CSM News Electronic Edition Volume 2, number 16 April 30, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [129.105.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. --------------------------- ========== Abstracts ========== Cytoplasmic Ca2+ and H+ concentrations determine cell fate in Dictyostelium discoideum Y. Kubohara and K. Okamoto Department of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371, Japan, and Department of Botany, Faculty of Science, Kyoto University, Kyoto 606-01, Japan FASEB J., in press A putative morphogen, named differentiation inducing factor (DIF), is essential for stalk cell differentiation in the cellular slime mold, Dictyostelium discoideum. To investigate the relationship between the signal molecule (DIF) and the concentrations of cytoplasmic calcium ions and proton, we have examined the effects of thapsigargin (Tg) and 5,5 dimethyl-2,4-oxazolidinedione (DMO) on cell differentiation of a mutant strain HM44, which is defective in DIF-production. Tg is a specific inhibitor of the Ca2+-ATPase present in endoplasmic and sarcoplasmic reticula, and rises the cytoplasmic calcium concentration. DMO is a reagent that decreases intracellular pH. When HM44 cells were incubated with Tg or DMO in the absence of DIF, a fraction of the cells were induced to stalk cells. If added together, these reagents induced stalk cell differentiation at high efficiency (70-80%), which was comparable to that attained with exogenous DIF. In the presence of the reagents, the efficiency was not much affected by lowering cell density, suggesting that the effect (stalk induction) of these reagents was not exerted through the stimulation of DIF-production. Thus, these results indicate that a rise in cytoplasmic calcium and proton concentrations triggers stalk cell differentiation possibly by mimicing the roles of DIF. ------------------------------------------------------------------- Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum Christina Schlatterer+, Frank Gollnick, Eckhard Schmidt, Rainer Meyer and Gerd Knoll Fakultat fur Biologie, Universitat Konstanz, D-78434 Konstanz J. Cell Science, in press Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+-concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+-indicator fura2. Stimulation of cells with doses of cAMP required to induce gene expression (=84 100 =B5M) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a [Ca2+]i-rise with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2-deoxy-cAMP that binds to the cAMP receptor induced an increase in [Ca2+]i, whereas the membrane-permeant analog 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the [Ca2+]i-change is mediated by the cell surface cAMP receptor. Since HC85 mutant cells which lack the Ga2 subunit of the G-protein that couples the receptor to phospholipase C also responded towards stimulation with cAMP, the Ca2+-influx does not seem to be triggered by the phosphoinositide signaling cascade. In order to find out whether such [Ca2+]i-changes could be a part of the differentiation process in vivo we determined the extracellular space in aggregates of cells as a basis for the estimation of the physiological cAMP concentration in the multicellular organism. Electron microscopy revealed only narrow gaps between the cells with the extracellular space comprising roughly 1% of the cell volume. From the estimation of the resulting concentration range of cAMP we consider it highly probable that cells in an aggregate encounter doses of cAMP that elicit a global [Ca2+]i-rise. This elevation could be a part of the signal transduction system involved in the processes of differentiation. -------------------------------------------------------------------- Differential localization of alpha-actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum. R. Furukawa and M. Fechheimer. Cell Motility and the Cytoskeleton, in press. Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, a-actinin and the 30 kD actin bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by co-localization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but dissociates from phagosomes soon after internalization (Furukawa, et al., Protoplasma 169, 18, 1992). Alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has dissociated. These results support the hypothesis that alpha-actinin and the 30 kd protein play distinct roles in cell structure and movement in Dictyostelium. -------------------------------------------------------------------- [End CSM News, volume 2, number 16] From fechheim@zookeeper.zoo.uga.edu Tue Apr 26 06:29:58 1994 Return-Path: Received: from zookeeper.zoo.uga.edu by worms.cmsbio.nwu.edu (4.1/SMI-ACNS-1.03) id AA00720; Tue, 26 Apr 94 06:29:56 CDT Received: from [128.192.13.70] by zookeeper.zoo.uga.edu (4.1) id AA06987; Tue, 26 Apr 94 07:35:55 EDT Date: Tue, 26 Apr 94 07:35:55 EDT Message-Id: <9404261135.AA06987@zookeeper.zoo.uga.edu> From: "Marcus Fechheimer" Reply-To: "Marcus Fechheimer" To: CSM-News@worms.cmsbio.nwu.edu Subject: csm newsletter contribution Status: RO Hi Rex- Please accept the attached for inclusion in the email newsletter. regards, mf R. Furukawa and M. Fechheimer. Differential localization of alpha-actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum. Cell Motility and the Cytoskeleton, in press. ABSTRACT: Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, a-actinin and the 30 kD actin bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by co-localization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but dissociates from phagosomes soon after internalization (Furukawa, et al., Protoplasma 169, 18, 1992). Alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has dissociated. These results support the hypothesis that alpha-actinin and the 30 kd protein play distinct roles in cell structure and movement in Dictyostelium.