A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

Mapping Intimacies ◽

10.1101/106930 ◽

2017 ◽

Author(s):

Stefan Harmansa ◽

Ilaria Alborelli ◽

Emmanuel Caussinus ◽

Markus Affolter

Keyword(s):

Protein Function ◽

Imaginal Disc ◽

Protein Localization ◽

Wing Disc ◽

Wing Imaginal Disc ◽

System A ◽

Polarized Cells ◽

Lateral Plane

AbstractInvestigating the role of protein localization is crucial to understand protein function in cells or tissues. However, in many cases the role of different subcellular fractions of given proteins along the apical-basal axis of polarized cells has not been investigated in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a nanobody-based toolbox to modify the localization and the dispersal of GFP-tagged proteins along the apical-basal axis of polarized cells. We show that the GrabFP system is an effective and easy-to-implement tool to mislocalize cytosolic and transmembrane GFP-tagged proteins and thereby functionally investigate protein localization along the apical-basal axis. We use the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.

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A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila

eLife ◽

10.7554/elife.22549 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Stefan Harmansa ◽

Ilaria Alborelli ◽

Dimitri Bieli ◽

Emmanuel Caussinus ◽

Markus Affolter

Keyword(s):

Fusion Proteins ◽

Imaginal Disc ◽

Protein Localization ◽

Wing Disc ◽

Drosophila Wing ◽

System A ◽

Polarized Cells ◽

Lateral Plane

The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.

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PPM1G promotes the progression of hepatocellular carcinoma via phosphorylation regulation of alternative splicing protein SRSF3

Cell Death and Disease ◽

10.1038/s41419-021-04013-y ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Dawei Chen ◽

Zhenguo Zhao ◽

Lu Chen ◽

Qinghua Li ◽

Jixue Zou ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Alternative Splicing ◽

Protein Function ◽

Vital Role ◽

Normal Tissues ◽

Mechanistic Analysis ◽

Highly Correlated ◽

Splicing Patterns

AbstractEmerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

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Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila

Cell Death and Disease ◽

10.1038/s41419-021-04091-y ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Dae-Wook Yang ◽

Jung-Wan Mok ◽

Stephanie B. Telerman ◽

Robert Amson ◽

Adam Telerman ◽

...

Keyword(s):

Cell Survival ◽

Topoisomerase Ii ◽

Wing Disc ◽

Organ Development ◽

Translationally Controlled Tumor Protein ◽

Protein Levels ◽

Eye Disc ◽

Mutual Regulation

AbstractRegulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Control of growth and patterning of the Drosophila wing imaginal disc by EGFR-mediated signaling

Development ◽

10.1242/dev.129.6.1369 ◽

2002 ◽

Vol 129 (6) ◽

pp. 1369-1376 ◽

Cited By ~ 2

Author(s):

Myriam Zecca ◽

Gary Struhl

Keyword(s):

Gene Expression ◽

Imaginal Disc ◽

Ectopic Expression ◽

Growth Factor Receptor ◽

Wing Disc ◽

Wing Imaginal Disc ◽

Egfr Signaling ◽

Drosophila Wing ◽

Compartment Boundary ◽

Egfr Ligand

The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal Growth Factor Receptor (EGFR) signaling, which heritably activates apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. We examine the source, identity and mode of action of the EGFR ligand(s) that specify these subdivisions. Of the three known ligands for the Drosophila EGFR, only Vein (Vn), but not Spitz or Gurken, is required for wing disc development. We show that Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression. However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where Vn-EGFR signaling is effective. After ap is heritably activated, the level of EGFR activity declines in D compartment cells as they proliferate and move ventrally, away from the source of the instructive ligand. We present evidence that this reduction is necessary for D and V compartment cells to interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing primordium.

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GITR-GITRL System, A Novel Player in Shock and Inflammation

The Scientific World JOURNAL ◽

10.1100/tsw.2007.106 ◽

2007 ◽

Vol 7 ◽

pp. 533-566 ◽

Cited By ~ 45

Author(s):

Ludovic Tibor Krausz ◽

Rodolfo Bianchini ◽

Simona Ronchetti ◽

Katia Fettucciari ◽

Giuseppe Nocentini ◽

...

Keyword(s):

Innate Immunity ◽

T Cells ◽

Inflammatory Responses ◽

Inflammatory Diseases ◽

Tumor Necrosis Factor Receptor ◽

Early Response ◽

System A ◽

Antigen Presenting

Glucocorticoid-induced TNFR-Related (GITR) protein is a member of the tumor necrosis factor receptor superfamily that modulates acquired and natural immune response. It is expressed in several cells and tissues, including T cells, natural killer cells, and, at lower levels, in cells of innate immunity. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting and endothelial cells. Recent evidence suggests that the GITR/GITRL system participates in the development of inflammatory responses, including shock, either due to early response of neutrophils and macrophages, or together with autoimmune/allergic pathogenesis. The pro-inflammatory role of the GITR/GITRL system is due to: 1) modulation of the extravasation process, 2) activation of innate immunity cells, 3) activation of effector T cells also favored by partial inhibition of suppressor T cells and modulation of dendritic function. This review summarizes thein vivorole of the GITR/GITRL system in inflammation and shock, explaining the mechanisms responsible for their effects, considering the interplay among the different cells of the immune system and transduction pathways activated by GITR and GITRL triggering. The hidden aspects about GITR/GITRL function, crucial for treatment planning of inflammatory diseases and shock by modulation of this system is stressed.

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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

eLife ◽

10.7554/elife.38709 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

David Li-Kroeger ◽

Oguz Kanca ◽

Pei-Tseng Lee ◽

Sierra Cowan ◽

Michael T Lee ◽

...

Keyword(s):

Protein Function ◽

Null Allele ◽

Protein Localization ◽

Dominant Marker ◽

Endogenous Gene ◽

Gene Trapping ◽

Cassette Exchange ◽

Protein Tag ◽

Integration Efficiency

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

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An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

10.1101/337337 ◽

2018 ◽

Author(s):

David Li-Kroeger ◽

Oguz Kanca ◽

Pei-Tseng Lee ◽

Sierra Cowan ◽

Michael Lee ◽

...

Keyword(s):

Protein Function ◽

Null Allele ◽

Protein Localization ◽

Dominant Marker ◽

Endogenous Gene ◽

Gene Trapping ◽

Cassette Exchange ◽

Protein Tag ◽

Integration Efficiency

AbstractWe generated new genetic tools to efficiently tag genes in Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on CRE recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette.This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications.

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Cyba-deficient mice display an increase in hematopoietic stem cells and an overproduction of immunoglobulins

Haematologica ◽

10.3324/haematol.2019.233064 ◽

2020 ◽

Vol 106 (1) ◽

pp. 142-153 ◽

Cited By ~ 1

Author(s):

Rodrigo Prieto-Bermejo ◽

Marta Romo-González ◽

Alejandro Pérez-Fernández ◽

Ignacio García-Tuñón ◽

Manuel Sánchez-Martín ◽

...

Keyword(s):

Protein Function ◽

Sharp Decrease ◽

Nadph Oxidases ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Stem Cell Populations ◽

Deficient Mice ◽

Transplant Experiments

The regulation of protein function by reversible oxidation is increasingly recognized as a key mechanism for the control of cellular signaling, modulating crucial biological processes such as cell differentiation. In this scenario, NADPH oxidases must occupy a prominent position. Our results show that hematopoietic stem and progenitor cells express three p22phox-dependent NADPH oxidases members (NOX1, NOX2 and NOX4). By deleting the p22phox coding gene (Cyba), here we have analyzed the importance of this family of enzymes during in vivo hematopoiesis. Cyba-/- mice show a myeloid bias, and an enrichment of hematopoietic stem cell populations. By means of hematopoietic transplant experiments we have also tried to dissect the specific role of the NADPH oxidases. While the absence of NOX1 or NOX2 provides a higher level of reconstitution, a lack of NOX4 rendered the opposite result, suggesting a functional specificity among the different NADPH oxidases. Cyba-/- cells showed a hampered activation of AKT1 and a sharp decrease in STAT5 protein. This is in line with the diminished response to IL-7 shown by our results, which could explain the overproduction of immunoglobulins observed in Cyba-/- mice.

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