Structure of Protein Related to Dan and Cerberus: Insights into the Mechanism of Bone Morphogenetic Protein Antagonism

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Cyclic Di-AMP Homeostasis in Bacillus subtilis: BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.     

Involvement of CaMK‐IIδ and gelsolin in Cd2+‐dependent cytoskeletal effects in mesangial cells

Cadmium is a toxic metal with pleiotropic effects on cell death and survival. The mesangial cell is particularly responsive to Cd's effects on kinase signaling pathways and cytoskeletal dynamics. Here we show that CaMK‐II is a participant in the cytoskeletal effects of Cd²⁺. A major mesangial cell isoform, CaMK‐IIδ, was identified in pellets of DNase I pull‐downs and cytosolic immunoprecipitates of G‐actin. CaMK‐IIδ was also present in Triton X‐100‐insoluble cytoskeletal preparations and translocated to the cytoskeleton in a concentration‐dependent manner in Cd‐treated cells. Translocation was suppressed by KN93, an inhibitor of CaMK‐II phosphorylation. In vitro actin polymerization studies indicated that recombinant CaMK‐IIδ sequestered actin monomer. Cytoskeletal preparations from Cd‐treated cells decrease the rate of polymerization, but KN93 co‐treatment prevents this effect. Over‐expressed CaMK‐IIδ also translocated to the cytoskeleton upon Cd exposure, and this was prevented by KN93. Conversely, siRNA silencing of CaMK‐IIδ increases the effect of cytoskeletal extracts on actin polymerization, and abrogates the effect of Cd. The actin capping and severing protein, gelsolin, translocates to the cytoskeleton in the presence of Cd²⁺, dependent upon the phosphorylation of CaMK‐II, and is recovered together with actin and CaMK‐IIδ in G‐actin pull‐downs and F‐actin sedimentation. Translocation is accompanied by generation of a 50 kDa gelsolin fragment whose appearance is prevented by KN93 and CaMK‐IIδ silencing. We conclude that cytoskeletal effects of Cd in mesangial cells are partially mediated by Cd‐dependent activation of CaMK‐IIδ, binding of CaMK‐IIδ and gelsolin to actin filaments, and cleavage of gelsolin. J. Cell. Physiol. 228: 78–86, 2013. © 2012 Wiley Periodicals, Inc.     

Reservoir to river passage of age‐0+ year fishes,indication of a dispersion pathway for a non‐native species

This study demonstrates passage of age‐0+ year individuals of pikeperch Sander lucioperca, common bream Abramis brama and non‐native tubenose goby Proterorhinus semilunaris from the Nové Mlýny Reservoir into the River Dyje (Danube River basin, Czech Republic) through the turbine of a hydropower facility. Most fishes had standard length (L_S) in the range 12–33 mm. Seasonal patterns corresponded with spawning activity, i.e. an early single spawning event for S. lucioperca, multiple spawning events for A. brama and continuous spawning with a later start and prolonged duration for P. semilunaris. Sander lucioperca, P. semilunaris and larger A. brama (>22 mm) drifted almost exclusively during the dark; smaller A. brama displayed no preference for light or dark. Proterorhinus semilunaris displayed significantly lower mortality than other species when passing through the turbine (3% compared to 18%). The passage of high numbers of P. semilunaris from the reservoir (estimated at 473 000 individuals per year), and their subsequent mass downstream drift, may have contributed to rapid population establishment along the River Dyje and the quick downstream expansion.     

Calcium-binding proteins and cytochrome oxidase activity in the turtle optic tectum with special reference to the tectofugal visual pathway

Using immunohistochemistry and a tracer technique we investigated the distribution in the optic tectum of turtles (Emys orbicularis and Testudo horsfieldi) of the calcium-binding proteins (CaBPr) parvalbumin (PV), calbindin (CB) and calretinin (CR) before and after labeling of the nucleus rotundus (Rot) with horseradish peroxidase. The optic tectum activity of the cytochrome oxidase (CO) was studied in parallel. In the principal link of the tectofugal visual pathway (central gray layer, SGC) in both chelonian species, the sparse PV-ir as well as CB- and CR-ir neurons were found significantly varying both in number and the intensity of immunoreactivity of their bodies and dendrites. In contrast, the superficial (SGFS) and deeper periventricular (SGP) tectal layers comprised numerous cells immunoreactive to all three CaBPr in different proportions. Only few retrogradely labeled tectorotundal SGC neurons expressed PV, CB or CR. The very large PV-ir neurons in SGC and SAC were not retrogradely labeled; morphologically they matched the efferent neurons with descending projections. SGC neurons of two chelonian species differed in the level of CO activity. Intense immunoreactivity to all three CaBPr and high CO activity were detected in both species in SGFS neuropil with some differences in sublaminar distribution patterns. The peculiarities of the CaBPr and CO activity distribution patterns in different segments of SGC neurons are discussed as related to the laminar organization of the turtle tectum and its retinal innervation. It is suggested that in the projection tectorotundal SGC neurons the CaBPr are concentrated mainly in their distal dendrites that contact retinal afferents in the superficial retinorecipient tectal layer.     

Temporal patterns in growth and survival of the round goby Neogobius melanostomus

Monthly, overwinter and annual instantaneous growth rates for round goby Neogobius melanostomus were calculated with maximal growth occurring in July and August and almost no growth observed between ice appearance (October) and melt (March). Annual absolute growth rates averaged 27·3 ± 1·9 mm for males and 19·8 ± 2·4 mm for females. The most parsimonious Cormack–Jolly–Seber model indicated that both the survival and recapture probabilities were dependent on sampling date, but not sex. Survival estimates remained high throughout the 13 month study with a median weekly survival probability of 0·920 (25 and 75% quartiles: 0·767 and 0·991), an overwinter survival probability of 99% and an annual survival rate of 67%. Survival probabilities were lowest for both sexes near the completion of the N. melanostomus reproductive season in July and August which supports existing evidence of higher mortality after reproduction, while challenging the paradigm that male N. melanostomus suffer comparatively higher mortality as a result of reproduction than females. Evidence indicating that growth and mortality rates are highest at the end of the reproductive season not only highlights seasonal variability in N. melanostomus natural history, but may also guide the control of this invasive species to periods when they are most vulnerable.     

Five in situ observations of live oarfish Regalecus glesne (Regalecidae) by remotely operated vehicles in the oceanic waters of the northern Gulf of Mexico

As part of the SERPENT Project, five observations of apparently healthy oarfish Regalecus glesne by remotely operated vehicles are reported from the northern Gulf of Mexico. Regalecus glesne were observed between 2008 and 2011 at depths from within the epipelagic and mesopelagic zones. These observations include the deepest verified record of R. glesne (463–492 m) and the first record of an arthropod ectoparasite (isopod).