Annabel Guichard

Annabel Guichard
University of California, San Diego | UCSD · Section of Cell and Developmental Biology

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51
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Publications

Publications (51)
Article
Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using inta...
Article
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A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticide...
Article
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Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways i...
Article
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Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this “active genetic” approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting...
Article
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Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present s...
Data
Blocking expression of Sec15, but not of the Rab11GEF Crag, prevents Rab11*YFP targeting to cell junctions in Drosophila salivary glands. (A-C) Rab11*YFP detected with a rabbit anti-GFP antibody in salivary glands. (A) Rab11*YFP selectively accumulates at the AJs in 1096GAL4>Rab11*YFP salivary glands. (B) Rab11* distribution is unchanged in 1096GAL...
Data
EF prevents Rab11* accumulation at AJs. Images from experiment described in Fig 1, panels E, F, H and I, were analyzed to quantify the effect of EF on junctional accumulation of Rab11*. Individual image crops from intercellular boundaries were generated. For each crop, average fluorescence was determined in ImageJ, and normalized to the average flu...
Data
Inhibition of Rab11 function in salivary glands leads to abnormal accumulation of D-Ecad around AJs, and intercellular gaps. (A-D) Salivary glands stained with an anti-D-Ecad antibody. (A) A wild-type salivary gland showing D-Ecad accumulation at AJs. (B) A SglGAL4>Rab11DN salivary gland, in which Rab11 inhibition in this tissue leads to D-Ecad acc...
Data
Arf6RNAi rescues normal apical D-Ecad levels in EF-expressing wing discs. Apical levels of D-Ecad in wing discs was measured using ImageJ. Arf6RNAi restores normal levels of apical D-Ecad in 1096GAL4>EF+Arf6RNAi discs (p<0.0001). Arf6RNAi does not notably affect apical levels of D-ECad. Surface areas of wings of the same genotypes were also measure...
Data
ET treatment reduces the levels of cadherins and Rab11 in HBMECs. (A-B) Western blot analysis of HBMECs cells. (A) Rab11A (~28 kD) and pan-Cadherin (~97 kD) levels are severely decreased in ET-treated cells (24hrs), while control actin (~42 kD) levels are only slightly reduced. (B) dcAMP, a cell-permeant stable analog of cAMP that is insensitive to...
Data
ET treatment reduces Rab11/Rip11 co-localization in MDCK cells. Co-localization between Rip11-GFP and DsRed-Rab11A in co-transfected MDCK cells measured by the Pearson's correlation coefficient (PCC) is reduced by ET treatment (n = 43, p<4.85X10-9). (TIF)
Data
Reduction of Epac -but not PKA- levels, suppresses the EF wing phenotype. (A-F) wings of the following genotypes: (A) Wild-type (+/+). (B) 1096GAL4>EF. (C) 1096GAL4>EpacRNAi. (D) 1096GAL4>EF+EpacRNAi. Inhibition of Epac expression potentlyreduces the EF phenotype. (E) PKA-C1B10/+ (B10 is a loss -of-function allele of PKA). (F) 1096GAL4>EF; PKA-C1B1...
Data
Activated Rab11 (Rab11*) preferentially accumulates at AJs in Drosophila wing imaginal discs. (A-C) Expression of Rab11wt in 1096GAL4>Rab11wtYFP wing discs. (A) Rab11wtYFP detected with a rabbit anti-GFP antibody appears as a peppered stain near the apical surface. (B) D-Ecad/GFP double stain. (C) corresponding D-Ecad stain marking AJs. (D-F) Expre...
Data
RNAi transgenes specifically block expression of cognate proteins Sec15, Crag, and Arf6. (A) A 1096GAL4>UAS-Sec15GFP salivary gland, stained with an anti-GFP antibody. (B) Sec15GFP expression in strongly inhibited by co-expression of a Sec15RNAi construct. (C) A 1096GAL4>UAS-CragHA salivary gland stained with an anti-HA antibody. (D) CragHA express...
Data
EF does not disrupt dRip11DN/Rab11 co-localization in salivary glands. (A-C) 1096GAL4>Rip11DN-GFP salivary glands, stained with (A) a rabbit anti GFP antibody, (B) a mouse anti Rab11 antibody, and (C) both antibodies, showing that Rab11 and Rip11DN-GFP co-localize in punctate vesicles. (D-F) 1096GAL4>Rip11DN+EF salivary glands stained with a rabbit...
Data
ET treatment reduces Sec15/Rab11* and Rab11*/Rip11 co-localization in HBMEC cells. (A-C) HBMECs, untreated. (D-F) HBMECs treated with ET for 6hours. Co-localization of Rab11* with Sec15 (B and E) and Rab11* with Rip11 (C and F) can be visualized following transfection of cells with Sec15-GFP. High-level expression of Sec15-GFP, and staining with an...
Article
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The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the t...
Data
Additional activities and in-depth analysis of NS1. (DOC)
Data
NS1(Vn) versus NS1(PR8) on other Hh target genes. Wing imaginal discs with no transgene (no tg) or ubiquitously expressing the indicated transgenes were stained with antibodies recognizing Collier (Col) (A-D), Ptc (E-H), Engrailed (En) (I-L), and Ci-155 (M-P). The moderately active NS1(Vn) has little, if any, effect on expression Col (B vs. A), Ptc...
Data
Analysis of Hh pathway components required for NS1-dependent dpp activation. Reducing the levels of Ci-155 by ubiquitously expressing Cos-2 blocks endogenous dpp-lacZ expression (A), and also blocks the ability of NS1 to enhance dpp-lacZ expression (B). In fused (fumH63) mutant discs, dpp-lacZ expression is broadened but not elevated (C). Expressio...
Data
Cytokine levels in infected mouse lungs. CXCL-10, TNF-α, and IL-1α levels were measured from extracts of mouse lungs infected with PR8-WT and PR8-A122V. CXCL-10 was significantly increased in mutant infected lungs compared to the WT (p = 0.043), whereas TNF-α, and IL-1α were not appreciably changed. 4–5 lungs were analyzed per group. (TIF)
Data
BMP2 expression levels in human cells is proportional to NS1 expression levels. (A) NL20 cells transfected with the indicated NS1-expressing plasmids show an induction of BMP2 expression proportional to NS1 expression. (B) The mean intensity of NS1 was quantified from 17–20 cells per group (*p = 0.021). (TIF)
Data
The A122V mutation disrupts NS1(Ud) binding to CPSF30. GST or GST-NS1 fusion proteins were incubated with extracts of 293T cells transfected with an N-terminal fragment of CPSF30 containing four of its zinc finger binding domains and tagged C-terminally with a V5 epitope. Proteins immune precipitated with glutathione beads were visualized on Wester...
Data
Secreted Dpp mediates the non-autonomous effect of NS1 on neighboring cells. Wing discs expressing the indicated transgenes with the ubiquitous 71B-GAL4 driver (A-F), or along the A/P border with the dpp-GAL4 driver (G, H), were assayed for the pattern of Dpp response by staining with a phospho-Smad1 antibody (pMad). Ubiquitous expression of a wild...
Data
NS1-R38A has reduced activity but can still modulate Hh signaling. (A) A wing with no transgene (no tg) with demarcated longitudinal veins L2-L5, M = margin. Wings expressing NS1(Vn)-R38A (C,E) have a reduced phenotype compared to wings expressing wild type NS1(Vn) (B,D) with either the moderate wing GAL4 driver, 71B (B,C) or the stronger driver, M...
Data
NS1 alters Notch target gene expression in a strain specific and A122V-dependent fashion. NS1 (Vn) reduces expression of Cut along the presumptive wing margin (B vs. A) and increases expression of Gbe-lacZ, a synthetic Notch reporter gene construct (E vs. D). Both of these effects of NS1 (Vn) are greatly reduced by the A122V mutation (C vs. B and F...
Data
The A122V mutation does not affect temporal regulation of viral proteins in infected tissue culture cells or affect weight loss in mice. (A) Temporal synthesis of two viral proteins, NP and M, was examined in A549 cells following a high-MOI single cycle infection with the PR8-WT and PR8-A122V viruses. No differences in the levels or kinetics of the...
Article
Many bacterial and viral pathogens block or subvert host cellular processes to promote successful infection. One host protein that is targeted by invading pathogens is the small GTPase RAB11, which functions in vesicular trafficking. RAB11 functions in conjunction with a protein complex known as the exocyst to mediate terminal steps in cargo transp...
Article
Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balan...
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Full-text available
Many of the cellular mechanisms underlying host responses to pathogens have been well conserved during evolution. As a result, Drosophila can be used to deconstruct many of the key events in host-pathogen interactions by using a wealth of well-developed molecular and genetic tools. In this review, we aim to emphasize the great leverage provided by...
Article
The anthrax toxins lethal toxin (LT) and edema toxin (ET) are essential virulence factors produced by Bacillus anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, d...
Article
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Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dep...
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Intracellular transport and processing of ligands is critical to the activation of signal transduction pathways that guide development. Star is an essential gene in Drosophila that has been implicated in the trafficking of ligands for epidermal growth factor (EGF) receptor signaling. The role of cytoplasmic motors in the endocytic and secretory pat...
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Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can be used for analyzing the mechanism of toxin action. In this study, we characterize the activities of two anthrax virulence factors, lethal factor (LF) and e...
Article
The activity of the TGF-alpha-like ligand Spitz in Drosophila depends on Rhomboid, a seven-transmembrane spanning protein that resides in the Golgi and acts as a serine protease to cleave Spitz, thereby releasing the soluble ligand. Several rhomboids in Drosophila have been implicated in the processing of TGF-alpha-like ligands, and consequent EGF...
Article
The adjacent knirps (kni) and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expre...
Article
The adjacent knirps (kni) and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expre...
Article
Full-text available
The Drosophila epidermal growth factor receptor (EGF-R) controls many critical cell fate choices throughout development. Several proteins collaborate to promote localized EGF-R activation, such as Star and Rhomboid (Rho), which act sequentially to ensure the maturation and processing of inactive membrane-bound EGF ligands. To gain insights into the...
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Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of ind...
Article
The Drosophila rhomboid (rho) gene participates in localized activation of EGF-receptor signaling in various developmental settings. The Rhomboid protein has been proposed to promote presentation and/or processing of the membrane-bound Spitz (mSpi) EGF-related ligand to generate an active diffusible form of the ligand. Here, we report on a new rhom...
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Full-text available
Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila EGF-Receptor, spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid (rho) and Star (S) encode membrane proteins. In this study,...
Article
RacGAP proteins have been shown to down-regulate members of the subfamily, small GTPases controlling actin network organisation. Only one RacGAP protein, RnRacGAP, has been identified in Drosophila. To examine RnRacGAP function, we generated transgenic strains expressing RnRacGAP under the control of the heat-shock promoter hsp70. In cellularising...
Article
The rotund (rn) gene in Drosophila melanogaster codes for a RacGTPase-activating protein, RnRacGAP. Cellular studies have shown that RacGAP proteins function as negative regulators of substrate Rac proteins which, in turn, control the localization and polymerization state of actin within the cell. Previous sequence analysis of rn genomic DNA and in...

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