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Providing high-level policy advice to developing countries with the purpose to shape national policies is a key function of the UN. Yet no official UN definition of policy advice exists and little is known on how much weight the UN gives to this support modality in contrast to capacity-building and implementation work. To address this gap, this article first articulates the case for the UN’s role in policy change. It then presents an empirical analysis of the policy advice landscape of the UN, providing a numerical estimate of the share of resources dedicated to policy advice and identifying five practical constraints on the UN’s policy advice function. Results suggest that, despite high expectations, the UN’s fieldwork is not strategically focused on policy advice. This article contributes to the underresearched field of UN development work and how it is, or should be, shaped by the UN’s multilateralism.

The synthesis, structure, and photocatalytic water splitting performance of two new titania (TiO 2 )/gold(Au)/Bombyx mori silk hybrid materials are reported. All materials are monoliths with diameters of up to ca. 4.5 cm. The materials are macroscopically homogeneous and porous with surface areas between 170 and 210 m 2/g. The diameter of the TiO 2 nanoparticles (NPs) – mainly anatase with a minor fraction of brookite – and the Au NPs are on the order of 5 and 7–18 nm, respectively. Addition of poly(ethylene oxide) to the reaction mixture enables pore size tuning, thus providing access to different materials with different photocatalytic activities. Water splitting experiments using a sunlight simulator and a Xe lamp show that the new hybrid materials are effective water splitting catalysts and produce up to 30 mmol of hydrogen per 24 h. Overall the article demonstrates that the combination of a renewable and robust scaffold such as B. mori silk with a photoactive material provides a promising approach to new monolithic photocatalysts that can easily be recycled and show great potential for application in lightweight devices for green fuel production., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 581

Member states of the United Nations (UN) agree that its development system needs substantial reform given its fragmentation and outdated structures, as well as new demands from the 2030 Agenda. Yet, a recent two-year reform process yielded no substantial reform decisions. Why did member states fail to endorse the necessary reforms despite almost unanimous recognition of the need for change? This paper describes member states’ conflicting positions on reforming the UN and analyses their failure to delegate authority to the UN development system. North and South, donors and recipients, are locked in a struggle for power and control, maximising bilateral influence at the expense of the benefits of multilateral cooperation. The paper contributes to the pool of UN studies, adding a decidedly political perspective of the reform process. It is based on diplomatic statements, negotiation drafts and interviews with UN diplomats.

The process of starch granule formation in leaves of Arabidopsis ( Arabidopsis thaliana) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe21/phs1a. The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules.

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.

The frequent production of the hepatotoxin microcystin and its impact on the life-style of bloom-forming cyanobacteria are poorly understood. Here we report that microcystin interferes with the assembly and the subcellular localization of RubisCO, inMicrocystis aeruginosaPCC7806. 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 inM. aeruginosaand relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of microcystin to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2fixation product 3-phosphoglycerate are significantly higher in the microcystin-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction ofM. aeruginosawith its heterotrophic microbial community.

Our introduction consists of four steps. In the first, we prepare the field by defining and discussing the core concepts of this book—privacy, data protection and cybersecurity. All three issues come with a distinct research tradition, but in the digitised society, these issues increasingly overlap, which arguably necessitates a fresh scientific approach. In the second step, we try to disentangle the complex net of interests and dynamics around these issues by proposing an actor-based approach, distinguishing three ideal-types of actors: citizens/users, the business sector and the state which we understand as forming a triangular relationship, a “menage a trois”, where all interests are also related to collective societal values such as security, efficiency, equality and trust. This concept is hardly novel, but it allows us to identify a distinct social science perspective on privacy and data protection as politically contested issues—a perspective which is currently largely missing in the literature. It also offers a framework for comparative studies, as for example societies can be expected to direct their regulatory efforts towards different balances of interests. In the third step, we focus particularly on cybersecurity and suggest on the basis of a literature review that discourse studies offer an interesting avenue for comparative study, advancing new research that goes beyond technicalities and connects cybersecurity with broader societal factors as identified above. In the fourth step, the introduction concludes with a brief overview of the individual contributions that we have organized in three sections.

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-Src1(1-646), 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.

Copper chemodosimeters: The copper(II)-promoted air oxidation of 1-3 to form 4-6 permits the highly selective colorimetric detection of Cu(2+) ions. The formation of copper(II) complexes of 4-6 proceeds rapidly, and the chemodosimeters 1-3 are viable at physiological pH.

Dictyostelium NE81 is the first protein found in a lower eukaryote with properties justifying its denomination as a lamin-like protein. Knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells., 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.

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.

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 Drosophila genome contains at least three loci for the Na,K-ATPase beta-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase beta-subunit, as its protein product co-precipitates with the Na,K-ATPase alpha-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the alpha-subunit and of the beta-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase beta-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major beta-subunit; the visual cells R1-R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, beta-subunits do not co-distribute exactly with the alpha-subunit at some developmental stages, supporting the concept that the alpha-subunit and beta-subunit can exist in the plasma membrane without being engaged in alpha/beta heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the alpha-subunit to septate junctions throughout development.

SUMMARYCockroach salivary glands are innervated by dopaminergic and serotonergic neurons. Both transmitters elicit saliva secretion. We studied the distribution pattern of neurons containing γ-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 GABAB 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.

SUMMARYThe paired salivary glands in the cockroach are composed of acini with ion-transporting peripheral P-cells and protein-secreting central C-cells, and a duct system for the modification of the primary saliva. Secretory activity is controlled by serotonergic and dopaminergic neurons, whose axons form a dense plexus on the glands. The spatial relationship of release sites for serotonin and dopamine to the various cell types was determined by anti-synapsin immunofluorescence confocal microscopy and electron microscopy. Every C-cell apparently has only serotonergic synapses on its surface. Serotonergic and dopaminergic fibres on the acini have their release zones at a distance of ∼0.5 μm from the P-cells. Nerves between acinar lobules may serve as neurohaemal organs and contain abundant dopaminergic and few serotonergic release sites. Some dopaminergic and serotonergic release sites reside in the duct epithelium, the former throughout the duct system, the latter only in segments next to acini. These findings are consistent with the view that C-cells respond exclusively to serotonin, P-cells to serotonin and dopamine, and most duct cells only to dopamine. Moreover, the data suggest that C-cells are stimulated by serotonin released close to their surface,whereas P-cells and most duct cells are exposed to serotonin/dopamine liberated at some distance.

γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABA A receptors or metabotropic GABA B receptors. GABA B receptors regulate, via G i/o G-proteins, ion channels, and adenylyl cyclases. In humans, GABA B receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABA B receptors in the American cockroach, Periplaneta americana . We have cloned cDNAs coding for putative GABA B receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABA B receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABA B receptors act as autoreceptors in this neuron. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

The differentiation of copper cells, which secrete stomach acid in Drosophila larvae, has been shown previously to be sensitive to the labialk3 mutation. Here we found that stomach acid secretion in adults was insensitive to labk3. The basis for this stage-specific effect was elucidated by characterizing the development, structure, and function of the adult midgut. First, we demonstrated by copper-dependent fluorescence and morphology that copper cells were present in the adult stomach. Fine-structure analysis of adult copper cells led to the identification of a previously unrecognized plasma membrane domain: apicolateral contacts between copper cells and their neighbors consisted of smooth septate junctions that were enriched in alphabeta-spectrin and ankyrin. Second, we demonstrated that adult copper cells were present in labk3/labvd1 (conditional/null) adults. The labial protein was expressed in adult labk3/labvd1 copper cells, but not in larvae. Thus the labk3 mutation had a stage-specific effect on midgut labial expression, but did not appear to affect protein function. Surprisingly, stomach acidification was dispensable during larval development, since labk3/labvd1 mutant larvae that lacked midgut acidification developed into fertile adults.

Ca2+ channels often have a restricted distribution over the elaborate endoplasmic reticulum (ER) in neuronal cells; however, the mechanisms maintaining Ca2+ channel position within the ER membrane are elusive. By means of confocal immunofluorescence microscopy, ryanodine receptor (RyR) Ca2+ channels were localized in honeybee photoreceptors, and the role of actin filaments in RyR positioning was examined. In control cells, RyRs were enriched at distinct microdomains of the ER next to the photoreceptive microvilli. After incubation with cytochalasin B or cytochalasin D, which results in a disruption of F-actin, RyRs were distributed through the entire ER, suggesting that RyRs are positioned within honeybee visual cells by interaction with the F-actin system.

Photoreceptor and accessory cells in the insect compound eye exhibit a characteristic architecture, probably established and maintained by the contribution of membrane-associated cytoskeletal elements. The present study identifies and localizes nonmuscle myosin-II in honeybee photoreceptors by use of an affinity-purified antibody against scallop muscle myosin-II heavy chain (MHC). Western blot analysis and immunofluorescence staining confirmed cross-reactivity of the antibody with honeybee muscle MHC. In the compound eye, the antibody identified a protein that comigrated with muscle MHC on sodium dodecylsulfate-polyacrylamide gels. Association with the cytoskeleton, ATP-dependent binding to exogenous actin filaments, and cross-reactivity with several other antibodies against MHC, including an antibody to Drosophila nonmuscle MHC, support the conclusion that the cross-reacting protein represents nonmuscle MHC. Confocal immunofluorescence microscopy on honeybee eyes showed that the motor protein was highly enriched at distinct regions of the photoreceptor surface next to the light-receptive compartment, the rhabdom. To determine the function of myosin-II in these cells, retinal tissue was incubated with 2,3-butanedione 2-monoxime (BDM), an inhibitor of myosin activity. BDM treatment resulted in an increase in surface curvature at precisely those membrane areas that exhibited intense immunoreactivity for MHC. Moreover, the positioning and alignment of the rhabdoms was altered after exposure to BDM. These results suggest that the activity of nonmuscle myosin-II in the visual cells exerts tension on a distinct surface region next to the rhabdom, contributes to the positioning of the rhabdom, and, thus, plays a role in maintaining the cellular architecture within the compound eye.

Physiological studies have provided evidence for the existence of ryanodine receptor (RyR) Ca2+ channels in compound eyes of insects. The present study identifies and localizes RyR in insect photoreceptors by use of an affinity-purified antibody against lobster muscle RyR. Western blotting and indirect immunofluorescence staining confirm cross-reactivity of the antibody with insect muscle RyR. In both honeybee and fly eyes, the antibody identifies a single protein that comigrates with muscle RyR on sodium dodecylsulfate (SDS) polyacrylamide gels demonstrating that RyR is present in this tissue. By confocal immunofluorescence microscopy on honeybee eyes, RyR is detected within the photoreceptors and shows a nonhomogeneous distribution over the endoplasmic reticulum (ER). Double labeling studies have demonstrated further that RyR is localized at distinct ER elements close to the light-sensitive microvilli and juxtaposed to adherens junctions. RyR has also been observed within the remaining soma of honeybee photoreceptors, being concentrated on ER cisternae close to mitochondria and the nonreceptive plasma membrane. For comparative purposes, the distribution of RyR has also been assayed in compound eyes of flies. In both Calliphora and Drosophila photoreceptors, the anti-RyR antibody provides punctate labeling throughout the cell body. The submicrovillar ER cisternae associated with the base of the microvilli, however, are only lightly labeled for RyR. These results suggest that RyR is involved with Ca2+ regulation in the nonreceptive cell area of both fly and honeybee photoreceptors, but that it may contribute to Ca2+ regulation close to the phototransduction compartment only in the latter cell. J. Comp. Neurol. 421:347–361, 2000. © 2000 Wiley-Liss, Inc.

Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, β-amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.

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.

Electron microscopic examination of pupal and adult blowfly (Calliphora erythrocephala) retina provides novel details on the biogenesis of the photoreceptor surface, particularly regarding the development of the microvillar rhabdomere and associated structures, such as the submicrovillar endoplasmic reticulum. Localization of the Na+, K(+)-ATPase on the surface of developing photoreceptors has also been examined by immunofluorescence confocal microscopy and immunogold electron microscopy. Na+, K(+)-ATPase has a nonpolarized distribution in midpupal photoreceptors that are determined by fate but that are not yet completely differentiated. Large amounts of Na+, K(+)-ATPase are synthesized and delivered to the cell surface throughout the second half of pupal life. At certain time points in late pupal development, specific membrane domains become cleared of Na+, K(+)-ATPase in the photoreceptors R1-R6. However, the distribution of Na+, K(+)-ATPase remains nonpolarized in R7/R8, even after eclosion. Because the membrane-associated cytoskeleton plays a direct role in the establishment and maintenance of membrane domains in a variety of systems, it is of interest to study the distribution of alpha-spectrin and its possible association with Na+, K(+)-ATPase. The localization of alpha-spectrin resembles the distribution pattern of Na+, K(+)-ATPase in midpupal and adult photoreceptors. However, changes in Na+, K(+)-ATPase localization in late pupal photoreceptors precede the redistribution of alpha-spectrin by several days. Biochemical studies of cellular membranes demonstrate further that Na+, K(+)-ATPase can be solubilized by Triton X-100, although alpha-spectrin remains in a macromolecular complex. These results indicate that the development and the maintenance of the polarized Na+, K(+)-ATPase distribution in blowfly photoreceptors are not tightly coupled to alpha-spectrin.

The ubiquitous InsP3/Ca(2+) signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca(2+) 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 [Ca(2+)] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP3-induced Ca(2+) release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP3/Ca(2+) signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca(2+) signals; (2) cyclosporin A and FK506, inhibitors of Ca(2+)/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca(2+) oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca(2+) responses does not involve Ca(2+) entry into the cells; (4) cyclosporin A increases InsP3-dependent Ca(2+) release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca(2+) responses, indicating that PKA and calcineurin act antagonistically on the InsP3/Ca(2+) signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP3/Ca(2+) signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.

The significance of amoebae for studies of nuclear architecture has considerably increased in the recent years. The availability of a protocol for isolation of nuclei in a quality sufficient for high-resolution light and electron microscopy is a prerequisite for such studies. Here we present a protocol for high enrichment of nuclei by sucrose density-gradient centrifugation. Moreover, we describe how to use these isolated nuclei as specimens for immunofluorescence and immune-electron microscopy of ultrathin sections.

Secretory cells in blowfly salivary gland are specialized via morphological and physiological attributes in order to serve their main function, i.e. the transport of solutes at a high rate in response to a hormonal stimulus, namely serotonin (5-HT). This study examines the way that 5-HT-insensitive precursor cells differentiate into morphologically complex 5-HT-responsive secretory cells. By means of immunofluorescence microscopy, immunoblotting and measurements of the transepithelial potential changes, we show the following. (1) The apical membrane of the secretory cells becomes organized into an elaborate system of canaliculi and is folded into pleats during the last pupal day and the first day of adulthood. (2) The structural reorganization of the apical membrane is accompanied by an enrichment of actin filaments and phosphorylated ERM protein (phospho-moesin) at this membrane domain and by the deployment of the membrane-integral part of vacuolar-type H+-ATPase (V-ATPase). These findings suggest a role for phospho-moesin, a linker between actin filaments and membrane components, in apical membrane morphogenesis. (3) The assembly and activation of V-ATPase can be induced immediately after eclosion by way of 8-CPT-cAMP, a membrane-permeant cAMP analogue. (4) 5-HT, however, produces the assembly and activation of V-ATPase only in flies aged for at least 2 h after eclosion, indicating that, at eclosion, the 5-HT receptor/adenylyl cyclase/cAMP signalling pathway is inoperative upstream of cAMP. (5) 5-HT activates both the Ca2+ signalling pathway and the cAMP signalling cascade in fully differentiated secretory cells. However, the functionality of these signalling cascades does not seem to be established in a tightly coordinated manner during cell differentation. * 5-HT : 5-hydroxytryptamine 8-CPT-cAMP : 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate cAMP : cyclic adenosine 3′,5′-monophosphate ERM : ezrin/radixin/moesin InsP3 : inositol 1,4,5-trisphosphate NHE-RF : Na+/H+ exchanger regulatory factor p.d. : pupal development p.e. : post-eclosion pERM : phosphorylated ezrin/radixin/moesin PKA : protein kinase A PKA-C : catalytic subunit of protein kinase A PKA-RII : regulatory subunit II of protein kinase A TEP : transepithelial potential V-ATPase : vacuolar-type H+-ATPase

Ca(2+) and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca(2+) 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)/Ca(2+) (Cv5-HT(2α)) or the cAMP pathway (Cv5-HT(7)), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca(2+) 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 [Ca(2+)]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca(2+) oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca(2+) oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca(2+) signalling pathway to 5-HT and the Cv5-HT(2α) receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca(2+) 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 Ca(2+) spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca(2+) oscillations in blowfly salivary glands.

The significance of amoebae for studies of nuclear architecture has considerably increased in the recent years. The availability of a protocol for isolation of nuclei in a quality sufficient for high-resolution light and electron microscopy is a prerequisite for such studies. Here we present a protocol for high enrichment of nuclei by sucrose density-gradient centrifugation. Moreover, we describe how to use these isolated nuclei as specimens for immunofluorescence and immune-electron microscopy of ultrathin sections.

Invertebrate microvillar photoreceptors contain an extensive, morphologically continuous endoplasmic reticulum (ER) that comprises several distinct subregions. Most prominent is the smooth submicrovillar ER, a sponge-like cisternal network underneath the photoreceptive microvillar membrane. The submicrovillar ER spatially separates the microvilli and a narrow space of submicrovillar cytoplasm from the remaining cell body, and, thus, defines a transduction compartment. In bee and locust photoreceptors, the shape and position of these submicrovillar ER cisternae is maintained by interaction with actin filaments. The structural layout of the ER is either rather static, or, in some invertebrate species, the ER undergoes dramatic rearrangements during illumination. The submicrovillar ER has a high Ca content in dark-adapted cells (47.5 mmol/kg dry weight in bee photoreceptors), and acts as a source and sink for Ca2+ mobilized by illumination. About 50% of the Ca content is released by a 3 s, non-saturating light stimulus, and an almost equimolar amount of Mg is taken up to maintain electroneutrality within the ER. Ca2+ release is initiated by Ins(1,4,5)P3. In addition, the submicrovillar ER contains a heparin-insensitive, caffeine- and ryanodine-sensitive Ca2+ release pathway in bee photoreceptors. Both the Ins(1,4,5)P3-dependent and the ryanodine-sensitive Ca2+ release mechanism are modulated by cytosolic Ca2+, but at different Ca2+ concentrations. The presence of two release pathways with different Ca2+ sensitivities may be a prerequisite for highly localized, exceptionally fast and large Ca2+ elevations during the illumination of invertebrate photoreceptors.

Immunohistochemical and physiological studies on various insect photoreceptors have demonstrated that the Na,K-ATPase (sodium pump) is restricted to the nonreceptive nonmicrovillar area of the plasma membrane. Here, we examined the distribution of the Na,K-ATPase in photoreceptor cells of the superposition-type compound eye in the moth Manduca sexta. Using immunofluorescent and immunogold cytochemistry, we show that the Na,K-ATPase is localized to both the nonmicrovillar and the microvillar parts of the plasma membrane. Manduca photoreceptors thus deviate from the common concept that the sodium pump and the molecular components of the photoreceptive machinery reside on different domains of the plasma membrane.