The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1_ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.

The Fat pathway controls both planar cell polarity (PCP) and organ growth [1, 2]. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP [1], whereas their slope can influence growth [3, 4]. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling [5-7]. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end-joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218 that is required for crossover formation and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C) that physically interact and promote homologous recombination. We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (gamma-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. gamma-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of gamma-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.

Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the o66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability.

The Hippo signaling pathway has a conserved role in growth control and is of fundamental importance during both normal development and oncogenesis. Despite rapid progress in recent years, key steps in the pathway remain poorly understood, in part due to the incomplete identification of components. Through a genetic screen, we identified the Drosophila Zyxin family gene, Zyx102 (Zyx), as a component of the Hippo pathway. Zyx positively regulates the Hippo pathway transcriptional co-activator Yorkie, as its loss reduces Yorkie activity and organ growth. Through epistasis tests, we position the requirement for Zyx within the Fat branch of Hippo signaling, downstream of Fat and Dco, and upstream of the Yorkie kinase Warts, and we find that Zyx is required for the influence of Fat on Warts protein levels. Zyx localizes to the sub-apical membrane, with distinctive peaks of accumulation at intercellular vertices. This partially overlaps the membrane localization of the myosin Dachs, which has similar effects on Fat-Hippo signaling. Co-immunoprecipitation experiments show that Zyx can bind to Dachs and that Dachs stimulates binding of Zyx to Warts. We also extend characterization of the Ajuba LIM protein Jub and determine that although Jub and Zyx share C-terminal LIM domains, they regulate Hippo signaling in distinct ways. Our results identify a role for Zyx in the Hippo pathway and suggest a mechanism for the role of Dachs: because Fat regulates the localization of Dachs to the membrane, where it can overlap with Zyx, we propose that the regulated localization of Dachs influences downstream signaling by modulating Zyx-Warts binding. Mammalian Zyxin proteins have been implicated in linking effects of mechanical strain to cell behavior. Our identification of Zyx as a regulator of Hippo signaling thus also raises the possibility that mechanical strain could be linked to the regulation of gene expression and growth through Hippo signaling.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) in complex with a sigma factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between sigma and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of sigma upon the transition to elongation. Furthermore, sigma can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with sigma also function to increase the sigma content of downstream elongation complexes. Our findings establish sigma-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.

In the past ten years, international initiatives have led to the development of large sets of genomic resources that allow comparative genomic studies between plant genomes at a high level of resolution. Comparison of map-based genomic sequences revealed shared intra-genomic duplications, providing new insights into the evolution of flowering plant genomes from common ancestors. Plant genomes can be presented as concentric circles, providing a new reference for plant chromosome evolutionary relationships and an efficient tool for gene annotation and cross-genome markers development. Recent palaeogenomic data demonstrate that whole-genome duplications have provided a motor for the evolutionary success of flowering plants over the last 50-70 million years.

The comparison of the chromosome numbers of today's species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5-12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.

In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54- and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the bar(au) gene that causes a golden-yellow (aurea) leaf color, so that the loss of the bar(au) gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the bar(au) gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/attP-flanked bar(au) gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked bar(au) gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the bar(au) gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

Bone morphogenetic protein (BMP) pathways control an array of developmental and homeostatic events, and must themselves be exquisitely controlled. Here, we identify Caenorhabditis elegans SMA-10 as a positive extracellular regulator of BMP-like receptor signaling. SMA-10 acts genetically in a BMP-like (Sma/Mab) pathway between the ligand DBL-1 and its receptors SMA-6 and DAF-4. We cloned sma-10 and show that it has fifteen leucine-rich repeats and three immunoglobulin-like domains, hallmarks of an LRIG subfamily of transmembrane proteins. SMA-10 is required in the hypodermis, where the core Sma/Mab signaling components function. We demonstrate functional conservation of LRIGs by rescuing sma-10(lf) animals with the Drosophila ortholog lambik, showing that SMA-10 physically binds the DBL-1 receptors SMA-6 and DAF-4 and enhances signaling in vitro. This interaction is evolutionarily conserved, evidenced by LRIG1 binding to vertebrate receptors. We propose a new role for LRIG family members: the positive regulation of BMP signaling by binding both Type I and Type II receptors.

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the beta-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP beta-subunit. We determined the 2.9-A resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-beta1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel beta sandwich fold composed of 11 antiparallel beta-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092-->Glu, observed in the Caenorhabditis elegans ortholog RPM-1.

The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect--a decrease in spontaneous reversals in locomotion--consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking.