Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization in Drosophila melanogaster

Myosin VI is a pointed-end–directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Are specialists at risk under environmental change? Neoecological,paleoecological and phylogenetic approaches

The question 'what renders a species extinction prone' is crucial to biologists. Ecological specialization has been suggested as a major constraint impeding the response of species to environmental changes. Most neoecological studies indicate that specialists suffer declines under recent environmental changes. This was confirmed by many paleoecological studies investigating longer-term survival. However, phylogeneticists, studying the entire histories of lineages, showed that specialists are not trapped in evolutionary dead ends and could even give rise to generalists. Conclusions from these approaches diverge possibly because (i) of approach-specific biases, such as lack of standardization for sampling efforts (neoecology), lack of direct observations of specialization (paleoecology), or binary coding and prevalence of specialists (phylogenetics); (ii) neoecologists focus on habitat specialization; (iii) neoecologists focus on extinction of populations, phylogeneticists on persistence of entire clades through periods of varying extinction and speciation rates; (iv) many phylogeneticists study species in which specialization may result from a lack of constraints. We recommend integrating the three approaches by studying common datasets, and accounting for range-size variation among species, and we suggest novel hypotheses on why certain specialists may not be particularly at risk and consequently why certain generalists deserve no less attention from conservationists than specialists.     

The histone deacetylase Rpd3p is required for transient changes in genomic expression in response to stress

Background  

Yeast responding to stress activate a large gene expression program called the Environmental Stress Response that consists of approximately 600 repressed genes and approximately 300 induced genes. Numerous factors are implicated in regulating subsets of Environmental Stress Response genes; however, a complete picture of Environmental Stress Response regulation remains unclear. We investigated the role of the histone deacetylase Rpd3p, previously linked to the upstream regions of many Environmental Stress Response genes, in producing Environmental Stress Response gene expression changes in response to stress.     

p120‐catenin is a binding partner and substrate for Group B Pak kinases

Pak5 is a member of the Group B p21‐activated kinases, which are effectors of the Rho family GTPases Cdc42 and Rac. Pak5 has been shown to promote cytoskeletal reorganization, inducing filopodia formation and neurite outgrowth in neuroblastoma cells. In this study, we used affinity chromatography followed by SDS–PAGE and mass spectrometry to identify potential downstream effectors of Pak5. Using this approach, we isolated p120‐catenin (p120), a known regulator of cytoskeletal reorganization and Rho GTPases. Using co‐immunoprecipitation assays we found that p120 preferentially interacts with Pak5 among the Group B Paks. Results from immunofluorescence studies revealed that Pak5 and p120 co‐localize in cells. Both Pak5 and constitutively active Pak4, the founding member of the Group B Paks, directly phosphorylate p120 in vitro. The phosphorylation was shown by Western blot and immunofluorescence to take place specifically on serine 288. This study is the first report of an upstream serine/threonine kinase that phosphorylates p120. J. Cell. Biochem. 110: 1244–1254, 2010. Published 2010 Wiley‐Liss, Inc.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent

Many eukaryotic genomes have experienced ancient whole-genome duplication (WGD) followed by massive gene loss. These eliminations were not random since some gene families were preferentially retained as duplicates. The gene balance hypothesis suggests that those genes with dosage reduction can imbalance their interacting partners or complex, resulting in decreased fitness. In Arabidopsis, the cytoplasmic ribosomal proteins (RP) are encoded by gene families with at least two members. We have focused our study on the two RPS6 genes in an attempt to understand why they have been retained as duplicates. We demonstrate that RPS6 function is vital for the plant. We also show that reducing the level of RPS6 accumulation (in the knock-out rps6a or rps6b single mutants, or in the double heterozygous RPS6A/rps6a,RPS6B/rps6b), confers a slow growth phenotype (haplodeficiency). Importantly, we demonstrate that the functions of two RPS6 genes are redundant and interchangeable. Finally, like in most other described Arabidopsis rp mutants, we observed that a reduced RPS6 level slightly alters the dorsoventral leaf patterning. Our results support the idea that the Arabidopsis RPS6 gene duplicates were evolutionarily retained in order to maintain an expression level necessary to sustain the translational demand of the cell, in agreement with the gene balance hypothesis.