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Institute
The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.
Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins.
Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.
Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins.
Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.
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.
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.
Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81
(2016)
The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.
Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81
(2016)
The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture.
Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms, and generally involves Sun-family proteins located at the inner nuclear envelope. There is still no common scheme for the outer nuclear membrane proteins interacting with Sun I in centrosome/nucleus attachment. Here we propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium. For the first time we provide a detailed microscopic analysis of the centrosomal and nuclear envelope localization of endogenous Dictyostelium Sun1 during interphase and mitosis. By immunogold electron microscopy we show that Sun1 is a resident of both nuclear membranes. Disruption of Sun1 function by overexpression of full-length GFP-Sun1 or a GFP-Sun-domain deletion construct revealed not only the established function in centrosome/nucleus attachment and maintenance of ploidy, but also a requirement of Sun1 for the association of the centromere cluster with the centrosome. Live-cell imaging visualized the occurrence of mitotic defects, and demonstrated the requirement of microtubules for dynamic distance changes between centrosomes and nuclei. FRAP analysis revealed at least two populations of Sun1, with an immobile fraction associated with the centrosome, and a mobile fraction in the nuclear envelope.
Dictyostelium centrosomes consist of a layered core structure surrounded by a microtubule-nucleating corona. At the G2/M transition, the corona dissociates and the core structure duplicates, yielding two spindle pole bodies. Finally, in telophase, the spindle poles mature into two new, complete centrosomes. CP55 was identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed that during interphase the majority of centrosomal GFP-CP55 is immobile, which indicates a structural task of CP55 at the centrosome. The CP55null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their partially disorganized Golgi apparatus.
The centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates, yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage.
Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism
(2012)
Lamins build the nuclear lamina and are required for chromatin organization, gene expression, cell cycle progression, and mechanical stabilization. Despite these universal functions, lamins have so far been found only in metazoans. We have identified protein NE81 in Dictyostelium, which has properties that justify its denomination as a lamin-like protein in a lower eukaryote. This is based on its primary structure, subcellular localization, and regulation during mitosis, and its requirement of the C-terminal CaaX box as a posttranslational processing signal for proper localization. Our knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells. All our results are in agreement with a role for NE81 in formation of a nuclear lamina. This function is corroborated by localization of Dictyostelium NE81 at the nuclear envelope in human cells. The discovery of a lamin-like protein in a unicellular organism is not only intriguing in light of evolution, it may also provide a simple experimental platform for studies of the molecular basis of laminopathies.
The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.
Background
Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER).
Results
Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of ß-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration.
Conclusion
This report shows, for the first time for an insect system, that cAMP can potentiate InsP3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport.
The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.
Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands
(2009)
Cockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing gamma-aminobutyric acid ( GABA) and their physiological role. Immunofluorescence revealed a GABA-immunoreactive axon that originates within the subesophageal ganglion at the salivary neuron 2 (SN2) and this extends within the salivary duct nerve towards the salivary gland. GABA-positive fibers form a network on most acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules. Co-staining with anti-synapsin revealed that some putative GABAergic terminals seem to make pre-synaptic contacts with GABA-negative release sites. Many putative GABAergic release sites are at some distance from other synapses and at distance from the acinar tissue. Intracellular recordings from isolated salivary glands have revealed that GABA does not affect the basolateral membrane potential of the acinar cells directly. When applied during salivary duct nerve stimulation, GABA enhances the electrical response of the acinar cells and increases the rates of fluid and protein secretion. The effect on electrical cell responses is mimicked by the GABA(B) receptor agonists baclofen and SKF97541, and blocked by the GABAB receptor antagonists CGP52432 and CGP54626. These findings indicate that GABA has a modulatory role in the control of salivation, acting presynaptically on serotonergic and/or dopaminergic neurotransmission.
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.
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.