A Dendritic Guidance Receptor Complex Brings Together Distinct Actin Regulators to Drive Efficient F-Actin Assembly and Branching

Proper morphogenesis of dendrites plays a fundamental role in the establishment of neural circuits. The molecular mechanism by which dendrites grow highly complex branches is not well understood. Here, using the C. elegans PVD neuron, we demonstrate that high-order dendritic branching requires actin polymerization driven by coordinated interactions between two membrane proteins, DMA-1 and HPO-30, and with their cytoplasmic interactors, the RacGEF TIAM-1 and the actin nucleation promotion factor WAVE Regulatory Complex (WRC). The dendrite branching receptor DMA-1 directly binds to the PDZ domain of TIAM-1, while the claudin-like protein HPO-30 directly interacts with the WRC. On dendrites, DMA-1 and HPO-30 form a receptor-associated signaling complex to bring TIAM-1 and the WRC to close proximity, leading to elevated assembly of F-actin needed to drive high-order dendrite branching. The synergistic activation of F-actin assembly by scaffolding distinct actin regulators might represent a general mechanism in promoting complex dendrite arborization

Distinct core promoter codes drive transcription initiation at key developmental transitions in a marine chordate

BACKGROUND: Development is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates. RESULTS: Our TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box. CONCLUSIONS: Our results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa

echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

Background: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results: We generated a number of new echinus alleles, some of which are likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases, which cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusions: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye

Plastid-nucleus communication involves calcium-modulated MAPK signalling

Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3 omega-mediated Ca2+-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca2+ signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain

Gold amides as anticancer drugs: synthesis and activity studies

Modular gold amide chemotherapeutics: Access to modern chemotherapeutics with robust and flexible synthetic routes that are amenable to extensive customisation is a key requirement in drug synthesis and discovery. A class of chiral gold amide complexes featuring amino acid derived ligands is reported herein. They all exhibit in vitro cytotoxicity against two slow growing breast cancer cell lines with limited toxicity towards normal epithelial cells

Plastid-nucleus communication involves calcium-modulated MAPK signalling

Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3 omega-mediated Ca2+-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca2+ signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain

Critical features of antigen-specific and allospecific recognition by cytotoxic T lymphocytes

In general, CD8+ T lymphocytes respond to a single class I MHC/peptide complex, a phenomenon known as MHC self-restriction . We previously reported that CD8+ CTLs from C57BL/6 mice were restricted to both H-2Kb and H-2Db when responding to the horse cyt c-derived peptide, p41--49. In Part I of this dissertation, we examine structural features of H-2Kb/cyt c and H-2Db/cyt c complexes responsible for dual recognition. B6.H-4.1c, a representative cloned CTL, was induced by similar concentrations of native peptide added to either H-2Kb- or H-2Db-expressing targets. Furthermore, the dissociation rates for both H-2Kb/cyt c and H-2D b/cyt c were comparable, but the optimal concentration of native peptide significantly increased for both H-2Kb/cyt c and H-2Db/cyt c in peptide competition assays. Based on computer-generated models, we proposed that the Pro 44-Gly45 sequence was critical for B6.H-4.1c recognition and therefore constructed single Ala substitution analogues of cyt c, designated p41--49/44A and p41--49/45A. Although p41--49/44A binds as well to H-2Kb and H-2Db as the native peptide, B6.H-4.1c recognition was ablated. The H-2Kb/p41--49/45A and H-2Db/p41--49/45A complexes were lysed, but required significantly higher peptide concentrations than the native peptide. Molecular models for cyt c, p41--49/44A and p41--49/45A demonstrated that disrupting the Pro44-Gly45 sequence altered peptide configuration and inhibited B6.H-4.1c recognition. Nonspecific immunosuppressive agents are critical in clinical transplantation success, but cause generalized T cell inhibition and leave patients susceptible to elevated rates of infection and malignancy. In part II of this dissertation, we propose that removing allospecific CTLs that selectively expand in vitro will ablate the alloreactive response. We show that five CD8+ Vbeta families in bm19 anti-C57BL/6 cultures proliferate, but cytolytic analysis implicates only Vbeta9+ and Vbeta12 + CTLs as responders against H-2Kb-expressing targets. Removal of both Vbeta9/12+ alloreactive CTLs did not affect the response to Kb-restricted antigens, nor to unrelated H-2d alloantigens. Depletion of both Vbeta9/12 + families significantly prolonged B6 allograft survival in bm19 mice. In addition, bm19 mice mounted a cell-mediated response against L. monocytogenes despite removing Vbeta9/12+ CTLs. These studies demonstrate the synergistic effect of two Vbeta families on alloreactivity and confirm that depletion of allograft-specific Vbeta+ CTLs prolong graft survival without disrupting host immune responsiveness

Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b

Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus) immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi) and extracellular fibrinogen-binding protein (Efb) bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen) together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions