partially merged. The presence of narrow constrictions at presumptive division sites argues that the constriction step of division proceeds normally. Our data suggest that cluA- cells may be blocked at a very late step in fission of the outer mitochondrial membrane. submitted by: Margaret Clarke, PhD [Margaret-Clarke@mail.omrf.ouhsc.edu] ---------------------------------------------------------------------------- - Chaperonin 60 and mitochondrial disease in Dictyostelium. Martha Kotsifas, Christian Barth, Arturo de Lozanne, Sui T. Lay, and Paul R. Fisher Journal of Muscle Research and Cell Motility. In Press. Abstract The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a 3-D model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities. submitted by: Paul Fisher [SMTP:fisher@lumi.micro.latrobe.edu.au] ---------------------------------------------------------------------------- - Multiple Signalling Pathways Connect Chemoattractant Receptors and Calcium Channels in Dictyostelium Thomas Nebl1, Martha Kotsifas1, Pauline Schaap2? and Paul R. Fisher1,* Journal of Muscle Research and Cell Motility. In Press. SUMMARY Dictyostelium mutants expressing aequorin were used to study and compare the roles of heterotrimeric G-proteins and the second messengers IP3 and cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals. The calcium responses of vegetative cells to folate were dramatically impaired in Gb and Ga4 null mutants but were restored with altered kinetics and temperature-sensitivity in Gb null mutants overexpressing wild type and temperature-sensitive Gb isoforms. Folic acid receptors thus mediate changes in [Ca2+]i via a Ga4bg-dependent pathway. Neither folate nor cAMP- induced [Ca2+]i signals were significantly altered in PLC null transformants, but [Ca2+]i changes elicited by both attractants were significantly prolonged in two stmF mutants lacking cGMP-specific phosphodiesterase activity. This confirms an important role of cGMP in regulating receptor-activated Ca2+ uptake and/or extrusion systems. This cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally down-regulated and all but disappeared by the time the cells reached full aggregation competence after 8 hours of starvation. The results suggest that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a complex manner via multiple signalling pathways, one that is G-protein- and cGMP-dependent (present at the vegetative and early poststarvation stage) and another that is G-protein-independent (dominant in fully aggregation-competent cells at ~ 8 hours post starvation). submitted by: Paul Fisher [SMTP:fisher@lumi.micro.latrobe.edu.au] ---------------------------------------------------------------------------- - [End Dicty News, volume 19, number 12]