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1.
Genome Res ; 34(3): 394-409, 2024 04 25.
Article in English | MEDLINE | ID: mdl-38508694

ABSTRACT

mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.


Subject(s)
Lymphocyte Activation , Protein Biosynthesis , RNA Stability , RNA, Messenger , Ribosomes , Ribosomes/metabolism , Animals , Mice , RNA, Messenger/metabolism , RNA, Messenger/genetics , CD4-Positive T-Lymphocytes/metabolism , Mice, Inbred C57BL , T-Lymphocytes/metabolism
2.
Nat Commun ; 14(1): 694, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36755036

ABSTRACT

Type I and III interferons (IFN-I/λ) are important antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDC) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to develop this response could be important to understand severe cases of COVID-19.


Subject(s)
COVID-19 , Interferon Type I , Humans , SARS-CoV-2/metabolism , Antiviral Agents/metabolism , Dendritic Cells/metabolism , Interferon Lambda
3.
Chembiochem ; 23(4): e202100640, 2022 02 16.
Article in English | MEDLINE | ID: mdl-34932835

ABSTRACT

A genetic assay permits simultaneous quantification of two interacting proteins and their bound fraction at the single-cell level using flow cytometry. Apparent in-cellula affinities of protein-protein interactions can be extracted from the acquired data through a titration-like analysis. The applicability of this approach is demonstrated on a diverse set of interactions with proteins from different families and organisms and with in-vitro dissociation constants ranging from picomolar to micromolar.


Subject(s)
Proteins/chemistry , Flow Cytometry , Humans , Protein Binding , Single-Cell Analysis
4.
Wiley Interdiscip Rev RNA ; 12(6): e1658, 2021 11.
Article in English | MEDLINE | ID: mdl-33949788

ABSTRACT

Eukaryotic gene expression is closely regulated by translation and turnover of mRNAs. Recent advances highlight the importance of translation in the control of mRNA degradation, both for aberrant and apparently normal mRNAs. During translation, the information contained in mRNAs is decoded by ribosomes, one codon at a time, and tRNAs, by specifically recognizing codons, translate the nucleotide code into amino acids. Such a decoding step does not process regularly, with various obstacles that can hinder ribosome progression, then leading to ribosome stalling or collisions. The progression of ribosomes is constantly monitored by the cell which has evolved several translation-dependent mRNA surveillance pathways, including nonsense-mediated decay (NMD), no-go decay (NGD), and non-stop decay (NSD), to degrade certain problematic mRNAs and the incomplete protein products. Recent progress in sequencing and ribosome profiling has made it possible to discover new mechanisms controlling ribosome dynamics, with numerous crosstalks between translation and mRNA decay. We discuss here various translation features critical for mRNA decay, with particular focus on current insights from the complexity of the genetic code and also the emerging role for the ribosome as a regulatory hub orchestrating mRNA decay, quality control, and stress signaling. Even if the interplay between mRNA translation and degradation is no longer to be demonstrated, a better understanding of their precise coordination is worthy of further investigation. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.


Subject(s)
Nonsense Mediated mRNA Decay , Ribosomes , Protein Biosynthesis , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/genetics , Ribosomes/metabolism
5.
Sci Rep ; 10(1): 20023, 2020 11 18.
Article in English | MEDLINE | ID: mdl-33208773

ABSTRACT

Glial cells are early sensors of neuronal injury and can store lipids in lipid droplets under oxidative stress conditions. Here, we investigated the functions of the RNA-binding protein, SPEN/SHARP, in the context of Parkinson's disease (PD). Using a data-mining approach, we found that SPEN/SHARP is one of many astrocyte-expressed genes that are significantly differentially expressed in the substantia nigra of PD patients compared with control subjects. Interestingly, the differentially expressed genes are enriched in lipid metabolism-associated genes. In a Drosophila model of PD, we observed that flies carrying a loss-of-function allele of the ortholog split-ends (spen) or with glial cell-specific, but not neuronal-specific, spen knockdown were more sensitive to paraquat intoxication, indicating a protective role for Spen in glial cells. We also found that Spen is a positive regulator of Notch signaling in adult Drosophila glial cells. Moreover, Spen was required to limit abnormal accumulation of lipid droplets in glial cells in a manner independent of its regulation of Notch signaling. Taken together, our results demonstrate that Spen regulates lipid metabolism and storage in glial cells and contributes to glial cell-mediated neuroprotection.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Lipid Droplets/chemistry , Neuroglia/cytology , Paraquat/toxicity , Parkinson Disease/prevention & control , RNA-Binding Proteins/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/drug effects , Drosophila melanogaster/metabolism , Herbicides/toxicity , Homeodomain Proteins/genetics , Male , Neuroglia/drug effects , Neuroglia/metabolism , Parkinson Disease/etiology , Parkinson Disease/metabolism , Parkinson Disease/pathology , RNA-Binding Proteins/genetics
6.
PLoS Biol ; 18(11): e3000940, 2020 11.
Article in English | MEDLINE | ID: mdl-33253165

ABSTRACT

It is unknown how growth in one tissue impacts morphogenesis in a neighboring tissue. To address this, we used the Drosophila ovarian follicle, in which a cluster of 15 nurse cells and a posteriorly located oocyte are surrounded by a layer of epithelial cells. It is known that as the nurse cells grow, the overlying epithelial cells flatten in a wave that begins in the anterior. Here, we demonstrate that an anterior to posterior gradient of decreasing cytoplasmic pressure is present across the nurse cells and that this gradient acts through TGFß to control both the triggering and the progression of the wave of epithelial cell flattening. Our data indicate that intrinsic nurse cell growth is important to control proper nurse cell pressure. Finally, we reveal that nurse cell pressure and subsequent TGFß activity in the stretched cells combine to increase follicle elongation in the anterior, which is crucial for allowing nurse cell growth and pressure control. More generally, our results reveal that during development, inner cytoplasmic pressure in individual cells has an important role in shaping their neighbors.


Subject(s)
Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Ovarian Follicle/cytology , Ovarian Follicle/metabolism , Animals , Biomechanical Phenomena , Cell Differentiation , Cell Polarity , Cell Shape , Cytoplasm/metabolism , Drosophila Proteins/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Microscopy, Atomic Force , Models, Biological , Oocytes/cytology , Oocytes/metabolism , Oogenesis , Pressure , Signal Transduction , Transforming Growth Factor beta/metabolism
7.
Mol Cell Proteomics ; 19(4): 701-715, 2020 04.
Article in English | MEDLINE | ID: mdl-32015065

ABSTRACT

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 µm After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.


Subject(s)
Flow Cytometry , Saccharomyces cerevisiae/metabolism , Two-Hybrid System Techniques , Fluorescence , Genes, Reporter , Peroxins/metabolism , Protein Binding , Protein Interaction Mapping , Proteome/metabolism , Reference Standards , Saccharomyces cerevisiae Proteins/metabolism , Single-Cell Analysis
8.
Nucleic Acids Res ; 47(21): 11164-11180, 2019 12 02.
Article in English | MEDLINE | ID: mdl-31602465

ABSTRACT

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , DNA-Binding Proteins/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Multiprotein Complexes/physiology , Sin3 Histone Deacetylase and Corepressor Complex/metabolism , Trans-Activators/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , DNA-Binding Proteins/genetics , Embryo, Nonmammalian , Histone-Lysine N-Methyltransferase/physiology , Multiprotein Complexes/metabolism , Protein Binding
9.
Nucleic Acids Res ; 46(15): 7686-7700, 2018 09 06.
Article in English | MEDLINE | ID: mdl-29931089

ABSTRACT

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.


Subject(s)
DEAD-box RNA Helicases/genetics , Gene Expression Profiling , MicroRNAs/genetics , Repressor Proteins/genetics , Cell Line, Tumor , DEAD-box RNA Helicases/metabolism , Humans , MCF-7 Cells , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/metabolism , Repressor Proteins/metabolism
10.
Int J Mol Sci ; 18(4)2017 Mar 24.
Article in English | MEDLINE | ID: mdl-28338624

ABSTRACT

Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.


Subject(s)
Caveolin 1/metabolism , Cell Nucleus/metabolism , Membrane Microdomains/metabolism , Nerve Growth Factor/pharmacology , Signal Transduction/drug effects , Animals , Antibodies, Monoclonal/immunology , CREB-Binding Protein/metabolism , Caveolin 1/antagonists & inhibitors , Caveolin 1/genetics , Caveolin 2/antagonists & inhibitors , Caveolin 2/genetics , Caveolin 2/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Mice , Nerve Tissue Proteins , PC12 Cells , Phosphorylation/drug effects , Protein Binding , Protein Transport/drug effects , RNA Interference , RNA, Small Interfering/metabolism , Rats , Receptor, Nerve Growth Factor/metabolism , Receptor, trkA/chemistry , Receptor, trkA/immunology , Receptor, trkA/metabolism , Receptors, Growth Factor , Receptors, Nerve Growth Factor/chemistry , Receptors, Nerve Growth Factor/immunology , Receptors, Nerve Growth Factor/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism
11.
PLoS One ; 9(4): e93718, 2014.
Article in English | MEDLINE | ID: mdl-24763198

ABSTRACT

The mitotic spindle is a microtubule-based structure that elongates to accurately segregate chromosomes during anaphase. Its position within the cell also dictates the future cell cleavage plan, thereby determining daughter cell orientation within a tissue or cell fate adoption for polarized cells. Therefore, the mitotic spindle ensures at the same time proper cell division and developmental precision. Consequently, spindle dynamics is the matter of intensive research. Among the different cellular models that have been explored, the one-cell stage C. elegans embryo has been an essential and powerful system to dissect the molecular and biophysical basis of spindle elongation and positioning. Indeed, in this large and transparent cell, spindle poles (or centrosomes) can be easily detected from simple DIC microscopy by human eyes. To perform quantitative and high-throughput analysis of spindle motion, we developed a computer program ACT for Automated-Centrosome-Tracking from DIC movies of C. elegans embryos. We therefore offer an alternative to the image acquisition and processing of transgenic lines expressing fluorescent spindle markers. Consequently, experiments on large sets of cells can be performed with a simple setup using inexpensive microscopes. Moreover, analysis of any mutant or wild-type backgrounds is accessible because laborious rounds of crosses with transgenic lines become unnecessary. Last, our program allows spindle detection in other nematode species, offering the same quality of DIC images but for which techniques of transgenesis are not accessible. Thus, our program also opens the way towards a quantitative evolutionary approach of spindle dynamics. Overall, our computer program is a unique macro for the image- and movie-processing platform ImageJ. It is user-friendly and freely available under an open-source licence. ACT allows batch-wise analysis of large sets of mitosis events. Within 2 minutes, a single movie is processed and the accuracy of the automated tracking matches the precision of the human eye.


Subject(s)
Caenorhabditis elegans/ultrastructure , Embryo, Nonmammalian/ultrastructure , Software , Spindle Apparatus/ultrastructure , Animals , Automation, Laboratory , Centrosome/ultrastructure , Humans , Microscopy, Polarization , Microscopy, Video
12.
Mol Cell Proteomics ; 12(7): 1939-52, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23579184

ABSTRACT

We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.


Subject(s)
Aptamers, Peptide/metabolism , Calcineurin/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Aptamers, Peptide/genetics , Cell Line, Tumor , HeLa Cells , Humans , Mutagenesis, Site-Directed , Rats , Two-Hybrid System Techniques
13.
J Virol ; 86(14): 7530-43, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22553336

ABSTRACT

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation.


Subject(s)
Eukaryotic Initiation Factor-3/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , RNA Stability , RNA, Messenger/metabolism , Trans-Activators/metabolism , Basic-Leucine Zipper Transcription Factors/biosynthesis , Basic-Leucine Zipper Transcription Factors/genetics , HEK293 Cells , Humans , Jurkat Cells , RNA Helicases , Retroviridae Proteins , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Viral Proteins/biosynthesis , Viral Proteins/genetics
14.
Cancer Res ; 72(8): 2006-16, 2012 Apr 15.
Article in English | MEDLINE | ID: mdl-22508697

ABSTRACT

Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , DNA Damage/physiology , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factor-3/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , BRCA1 Protein/metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Survival , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Immunoblotting , Immunoprecipitation , Microscopy, Confocal
15.
Mol Biol Cell ; 18(12): 4899-910, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17898081

ABSTRACT

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.


Subject(s)
Actins/metabolism , Hyaluronan Receptors/metabolism , Integrin beta3/metabolism , Osteoclasts/metabolism , Animals , Bone Resorption , Intracellular Membranes/metabolism , Mice , Mice, Knockout , Signal Transduction , Wiskott-Aldrich Syndrome Protein/deficiency , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/metabolism
16.
J Virol ; 79(13): 8629-36, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15956605

ABSTRACT

CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and co-immunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucine motif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.


Subject(s)
CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/virology , Gene Products, nef/immunology , HIV-1/physiology , Antigens, CD/immunology , Bacterial Proteins/analysis , Binding Sites , Cell Line , Genes, Reporter , HIV-1/genetics , HIV-1/immunology , Humans , Kidney , Luminescent Measurements/methods , Luminescent Proteins/analysis , nef Gene Products, Human Immunodeficiency Virus
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