Dicty News Electronic Edition Volume 10, number 6 February 22, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== The Hybrid Histidine Kinase dhkB Regulates Spore Germination in Dictyostelium discoideum Michael J. Zinda and Charles K. Singleton Vanderbilt University Developmental Biology, in press Spore germination is a defined developmental process that marks a critical point in the life cycle of Dictyostelium discoideum. Upon germination the environmental conditions must be conducive to cell growth to ensure survival of emerged amoebae. However, the signal transduction pathways controlling the various aspects of spore germination in large part remain to be elucidated. We have used degenerate PCR to identify dhkB, a two-component histidine kinase, from Dictyostelium discoideum. dhkB is predicted to be a transmembrane hybrid sensor kinase. The dhkB null cells develop with normal timing to give what seem to be mature fruiting bodies by 22 to 24 hours. However over the next several hours, the ellipsoidal and encapsulated spores proceed to swell and germinate in situ within the sorus and thus do not respond to the normal inhibitors of germination present within the sorus. The emerged amoebae dehydrate due to the high osmolarity within the sorus, and by 72 hours 4% or less of the amoebae remain as spores while most cells are now nonviable. Precocious germination is suppressed by ectopic activation of or expression of cAMP-dependent protein kinase A. Additionally, at 24 hours the intracellular concentration of cAMP of dhkB- spores is 40% that of dhkB+ spores. The results indicate that DHKB regulates spore germination, and a functional DHKB sensor kinase is required for the maintenance of spore dormancy. DHKB probably acts by maintaining an active PKA that in turn is inhibitory to germination. ------------------------------------------------------------------------- Chemotactic and osmotic signals share a cGMP transduction pathway in Dictyostelium discoideum Hidekazu Kuwayama and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands FEBS Letters, in press In the ameboid eukaryote Dictyostelium discoideum, chemotactic stimulation by cAMP induces an increase of intracellular cGMP and subsequently the phosphorylation of myosin heavy chain II. Resistance to high osmotic stress also requires transient increases of intracellular cGMP and phosphorylation of myosin heavy chain II, although the kinetics is much slower than for chemotaxis. To examine if chemotaxis and osmotic stress share common signalling components we systematically analyzed the osmotic cGMP response and survival in chemotactic mutants with altered cGMP signaling. Null-mutants with deletions of cell surface cAMP receptors or the associated GTP-binding proteins G 2 and G show no cAMP-induced cGMP response and chemotaxis; in contrast, osmotic stress induces the normal cGMP accumulation and survival. The same result was obtained with the non-chemotactic mutant KI-10, which lacks the activation of guanylyl cyclase by cAMP. This indicates that these components are required for chemotaxis but not osmotic cGMP signaling and survival. The potential guanylyl cyclase null mutant KI-8 shows no chemotaxis, no osmotic cGMP increase and reduced survival in high osmolarity. Two types of cGMP-binding protein mutants, KI-4 and KI-7, also show reduced torelance during high osmotic stress. Taken together, these observations clarify that chemotactic and osmotic signals are detected by different mechanisms, but share a cGMP signaling pathway. ------------------------------------------------------------------------- Use of a fusion protein between GFP and an actin binding domain to visualize transient F-actin structures associated with cell motility and surface adherence. Ka Ming Pang, Eunkyung Lee and David A. Knecht Department of Molecular and Cell Biology,University of Connecticut, Storrs, CT 06269,USA Current Biology in press Abstract Many important cellular processes involve changes in the quantity, location and the organization of actin filaments. We have constructed a green fluorescent protein-actin binding domain fusion protein (GFP-ABD) which allows the visualization of these changes in live cells. GFP was fused to the N-terminus of the 25kD highly conserved actin binding domain from the actin cross-linking protein ABP-120. In live Dictyostelium cells expressing GFP-ABD, the three dimensional architecture of the actin cortex was clearly visualized. The fluorescence pattern of GFP-ABD coincides with that of rhodamine- phalloidin indicating that this probe specifically binds F-actin in cells. On the ventral surface of non-polarized vegetative cells, a broad ring of actin was periodically assembled and then contracted. In polarized cells transient punctate F-actin structures were evident. Cells cycled between these two morphologies. During pseudopod formation, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in local filament density. This GFP probe specifically binds F-actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously. ------------------------------------------------------------------------- Formation of F-actin aggregates in cells treated with actin stabilizing drugs Eunkyung Lee, Eric A. Shelden and David A. Knecht Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269 Cell Motil. Cytoskel., January 1998 ABSTRACT We have electroporated Dictyostelium amoebae with fluorescent phalloidin in order to visualize the localization and behavior of F-actin filaments in living cells. Immediately after electroporation with phalloidin, cells became round and showed bright staining in the cortical region. Over time, the cortical staining disappeared and was replaced by a large aggregate of actin filaments. The aggregates were predominantly localized to the apical posterior of actively moving cells, and in the middle of dividing cells or stationary AX4 cells. Mutants lacking myosin II or ABP-120 also formed actin aggregates, however the rate of formation of aggregates was slower in myosin II mutant cells. In order to further investigate this phenomenon, we have used jasplakinolide, a membrane permeable drug that also stabilizes F-actin filaments. Cells treated with jasplakinolide formed actin aggregates in a concentration dependent manner. Drug treatment led to an increase in the proportion of actin associated with the cytoskeleton. Jasplakinolide treated cells were still motile, however their rate of movement was less than that of untreated cells. Cytochalasin B and nocodazole had inhibitory effects on aggregate formation, while azide blocked the process completely. We hypothesize that aggregates are formed from the cortical flow of F-actin filaments. These filaments would normally be depolymerized, but are artificially stabilized by phalloidin or jasplakinolide binding. The localization of the aggregate is likely to be an indication of the direction of cortical flow. ------------------------------------------------------------------------- A Role for Dictyostelium racE in Cortical Tension and Cleavage Furrow Progression Noel Gerald(1), Jianwu Dai(1), H. Ping Ting-Beall(2) and Arturo De Lozanne(1*) 1Department of Cell Biology, box 3709, Duke University Medical Center Durham, NC 27710 2Department of Mechanical Engineering and Materials Science Duke University, Durham, NC 27708 *To whom correspondence should be addressed: phone (919) 681-6851 fax (919) 681-7978, email: a.delozanne@cellbio.duke.edu J. Cell Biol., in press. ABSTRACT The small GTPase racE is essential for cytokinesis in Dictyostelium. We found that this requirement is restricted to cells grown in suspension. When attached to a substrate, racE null cells form an actomyosin contractile ring and complete cytokinesis normally. Nonetheless, racE null cells fail completely in cytokinesis when in suspension. To understand this conditional requirement for racE, we developed a method to observe cytokinesis in suspension. Using this approach we found that racE null cells attempt cytokinesis in suspension by forming a contractile ring and cleavage furrow. However, the cells form multiple blebs and fail in cytokinesis by regression of the cleavage furrow. We believe this phenotype is caused by the extremely low level of cortical tension found in racE null cells compared to wild-type cells. The reduced cortical tension of racE null cells is not caused by a decrease in their content of F-actin. Instead, mitotic racE null cells contain abnormal F-actin aggregates. These results suggest that racE is essential for the organization of the cortical cytoskeleton to maintain proper cortical integrity. This function of racE is independent of attachment to a substrate but can be bypassed by other signaling pathways induced by adhesion to a substrate. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 6]