Dicty News Electronic Edition Volume 21, number 2 July 18, 2003 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 ============= Dictyostelium SAPKalpha, a novel stress-activated MEK kinase-like kinase, is important for the proper regulation of the cytoskeleton Binggang Sun, Hui Ma, and Richard A. Firtel Section of Cell and Developmental Biology Division of Biological Sciences and Center for Molecular Genetics University of California, San Diego 9500 Gilman Drive La Jolla CA 92093-0634 Molecular Biology of the Cell, in press MAP kinase cascades regulate various cellular functions including growth, cell differentiation, development, and stress responses. We have identified a new Dictyostelium kinase (SAPKalpha), which is related to members of the MLK class of MEK kinases. SAPKalpha is activated by osmotic stress, heat shock, and detachment from the substratum and by a membrane-permeable cGMP analog, a known regulator of stress responses in Dictyostelium. SAPKalpha is important for cellular resistance to stresses, as SAPKalpha null cells exhibit reduced viability in response to osmotic stress. We found that SAPKalpha mutants affect cellular processes requiring proper regulation of the actin cytoskeleton, including cell motility, morphogenesis, cytokinesis, and cell adhesion. Overexpression of SAPKalpha results in highly elevated basal and chemoattractant-stimulated F-actin levels and strong aggregation and developmental defects, including a failure to polarize and chemotax and abnormal morphogenesis. These phenotypes require a kinase-active SAPKalpha. SAPKalpha null cells exhibit reduced chemoattractant-stimulated F-actin levels, cytokinesis, developmental and adhesion defects, and a motility defect that is less severe than that exhibited by SAPKalpha overexpressing cells. SAPKalpha co-localizes with F-actin in F-actin-enriched structures, including membrane ruffles and pseudopodia during chemotaxis. Although SAPKalpha is required for these F-actin-mediated processes, it is not detectably activated in response to chemoattractant stimulation. Submitted by: Rick Firtel [rafirtel@ucsd.edu] ----------------------------------------------------------------------------- Feedback control in intracellular signaling pathways: Regulating chemotaxis in Dictyostelium discoideum Pablo A. Iglesias Electrical & Computer and Biomedical Engineering The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218 pi@jhu.edu European J. Control, in press Positive and negative feedback loops are used throughout engineering to tradeoff between amplification and robustness. We show how the social amoebae Dictyostelium discoideum uses these same principles to aggregate during starvation, allowing it to survive difficult environmental conditions. Submitted by: Pablo Iglesias [pi@jhu.edu] ----------------------------------------------------------------------------- PIR121 Regulates Pseudopod Dynamics and SCAR Activity in Dictyostelium Simone L. Blagg, Michael Stewart, Christine Sambles and Robert H. Insall* Current Biology, in press. School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT UK BACKGROUND The WASP/SCAR family of adaptor proteins coordinates actin reorganization by coupling different signalling molecules, including Rho-family GTPases, to the activation of the Arp2/3 complex. WASP binds directly to Cdc42 through its GTPase-binding domain (GBD), but SCAR does not contain a GBD and no direct binding has been found. However, SCAR has recently been found to copurify with four other proteins in a complex. One of these, PIR121, binds directly to Rac. RESULTS We have identified four of the members of this complex in Dictyostelium, and disrupted the pirA gene, which encodes PIR121. The resulting mutant cells are unusually large, maintain an excessive proportion of their actin in a polymerized state, and display severe defects in movement and chemotaxis. They also continually extend new pseudopods by widening and splitting existing leading edges, rather than by initiating new pseudopods. Comparing these cells to scar null mutants shows behaviour that is broadly consistent with overactivation of SCAR. Deletion of the pirA gene in a scar- mutant resulted in cells resembling their scar- parents with no obvious changes, confirming that PIR121 mainly acts through SCAR in vivo. Surprisingly, given their hyperactive phenotype, we find that pirA- mutants contain very little intact SCAR protein, despite normal levels of mRNA, suggesting a post-transcriptional downregulation of activated SCAR. CONCLUSIONS Our results demonstrate a genetic connection between the pirA and scar genes. PIR121 appears to inhibit the activity of SCAR in the absence of activating signals. The location of the newly formed protrusions indicates that unregulated SCAR is acting at the edges of existing pseudopods, not elsewhere in the cell. We suggest that active SCAR protein released from the inhibitory complex is rapidly removed, and this is an important and novel mechanism for controlling actin dynamics. Submitted by: Simone Blagg [slb761@bham.ac.uk] =============================================================================== [End Dicty News, volume 21, number 2]