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A lamin in lower eukaryotes?
(2012)
Lamins are the major components of the nuclear lamina and serve not only as a mechanical support, but are also involved in chromatin organization, epigenetic regulation, transcription and mitotic events. Despite these universal tasks, lamins have so far been found only in metazoans. Yet, recently we have identified Dictyostelium NE81 as the first lamin-like protein in a lower eukaryote. Based on the current knowledge, we draw a model for nuclear envelope organization in Dictyostelium in this Extra View and we review the experimental data that justified this classification. Furthermore we provide unpublished data underscoring the requirement of posttranslational CaaX-box processing for proper protein localization at the nuclear envelope. Sequence comparison of NE81 sequences from four Dictyostelia with bona fide lamins illustrates the evolutional relationship between these proteins. Under certain conditions these usually unicellular social amoebae congregate to form a multicellular body. We propose that the evolution of the lamin-like NE81 went along with the invention of multicellularity.
Ca2+ and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca2+ and cAMP signals. In salivary glands of the blowfly Calliphora vicina fluid secretion is stimulated by serotonin (5-hydroxytryptamine, 5-HT) via activation of two different 5-HT receptors coupled to the InsP(3)/Ca2+ (Cv5-HT2 alpha) or the cAMP pathway (Cv5-HT7), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca2+ release to InsP(3). Here we study the effects of the CAMP signalling pathway on 5-HT-induced oscillations in transepithelial potential (TEP) and in intracellular [Ca2+]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca2+ oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca2+ oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca2+ signalling pathway to 5-HT and the Cv5-HT2 alpha, receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca2+ oscillations in cells loaded with subthreshold concentrations of InsP(3), (5) inhibition of protein kinase A by H-89 abolishes 5-HT-induced TEP and Ca2+ spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca2+ oscillations in blowfly salivary glands.
We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-alpha-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.
Calcineurin activity augments cAMP/PKA-dependent activation of V-ATPase in blowfly salivary glands
(2010)
We have examined the role of the Ca2+-dependent protein phosphatase 2B (calcineurin) in the regulation of the vacuolar H+-ATPase (V-ATPase) in blowfly salivary glands. In response to the neurohormone serotonin [5-hydroxytryptamine (5-HT)] and under the mediation of the cAMP/PKA signaling pathway, the secretory cells assemble and activate V-ATPase molecules at the apical membrane. We demonstrate that the inhibition of calcineurin activity by cyclosporin A, by FK- 506, or by prevention of the elevation of Ca2+ diminishes the 5-HT-induced assembly and activation of V-ATPase. The effect of calcineurin on V-ATPase is mediated by the cAMP/PKA signaling pathway, with calcineurin acting upstream of PKA, because 1) cyclosporin A does not influence the 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP)-induced activation of V-ATPase, and 2) the 5-HT-induced rise in cAMP is highly reduced in the presence of cyclosporin A. Moreover, a Ca2+ rise evoked by the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid leads to an increase in intracellular cAMP concentration and a calcineurin-mediated PKA- dependent activation of V-ATPase. We propose that calcineurin activity mediates cross talk between the inositol 1,4,5- trisphosphate/Ca2+ and the cAMP/PKA signaling pathways, thereby augmenting the 5-HT-induced rise in cAMP and thus the cAMP/PKA-mediated activation of V-ATPase.
The ubiquitous InsP(3)/Ca2+ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca2+ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca2+] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP(3)-induced Ca2+ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP(3)/Ca2+ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca2+ signals; (2) cyclosporin A and FK506, inhibitors of Ca2+/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca2+ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca2+ responses does not involve Ca2+ entry into the cells; (4) cyclosporin A increases InsP(3)-dependent Ca2+ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca2+ responses, indicating that PKA and calcineurin act antagonistically on the InsP(3)/Ca2+ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP(3)/Ca2+ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations. (C) 2014 Elsevier Ltd. All rights reserved.
Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. in the blowfly (Calliphora vicina) salivary gland, V- ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the CAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that CAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V, components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus