Refine
Has Fulltext
- no (9)
Year of publication
- 2012 (9) (remove)
Language
- English (9)
Is part of the Bibliography
- yes (9)
Keywords
- Calliphora vicina (2)
- Centrosome (2)
- Corona (2)
- Dictyostelium (2)
- Microtubules (2)
- Nucleus (2)
- cAMP (2)
- Adenylyl cyclase (1)
- Ca2+ (1)
- Crosstalk (1)
Institute
Unique properties of eukaryote-type actin and profilin horizontally transferred to cyanobacteria
(2012)
A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 mu m in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 510 lam and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.
The blowfly salivary gland - A model system for analyzing the regulation of plasma membrane V-ATPase
(2012)
Vacuolar H+-ATPases (V-ATPases) are heteromultimeric proteins that use the energy of ATP hydrolysis for the electrogenic transport of protons across membranes. They are common to all eukaryotic cells and are located in the plasma membrane or in membranes of acid organelles. In many insect epithelia, V-ATPase molecules reside in large numbers in the apical plasma membrane and create an electrochemical proton gradient that is used for the acidification or alkalinization of the extracellular space, the secretion or reabsorption of ions and fluids, the import of nutrients, and diverse other cellular activities. Here, we summarize our results on the functions and regulation of V-ATPase in the tubular salivary gland of the blowfly Calliphora vicina. In this gland, V-ATPase activity energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). Because of particular morphological and physiological features, the blowfly salivary glands are a superior and exemplary system for the analysis of the intracellular signaling pathways and mechanisms that modulate V-ATPase activity and solute transport in an insect epithelium.
Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP(3))/Ca2+ and cyclic adenosine 3',5'-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2 alpha, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2 alpha-transfected mammalian cells with 5-HT elevates cytosolic [Ca2+] in a dose-dependent manner (EC50 = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP](i) (EC50 = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT2 alpha or Cv5-HT7 in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv(5)-HT2 alpha receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT7 receptor, and clozapine (1 mu M) antagonizes the effects of 5-HT via Cv5-HT7 in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca2+- and cAMP-signalling cascades. Citation: Roser C, Jordan N, Balfanz S, Baumann A, Walz B, et al. (2012) Molecular and Pharmacological Characterization of Serotonin 5-HT2a and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina.
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.
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.
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.
Crosstalk between intracellular signalling pathways is a functionally important and widespread phenomenon in cell physiology across phyla. In the salivary gland of the blowfly, serotonin induces fluid secretion via parallel activation of both the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways, which interact on multiple levels. We have determined the molecular identity of a link between both pathways that mediates a Ca2+-dependent rise of intracellular cAMP. Whereas hydrolysis of cAMP via phosphodiesterases is largely independent of Ca2+, cAMP synthesis by adenylyl cyclases (AC) is potentiated in a Ca2+/calmodulin (Ca2+/CaM)-dependent manner. The existence of a Ca2+/CaM-dependent AC is supported by physiological data and a molecular approach. We have cloned Cv rutabaga cDNA, encoding the first blowfly AC, and confirmed its expression in the salivary gland via reverse transcription followed by polymerase chain reaction. The putative gene product of Cv rutabaga is a Ca2+/CaM-dependent type I AC and shows highest homology to Rutabaga from Drosophila. Thus, a Ca2+/CaM-dependent AC serves as a link between the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways in the salivary gland of the blowfly and might be important for the amplification and optimization of the secretory response.
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.