Dicty News Electronic Edition Volume 18, number 10 June 1, 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. =========================================== DictyBase and Stock Center Funded By NIH =========================================== We are pleased to announce that the National Institutes of Health has notified us that they will be funding both DictyBase and The Dictyostelium stock center. The support of the Dictyostelium community has been instrumental in obtaining the support for these resources. We look forward to working with you to establish these resources. Rex Chisholm, DictyBase Jakob Franke and Rich Kessin, Stock Center ============= Abstracts ============= The BEACH Family of Proteins: Phylogenetic & Functional Analysis of Six Dictyostelium BEACH Proteins. Ning Wang, Wei-I Wu, and Arturo De Lozanne Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. a.delozanne@mail.utexas.edu Journal of Cellular Biochemistry, in press. ABSTRACT The BEACH-domain containing proteins constitute a new family of proteins found in all eukaryotes. The function of these proteins, which include the Chediak-Higashi syndrome protein, Neurobeachin, LvsA and FAN, is still poorly understood. To understand the diversity of this novel protein family, we analyzed a large array of BEACH-family protein sequences from several organisms. Comparison of all these sequences suggests that they can be classified into five distinct groups that may represent five distinct functional classes. In Dictyostelium we identified six proteins in this family, named LvsA-F, that belong to four of those classes. To test the function of these proteins in Dictyostelium we created disruption mutants in each of the lvs genes. Phenotypic analyses of these mutants indicate that LvsA is required for cytokinesis and osmoregulation and LvsB functions in lysosomal traffic. The LvsC-F proteins are not required for these or other processes such as growth and development. These results strongly support the concept that BEACH proteins from different classes have distinct cellular functions. Having six distinct BEACH proteins, Dictyostelium should be an excellent model system to dissect the molecular function of this interesting family of proteins. ----------------------------------------------------------------------------- Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development Estella Wong*, Chunzhong Yang*, Jun Wang*, Danny Fuller, William F. Loomis, and Chi-Hung Siu* 1 *Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada; and Center for Molecular Genetics, Department of Biology, University of California at San Diego, La Jolla, California 92037 Development, in press. SUMMARY The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells lacking DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by ~6 hours. The yield of spores was reduced by ~50%. The proportion of prestalk cells in cadA- slugs showed a 2.5-fold increase over the parental strain. When cadA- cells were transfected with pcotB::GFP to label prespore cells, aberrant cell sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA- cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation. ----------------------------------------------------------------------------- Spatial and temporal regulation of 3-phosphoinositides by PI3 kinase and PTEN mediates chemotaxis Satoru Funamoto, Ruedi Meili1, Susan Lee, Lisa Parry, and Richard A. Firtel Cell, in press. Abstract We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidyl inositol-3 kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, while PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, we show that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis. ----------------------------------------------------------------------------- Evidence for a novel, strongly bound acto-S1 complex carrying ADP and phosphate stabilized in the G680V mutant of Dictyostelium myosin II. Taro Q. P. Uyeda0, Kiyotaka Tokuraku and Bruce Patterson0 0 Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA Biochemistry, in press. Abstract Gly 680 of Dictyostelium myosin II sits at a critical position within the reactive thiol helices. We have previously shown that G680V mutant subfragment 1 largely remains in strongly actin-bound states in the presence of ATP. We speculated that acto-G680V subfragment 1 complexes accumulate in the A0M0ADP0Pi state, based on the biochemical phenotypes conferred by mutations which suppress the G680V mutation in vivo [Wu, Y. et al. (1999) Genetics, 153, 107-116]. Here, we report further characterization of the interaction between actin and G680V subfragment 1. Light scattering data demonstrates that the majority of G680V subfragment 1 is bound to actin in the presence of ATP. These acto-G680V subfragment 1 complexes in the presence of ATP do not efficiently quench the fluorescence of pyrene-actin, unlike those in rigor complexes or in the presence of ADP alone. Kinetic analyses demonstrated that phosphate release, but not ATP hydrolysis or ADP release, is very slow and rate limiting in the acto-G680V subfragment 1 ATPase cycle. Single turnover kinetic analysis demonstrates that, during ATP hydrolysis by the acto-G680V subfragment 1 complex, quenching of pyrene fluorescence significantly lags the increase of light scattering. This is unlike the situation with wild-type subfragment 1, where the two signals have similar rate constants. These data support the hypothesis that the main intermediate during ATP hydrolysis by acto-G680V subfragment 1 is an acto- subfragment 1 complex carrying ADP and Pi, which scatters light but does not quench the pyrene fluorescence and so has a different conformation from the rigor complex. ----------------------------------------------------------------------------- Pseudopodium Dynamics and Rapid Cell Movement in Dictyostelium Ras Pathway Mutants Jonathan R. Chubb*, Andrew Wilkins*, Deborah J. Wessels+, David R. Soll+ and Robert Insall* Cell Motil. Cytoskel., in press. Abstract Loss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility which underlie these phenotypes using computer-assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS- cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB- cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F-actin in both mutant cell lines are significantly elevated with respect to the wild-type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes. ----------------------------------------------------------------------------- Myosin II dynamics in Dictyostelium: Determinants for filament assembly and translocation to the cell cortex during chemoattractant responses Stephanie Levi, Mark V. Polyakov, and Thomas T. Egelhoff Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, OH Cell Motility and the Cytoskeleton, IN PRESS ABSTRACT In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G-protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation, and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP-myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP-myosin fusions, and with latrunculin A-treated cells demonstrated that F-actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A "headless" GFP-myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor-based motility may drive translocation. This lack of localization contrasts with earlier work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant-stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant-regulated myosin II filament assembly and translocation. ----------------------------------------------------------------------------- A novel PCR-mediated method for one-tube generation of a gene disruption construct Hidekazu Kuwayama1, Shinji Obara1, Takahiro Morio1, Mariko Katoh1, Satoru Kuhara2 and Yoshimasa Tanaka1. 1. Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. 2. Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka, 812-8581, Japan. Biotechnology letters, in press ABSTRACT We report a novel PCR-based method for generating a gene disruption construct, which requires no purification of PCR fragments and enables the whole procedure to be completed in one tube very rapidly. The procedure starts with PCR amplification of both the 5' and 3' regions of a particular gene in one tube. Then, exonuclease I is added to the tube to remove the residual primers. After heat inactivation of the enzyme, a marker cassette DNA fragment is added and fusion PCR is performed to build up a gene disruption construct. The gene disruption construct is subsequently amplified with the outermost primers in the amount necessary for transformation. In order to distinguish the gene disruption construct from the remaining intact gene allele, the outermost primers are designed to have GC-rich tag sequences that anneal at a higher temperature, ensuring the specific amplification of the gene disruption construct. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 10]