Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by calmodulin and predicted serine phosphorylations

Kir/Gem, together with Rad, Rem and Rem2, is a member of the RGK small GTP-binding protein family. These multifunctional proteins regulate voltage-gated calcium channel (VGCC) activity and cell-shape remodeling. Calmodulin and 14-3-3 binding modulate the functions of RGK proteins. Intriguingly, abolishing the binding of calmodulin or calmodulin and 14-3-3 results in nuclear accumulation of RGK proteins. Under certain conditions, the Ca(v)beta3-subunit of VGCCs can be translocated into the nucleus along with the RGK proteins, resulting in channel inactivation. The mechanism by which nuclear localization of RGK proteins is accomplished and regulated, however, is unknown. Here, we identify specific nuclear localization signals (NLS) in Kir/Gem that are both required and sufficient for nuclear transport. Importin alpha5 binds to Kir/Gem, and its depletion using RNA interference impairs nuclear translocation of this RGK protein. Calmodulin and predicted phosphorylations on serine residues within or in the vicinity of a C-terminal bipartite NLS regulate nuclear translocation by interfering with the association between importinalpha5 and Kir/Gem. These predicted phosphorylations, however, do not affect Kir/Gem-mediated calcium channel downregulation but rather, as shown in the accompanying paper (Mahalakshmi RN, Ng MY, Guo K, Qi Z, Hunziker W, Béguin P. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 2007; doi:10.1111/j.1600-0854.2007.00599.x), interfere with cell-shape remodeling.     

Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport

The members of the RGK small GTP-binding protein family, Kir/Gem, Rad, Rem and Rem2, are multifunctional proteins that regulate voltage-gated calcium channel activity and cell shape remodeling. Calmodulin (CaM) or CaM 14-3-3 are regulators of RGK functions and their association defines the subcellular localization of RGK proteins. Abolition of CaM association results in the accumulation of RGK proteins in the nucleus, whereas 14-3-3 binding maintains them in the cytoplasm. Kir/Gem possesses nuclear localization signals (NLS) that mediate nuclear accumulation through an importin alpha5-dependent pathway (see Mahalakshmi RN, Nagashima K, Ng MY, Inagaki N, Hunziker W, Béguin P. Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by Calmodulin and predicted service phosphorylations. Traffic 2007; doi: 10.1111/j.1600-0854.2007.00598.x). Because the extent of nuclear localization depends on the RGK protein and the cell type, the mechanism and regulation of nuclear transport may differ. Here, we extend our analysis to the other RGK members and show that Rem also binds importin alpha5, whereas Rad associates with importins alpha3, alpha5 and beta through three conserved NLS. Predicted phosphorylation of a serine residue within the bipartite NLS affects, as observed for Kir/Gem, nuclear accumulation of Rem, but not that of Rad or Rem2. We also identify an additional regulatory phosphorylation for all RGK proteins that prevents binding of 14-3-3 and thereby interferes with their cytosolic relocalization by 14-3-3. Functionally, nuclear localization of RGK proteins contributes to the suppression of RGK-mediated cell shape remodeling. Importantly, we show that endogenous RGK proteins are localized predominantly in the nucleus of individual cells of the brain cortex 'in situ' as well as in primary hippocampal cells, indicating that transport between the nucleus and their site of action in the cytoplasm (i.e., cytoskeleton, endoplasmic reticulum or plasma membrane) is of physiological relevance for the regulation of RGK protein function.     

Miocene methane‐derived carbonates from southwestern Washington,USA and a model for silicification at seeps

Kuechler, R.R., Birgel, D, Kiel, S, Freiwald, A, Goedert, J.L., Thiel, V & Peckmann, J. 2011: Miocene methane‐derived carbonates from southwestern Washington, USA and a model for silicification at seeps. Lethaia, Vol. 45, pp. 259–273. Exotic limestone masses with silicified fossils, enclosed within deep‐water marine siliciclastic sediments of the Early to Middle Miocene Astoria Formation, are exposed along the north shore of the Columbia River in southwestern Washington, USA. Samples from four localities were studied to clarify the origin and diagenesis of these limestone deposits. The bioturbated and reworked limestones contain a faunal assemblage resembling that of modern and Cenozoic deep‐water methane‐seeps. Five phases make up the paragenetic sequence: (1) micrite and microspar; (2) fibrous, banded and botryoidal aragonite cement, partially replaced by silica or recrystallized to calcite; (3) yellow calcite; (4) quartz replacing carbonate phases and quartz cement; and (5) equant calcite spar and pseudospar. Layers of pyrite frequently separate different carbonate phases and generations, indicating periods of corrosion. Negative δ¹³C_carbonate values as low as ?37.6‰ V‐PDB reveal an uptake of methane‐derived carbon. In other cases, δ¹³C_carbonate values as high as 7.1‰ point to a residual, ¹³C‐enriched carbon pool affected by methanogenesis. Lipid biomarkers include ¹³C‐depleted, archaeal 2,6,10,15,19‐pentamethylicosane (PMI; δ¹³C: ?128‰), crocetane and phytane, as well as various iso‐ and anteiso‐carbon chains, most likely derived from sulphate‐reducing bacteria. The biomarker inventory proves that the majority of the carbonates formed as a consequence of sulphate‐dependent anaerobic oxidation of methane. Silicification of fossils and early diagenetic carbonate cements as well as the precipitation of quartz cement – also observed in other methane‐seep limestones enclosed in sediments with abundant diatoms or radiolarians – is a consequence of a preceding increase of alkalinity due to anaerobic oxidation of methane, inducing the dissolution of silica skeletons. Once anaerobic oxidation of methane has ceased, the pH drops again and silica phases can precipitate. □Biomarkers, carbonates, isotopes, methane, Miocene, silicification, Washington.     

Co-expression tools for plant biology: opportunities for hypothesis generation and caveats

Gene co‐expression analysis has emerged in the past 5 years as a powerful tool for gene function prediction. In essence, co‐expression analysis asks the question ‘what are the genes that are co‐expressed, that is, those that show similar expression profiles across many experiments, with my gene of interest?’. Genes that are highly co‐expressed may be involved in the biological process or processes of the query gene. This review describes the tools that are available for performing such analyses, how each of these perform, and also discusses statistical issues including how normalization of gene expression data can influence co‐expression results, calculation of co‐expression scores and P values, and the influence of data sets used for co‐expression analysis. Finally, examples from the literature will be presented, wherein co‐expression has been used to corroborate and discover various aspects of plant biology.     

Xeml Lab: a tool that supports the design of experiments at a graphical interface and generates computer-readable metadata files, which capture information about genotypes, growth conditions, environmental perturbations and sampling strategy

Data mining depends on the ability to access machine-readable metadata that describe genotypes, environmental conditions, and sampling times and strategy. This article presents Xeml Lab. The Xeml Interactive Designer provides an interactive graphical interface at which complex experiments can be designed, and concomitantly generates machine-readable metadata files. It uses a new eXtensible Mark-up Language (XML)-derived dialect termed XEML. Xeml Lab includes a new ontology for environmental conditions, called Xeml Environment Ontology. However, to provide versatility, it is designed to be generic and also accepts other commonly used ontology formats, including OBO and OWL. A review summarizing important environmental conditions that need to be controlled, monitored and captured as metadata is posted in a Wiki ( http://www.codeplex.com/XeO ) to promote community discussion. The usefulness of Xeml Lab is illustrated by two meta-analyses of a large set of experiments that were performed with Arabidopsis thaliana during 5 years. The first reveals sources of noise that affect measurements of metabolite levels and enzyme activities. The second shows that Arabidopsis maintains remarkably stable levels of sugars and amino acids across a wide range of photoperiod treatments, and that adjustment of starch turnover and the leaf protein content contribute to this metabolic homeostasis.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

Decadal vegetation changes in a northern peatland, greenhouse gas fluxes and net radiative forcing

Thawing permafrost in the sub‐Arctic has implications for the physical stability and biological dynamics of peatland ecosystems. This study provides an analysis of how permafrost thawing and subsequent vegetation changes in a sub‐Arctic Swedish mire have changed the net exchange of greenhouse gases, carbon dioxide (CO₂) and CH₄ over the past three decades. Images of the mire (ca. 17 ha) and surroundings taken with film sensitive in the visible and the near infrared portion of the spectrum, [i.e. colour infrared (CIR) aerial photographs from 1970 and 2000] were used. The results show that during this period the area covered by hummock vegetation decreased by more than 11% and became replaced by wet‐growing plant communities. The overall net uptake of C in the vegetation and the release of C by heterotrophic respiration might have increased resulting in increases in both the growing season atmospheric CO₂ sink function with about 16% and the CH₄ emissions with 22%. Calculating the flux as CO₂ equivalents show that the mire in 2000 has a 47% greater radiative forcing on the atmosphere using a 100‐year time horizon. Northern peatlands in areas with thawing sporadic or discontinuous permafrost are likely to act as larger greenhouse gas sources over the growing season today than a few decades ago because of increased CH₄ emissions.     

Z-ring-independent interaction between a subdomain of FtsA and late septation proteins as revealed by a polar recruitment assay

FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.