Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila

The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C). The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.     

Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts.

Vnd/NK-2 protein was detected in 11 neuroblasts per hemisegment in Drosophila embryos, 9 medial and 2 intermediate neuroblasts. Fragments of DNA from the 5'-flanking region of the vnd/NK-2 gene were inserted upstream of an enhancerless betagalactosidase gene in a P-element and used to generate transgenic fly lines. Antibodies directed against Vnd/NK-2 and beta-galactosidase proteins then were used in double-label experiments to correlate the expression of beta-galactosidase and Vnd/NK-2 proteins in identified neuroblasts. DNA region A, which corresponds to the -4.0 to -2.8-kb fragment of DNA from the 5'-flanking region of the vnd/NK-2 gene was shown to contain one or more strong enhancers required for expression of the vnd/NK-2 gene in ten neuroblasts. DNA region B (-5.3 to -4.0 kb) contains moderately strong enhancers for vnd/NK-2 gene expression in four neuroblasts. Hypothesized DNA region C, whose location was not identified, contains one or more enhancers that activate vnd/NK-2 gene expression only in one neuroblast. These results show that nucleotide sequences in at least three regions of DNA regulate the expression of the vnd/NK-2 gene, that the vnd/NK-2 gene can be activated in different ways in different neuroblasts, and that the pattern of vnd/NK-2 gene expression in neuroblasts of the ventral nerve cord is the sum of partial patterns.     

Cuticular hydrocarbon analysis of an awake behaving fly using direct analysis in real-time time-of-flight mass spectrometry

In mammals and insects, pheromones strongly influence social behaviors such as aggression and mate recognition. In Drosophila melanogaster, pheromones in the form of cuticular hydrocarbons play prominent roles in courtship. GC/MS is the primary analytical tool currently used to study Drosophila cuticular hydrocarbons. Although GC/MS is highly reproducible and sensitive, it requires that the fly be placed in a lethal solution of organic solvent, thereby impeding further behavioral studies. We present a technique for the analysis of hydrocarbons and other surface molecules from live animals by using direct analysis in real-time (DART) MS. Cuticular hydrocarbons were sampled from the surface of a restrained, awake behaving fly by using several brief, carefully controlled depressions of the abdomen with a small steel probe. DART mass spectral analysis of the probe detected ions with mass-to-charge ratio (m/z) of the protonated molecule corresponding to many of the previously identified unsaturated hydrocarbons. Six additional cuticular hydrocarbons also were identified. Consistent with previous GC/MS studies, male and female differences in chemical composition were evident. Spatial differences in the expression profile also were observed on males. Sampling from an individual female first as a virgin and then 45 and 90 min after successful copulation showed that mass signals likely to correspond to cis-vaccenyl acetate, tricosene, and pentacosene increased in relative intensity after courtship. This method provides near-instantaneous analysis of an individual animal's chemical profile in parallel with behavioral studies and could be extended to other models of pheromone-mediated behavior.     

Low genomic diversity in tropical oceanic N2-fixing cyanobacteria

High levels of genomic and allelic microvariation have been found in major marine planktonic microbial species, including the ubiquitous open ocean cyanobacterium, Prochlorococcus marinus. Crocosphaera watsonii is a unicellular cyanobacterium that has recently been shown to be important in oceanic N2 fixation and has been reported from the Atlantic and Pacific oceans in both hemispheres, and the Arabian Sea. In direct contrast to the current observations of genomic variability in marine non-N2-fixing planktonic cyanobacteria, which can range up to >15% nucleotide sequence divergence, we discovered that the marine planktonic nitrogen-fixing cyanobacterial genus Crocosphaera has remarkably low genomic diversity, with <1% nucleotide sequence divergence in several genes among widely distributed populations and strains. The cultivated C. watsonii WH8501 genome sequence was virtually identical to DNA sequences of large metagenomic fragments cloned from the subtropical North Pacific Ocean with <1% sequence divergence even in intergenic regions. Thus, there appears to be multiple strategies for evolution, adaptation, and diversification in oceanic microbial populations. The C. watsonii genome contains multiple copies of several families of transposases that may be involved in maintaining genetic diversity through genome rearrangements. Although genomic diversity seems to be the rule in many, if not most, marine microbial lineages, different forces may control the evolution and diversification in low abundance microorganisms, such as the nitrogen-fixing cyanobacteria.     

Bell-like homeodomain selectively regulates the high-irradiance response of phytochrome A

Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A.     

Transport proteins NHA1 and NHA2 are essential for survival,but have distinct transport modalities

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na⁺/H⁺ exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na⁺ stress; survival on K⁺ intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na⁺/H⁺ exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl⁻ conductance and acts as a H⁺-Cl⁻ cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4′-diiso-thiocyano-2,2′-disulfonic acid stilbene and 4,4′-dibenzamido-2,2′-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.     

Methylation of CpG sites in BCL2 major breakpoint region and the increase of BCL2/JH translocation with aging

The BCL2 breakage mechanism has been shown to be highly dependent on DNA methylation at the major breakpoint region (MBR) CpG sites. We recently described an increased frequency of BCL2/ JH translocation with aging. It is known that methylation levels change with aging. The present study aimed to determine whether methylation alterations at CpG sites of BCL2 MBR were the cause of increased breakages with aging. We analyzed the methylation levels of three CpG sites on the region by pyrosequencing and studied if methylation levels and/or polymorphisms affecting CpG sites were associated with an increase of translocations. We observed that although the methylation levels of MBR CpG sites were higher in individuals with BCL2/JH translocation, in contrast to our expectations, these levels decreased with the age. Moreover, we show that polymorphisms at those CpG sites leading to absence of methylation seem to be a protective factor for the apparition of translocations.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-015-9834-5) contains supplementary material, which is available to authorized users.     

Hip, an HP1-interacting protein, is a haplo- and triplo-suppressor of position effect variegation

The Drosophila heterochromatin protein 1 (HP1) regulates epigenetic gene silencing and heterochromatin formation by promoting and maintaining chromatin condensation. Here we report the identification and characterization of an HP1-interacting protein (Hip). Hip interacts with HP1 in vitro and is associated with HP1 in vivo. This interaction is mediated by at least three independent but similar HP1-binding modules of the Hip protein. Hip and HP1 completely colocalize in the pericentric heterochromatin, and both haplo- and triplo-dosage mutations act as dominant suppressors of position effect variegation. These findings identify a player in heterochromatinization and suggest that Hip cooperates with HP1 in chromatin remodeling and gene silencing.     

Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli

SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.Posttranslational modifications (PTMs) are key mechanisms used by both prokaryotes and eukaryotes to regulate protein activity specifically, locally, and temporally. Ubiquitin and ubiquitin-like proteins (UBLs) constitute a specific class of small protein modifiers that can be covalently attached to a target protein via the formation of an isopeptide bond in a reversible manner. Small ubiquitin-related modifier (SUMO), one of these UBLs, is an essential PTM in eukaryotic cells that is involved in various cellular functions including gene expression regulation, DNA repair, intracellular transport, and response to viral and bacterial infections (1–5). The human genome encodes three different functional SUMO isoforms (SUMO1, SUMO2, and SUMO3) that are conjugated to distinct but overlapping sets of target proteins (1, 2, 6). Conjugation of SUMO to its targets in humans requires an E1-activating enzyme (the SAE1/SAE2 heterodimer), an E2-conjugating enzyme (Ubc9), and several E3 SUMO enzymes. Once conjugated to its target, SUMO can be deconjugated by several different SUMO isopeptidases that tightly regulate the SUMOylation levels of proteins (7).Since the discovery of SUMO two decades ago, much effort has been dedicated to the identification of SUMO-conjugated proteins in different organisms including yeast, plants, and mammals (8). However, isolation of SUMOylated proteins has proven to be challenging. Indeed, for most SUMO substrates, only a small proportion of the total amount of protein is SUMO-modified. In addition, the high activity of SUMO isopeptidases in cell lysates results in the rapid loss of SUMO conjugation in the absence of appropriate inhibitors. Thus, the most common approach used to isolate SUMOylated proteins is based on the expression of His-tagged versions of SUMO allowing the purification of SUMO-conjugated proteins by nickel chromatography under denaturing conditions (8, 9). Denaturing conditions inactivate SUMO isopeptidases and also prevent contamination by proteins interacting noncovalently with SUMO via specific domains such as SUMO-interacting motifs (SIMs) (2). Once SUMOylated proteins have been isolated, their analysis by mass spectrometry (MS) has been widely used to identify SUMO-modified proteins and, albeit less successfully, SUMO-conjugation sites.Mapping the exact lysine residue to which SUMO is attached in modified proteins is a critical step to get further insight into the function of SUMOylation. Indeed, the identification of SUMO sites allows the generation of non-SUMOylatable mutants and the study of associated phenotypes. Identification of SUMO sites by MS is not straightforward (8). Unlike ubiquitin, which leaves a small diglycine (GG) signature tag on the modified lysine residue after trypsin digestion, SUMO leaves a larger signature that severely hampers the identification of modified peptides.In addition to the identification of the SUMO site per se, a comparison of the SUMOylation status of sites in different cell-growth conditions is critical for better characterizing the biological implications of SUMOylation. For example, analysis of SUMOylation changes induced after heat shock, arsenic treatment, inhibition of the proteasome, or during the cell cycle has led to numerous insights into the role of SUMOylation in cell physiology (refs. 10–14 and reviewed in ref. 2). Here, we devised a performant approach which combines the use of SUMO variants, peptide immunocapture, and quantitative proteomics for high-throughput identification of SUMO sites. We then show that our approach is able to characterize global changes in the cell SUMOylome in response to a given stimulus, such as exposure to a bacterial toxin, listeriolysin O (LLO).     

The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. At the core of the Hippo pathway is a kinase cascade extending from the Hippo (Hpo) tumor suppressor to the Yorkie (Yki) oncoprotein. The Hippo kinase cascade, in turn, is regulated by apical membrane-associated proteins such as the FERM domain proteins Merlin and Expanded (Ex), and the WW- and C2-domain protein Kibra. How these apical proteins are themselves regulated remains poorly understood. Here, we identify the transmembrane protein Crumbs (Crb), a determinant of epithelial apical-basal polarity in Drosophila embryos, as an upstream component of the Hippo pathway in imaginal disk growth control. Loss of Crb leads to tissue overgrowth and target gene expression characteristic of defective Hippo signaling. Crb directly binds to Ex through its juxtamembrane FERM-binding motif (FBM). Loss of Crb or mutation of its FBM leads to mislocalization of Ex to basolateral domain of imaginal disk epithelial cells. These results shed light on the mechanism of Ex regulation and provide a molecular link between apical-basal polarity and tissue growth. Furthermore, our studies implicate Crb as a putative cell surface receptor for Hippo signaling by uncovering a transmembrane protein that directly binds to an apical component of the Hippo pathway.     

Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1

Synaptic vesicle fusion at many synapses has been kinetically separated into two distinct Ca²⁺-dependent temporal components consisting of a rapid synchronous phase followed by a slower asynchronous component. Mutations in the synaptic vesicle Ca²⁺ sensor Synaptotagmin 1 (Syt 1) reduce synchronous neurotransmission while enhancing the slower asynchronous phase of release. Syt 1 regulation of vesicle fusion requires interactions mediated by its tandem cytoplasmic C2 domains (C2A and C2B). Although Ca²⁺ binding by Syt 1 is predicted to drive synchronous release, it is unknown if Ca²⁺ interactions with either C2 domain is required for suppression of asynchronous release. To determine if Ca²⁺ binding by Syt 1 regulates these two phases of release independently, we performed electrophysiological analysis of transgenically expressed Syt 1 mutated at Ca²⁺ binding sites in C2A or C2B in the background of Drosophila Syt 1-null mutants. Transgenic animals expressing mutations that disrupt Ca²⁺ binding to C2A fully restored the synchronous phase of neurotransmitter release, whereas the asynchronous component was not suppressed. In contrast, rescue with Ca²⁺-binding mutants in C2B displayed little rescue of the synchronous release component, but reduced asynchronous release. These results suggest that the tandem C2 domains of Syt 1 play independent roles in neurotransmission, as Ca²⁺ binding to C2A suppresses asynchronous release, whereas Ca²⁺ binding to C2B mediates synchronous fusion.     

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.Specialized biological processes are carried out in specific organelles and subcellular compartments. For example, mitochondria are the site of oxidative respiration, neurons pass electrical or chemical signals to others through synapses, and apical and basolateral domains of epithelial cells are critical for their polarized functions. Understanding how these structures underlie specialized functions requires the comprehensive identification of proteins within spatially defined cellular domains.A common strategy to study the localization of a particular protein is to generate green fluorescent protein (GFP) fusion proteins. However, it is time-consuming and labor-intensive to investigate protein localization at a large scale using GFP tagging, especially in vivo. Therefore, highly sensitive mass spectrometry (MS) approaches have been developed to systematically characterize the proteome of subcellular compartments. However, using MS approaches to characterize the proteome of subcellular domains has been limited by purification methods and is commonly associated with numerous false positives and false negatives due to contamination and loss of components during purification, respectively. For example, mitochondria are composed of an outer membrane and an inner membrane, generating two subcompartmental regions: the intermembrane space and the matrix located within the inner membrane. Because the ultrastructure of mitochondria is often disrupted during isolation processes, the isolation of specific subcompartmental regions of mitochondria is prone to contamination.Recently, a method based on an engineered ascorbate peroxidase (APEX) has been developed and shown to function in cultured mammalian cells for proteomic mapping (1). Upon activation, the APEX enzyme turns a biotin-phenol substrate into a highly reactive radical that covalently tags neighboring proteins on electron-rich amino acids such as tyrosine. Biotinylated endogenous proteins can then be isolated and identified by MS. Thus, APEX labeling can be applied to bypass organelle purification steps, offering an alternative approach for systematic proteomic characterization in live cells. Here we report that the approach can be applied to characterize the subcellular proteome in live tissues and map the mitochondrial matrix proteome of Drosophila muscle. In addition to characterizing a number of uncharacterized putative mitochondrial proteins, we establish MitoMax, a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation.