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Cep192, a novel missing link between the centrosomal core and corona in Dictyostelium amoebae
(2021)
The Dictyostelium centrosome is a nucleus-associated body with a diameter of approx. 500 nm. It contains no centrioles but consists of a cylindrical layered core structure surrounded by a microtubule-nucleating corona. At the onset of mitosis, the corona disassembles and the core structure duplicates through growth, splitting, and reorganization of the outer core layers. During the last decades our research group has characterized the majority of the 42 known centrosomal proteins. In this work we focus on the conserved, previously uncharacterized Cep192 protein. We use superresolution expansion microscopy (ExM) to show that Cep192 is a component of the outer core layers. Furthermore, ExM with centrosomal marker proteins nicely mirrored all ultrastructurally known centrosomal substructures. Furthermore, we improved the proximity-dependent biotin identification assay (BioID) by adapting the biotinylase BioID2 for expression in Dictyostelium and applying a knock-in strategy for the expression of BioID2-tagged centrosomal fusion proteins. Thus, we were able to identify various centrosomal Cep192 interaction partners, including CDK5RAP2, which was previously allocated to the inner corona structure, and several core components. Studies employing overexpression of GFP-Cep192 as well as depletion of endogenous Cep192 revealed that Cep192 is a key protein for the recruitment of corona components during centrosome biogenesis and is required to maintain a stable corona structure.
The synthesis, structure, and photocatalytic water splitting performance of two new titania (TiO2)/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 TiO2 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.
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: The hypopharyngeal gland of worker bees contributes to the production of the royal jelly fed to queens and larvae. The gland consists of thousands of two-cell units that are composed of a secretory cell and a duct cell and that are arranged in sets of about 12 around a long collecting duct. Results: By fluorescent staining, we have examined the morphogenesis of the hypopharyngeal gland during pupal life, from a saccule lined by a pseudostratified epithelium to the elaborate organ of adult worker bees. The hypopharyngeal gland develops as follows. (1) Cell proliferation occurs during the first day of pupal life in the hypopharyngeal gland primordium. (2) Subsequently, the epithelium becomes organized into rosette-like units of three cells. Two of these will become the secretory cell and the duct cell of the adult secretory units; the third cell contributes only temporarily to the development of the secretory units and is eliminated by apoptosis in the second half of pupal life. (3) The three-cell units of flask-shaped cells undergo complex changes in cell morphology. Thus, by mid-pupal stage, the gland is structurally similar to the adult hypopharyngeal gland. (4) Concomitantly, the prospective secretory cell attains its characteristic subcellular organization by the invagination of a small patch of apical membrane domain, its extension to a tube of about 100 mu m in length (termed a canaliculus), and the expansion of the tube to a diameter of about 3 mu m. (6) Finally, the canaliculus-associated F-actin system becomes reorganized into rings of bundled actin filaments that are positioned at regular distances along the membrane tube. Conclusions: The morphogenesis of the secretory units in the hypopharyngeal gland of the worker bee seems to be based on a developmental program that is conserved, with slight modification, among insects for the production of dermal glands. Elaboration of the secretory cell as a unicellular seamless epithelial tube occurs by invagination of the apical membrane, its extension likely by targeted exocytosis and its expansion, and finally the reorganisation of the membrane-associated F-actin system. Our work is fundamental for future studies of environmental effects on hypopharyngeal gland morphology and development.
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 honeybee hypopharyngeal gland consists in numerous units, each comprising a secretory cell and a canal cell. The secretory cell discharges its products into a convoluted tubular membrane system, the canaliculus, which is surrounded at regular intervals by rings of actin filaments. Using probes for various membrane components, we analyze the organization of the secretory cells relative to the apicobasal configuration of epithelial cells. The canaliculus was defined by labeling with an antibody against phosphorylated ezrin/radixin/moesin (pERM), a marker protein for the apical membrane domain of epithelial cells. Anti-phosphotyrosine visualizes the canalicular system, possibly by staining the microvillar tips. The open end of the canaliculus leads to a region in which the secretory cell is attached to the canal cell by adherens and septate junctions. The remaining plasma membrane stains for Na,K-ATPase and spectrin and represents the basolateral domain. We also used fluorophore-tagged phalloidin, anti-phosphotyrosine and anti-pERM as probes for the canaliculus in order to describe fine-structural changes in the organization of the canalicular system during the adult life cycle. These probes in conjunction with fluorescence microscopy allow the fast and detailed three-dimensional analysis of the canalicular membrane system and its structural changes in a developmental mode or in response to environmental factors.
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.
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.
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.