Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.     

Calcineurin function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle

Mutations in the Drosophila calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle to direct flight muscle. Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2.     

Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1s in Yunnan, China

Background and Aims

Rhododendron (Ericaceae) is a large woody genus in which hybridization is thought to play an important role in evolution and speciation, particularly in the Sino-Himalaya region where many interfertile species often occur sympatrically. Rhododendron agastum, a putative hybrid species, occurs in China, western Yunnan Province, in mixed populations with R. irroratum and R. delavayi.

Methods

Material of these taxa from two sites 400 km apart (ZhuJianYuan, ZJY and HuaDianBa, HDB) was examined using cpDNA and internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) loci, to test the possibility that R. agastum was in fact a hybrid between two of the other species. Chloroplast trnL-F and trnS-trnG sequences together distinguished R. irroratum, R. delavayi and some material of R. decorum, which is also considered a putative parent of R. agastum.

Key Results

All 14 R. agastum plants from the HDB site had the delavayi cpDNA haplotype, whereas at the ZJY site 17 R. agastum plants had this haplotype and four had the R. irroratum haplotype. R. irroratum and R. delavayi are distinguished by five unequivocal point mutations in their ITS sequences; every R. agastum accession had an additive pattern (double peaks) at each of these sites. Data from AFLP loci were acquired for between ten and 21 plants of each taxon from each site, and were analysed using a Bayesian approach implemented by the program NewHybrids. The program confirmed the identity of all accessions of R. delavayi, and all R. irroratum except one, which was probably a backcross. All R. agastum from HDB and 19 of 21 from ZJY were classified as F₁ hybrids; the other two could not be assigned a class.

Conclusions

Rhododendron agastum represents populations of hybrids between R. irroratum and R. delavayi, which comprise mostly or only F₁s, at the two sites examined. The sites differ in that at HDB there was no detected variation in cpDNA type or hybrid class, whereas at ZJY there was variation in both.     

Analysis of PHA-1 Reveals a Limited Role in Pharyngeal Development and Novel Functions in Other Tissues

PHA-1 encodes a cytoplasmic protein that is required for embryonic morphogenesis and attachment of the foregut (pharynx) to the mouth (buccal capsule). Previous reports have in some cases suggested that PHA-1 is essential for the differentiation of most or all pharyngeal cell types. By performing mosaic analysis with a recently acquired pha-1 null mutation (tm3671), we found that PHA-1 is not required within most or all pharyngeal cells for their proper specification, differentiation, or function. Rather, our evidence suggests that PHA-1 acts in the arcade or anterior epithelial cells of the pharynx to promote attachment of the pharynx to the future buccal capsule. In addition, PHA-1 appears to be required in the epidermis for embryonic morphogenesis, in the excretory system for osmoregulation, and in the somatic gonad for normal ovulation and fertility. PHA-1 activity is also required within at least a subset of intestinal cells for viability. To better understand the role of PHA-1 in the epidermis, we analyzed several apical junction markers in pha-1(tm3671) homozygous embryos. PHA-1 regulates the expression of several components of two apical junction complexes including AJM-1–DLG-1/discs large complex and the classical cadherin–catenin complex, which may account for the role of PHA-1 in embryonic morphogenesis.     

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

We have examined the effect of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor gene expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5''-TTAT-3'' canonical core motifs than the probe containing the 5''-TTAC-3'' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes. [BMB Reports 2014; 47(2): 92-97]     

The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

Ribosomal DNA organization before and after magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.     

The On-Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca²⁺ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca²⁺. Our results indicate that loss of Ca²⁺ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo–formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

Unique and Shared Functions of Nuclear Lamina LEM Domain Proteins in Drosophila

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LAP2, emerin, MAN1 domain, LEM-D) proteins are components of the nuclear lamina, identified by a shared ∼45-amino-acid motif that binds Barrier-to-autointegration factor (BAF), a chromatin-interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks), and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. Here, we generated null alleles of bocks, finding that loss of Bocks causes no overt adult phenotypes. Next, we defined phenotypes associated with lem-d double mutants. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, suggesting that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. Our findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins.     

Planar cell polarity and tissue design: Shaping the Drosophila wing membrane

Planar cell polarity (PCP) describes the orientation of a cell within the plane of an epithelial cell layer. During tissue development, epithelial cells normally align their PCP so that they face in the same direction. This alignment allows cells to move in a common direction, or to generate structures with a common orientation. A classic system for studying the coordination of epithelial PCP is the developing Drosophila wing. The alignment of epithelial PCP during pupal wing development allows the production of an array of cell hairs that point towards the wing tip. Multiple studies have established that the Frizzled (Fz) PCP signaling pathway coordinates wing PCP. Recently, we have found that the same pathway also controls the formation of ridges on the Drosophila wing membrane. However, in contrast to hair polarity, ridge orientation differs between the anterior and posterior wing. How can the Fz PCP pathway generate a different relationship between hair and ridge orientation in different parts of the wing? In this Extra View article, we discuss membrane ridge development drawing upon our recent PLoS Genetics paper and other, published and unpublished, data. We also speculate upon how our findings impact the ongoing debate concerning the interaction of the Fz PCP and Fat/Dachsous pathways in the control of PCP.     

Increase in Alphaproteobacteria in association with a polychaete,Capitella sp. I,in the organically enriched sediment

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, N^nd-p, was isolated as a lethal in combination with an e(r) allele, e(r)^p2. Both mutants are viable as single mutants. N^nd-p is caused by a P-element insertion in the 5′ UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that N^nd-p is a hypomorphic allele of N. The three viable notchoid alleles, N^nd-p, N^nd-1 and N^nd-3, are lethal in combination with e(r)⁻ alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the N e(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dx e(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)⁻ mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.     

A Cytoplasmic Suppressor of a Nuclear Mutation Affecting Mitochondrial Functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the “other” genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko^25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko^25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko^25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease.