In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics
2016; Elsevier BV; Volume: 15; Issue: 5 Linguagem: Inglês
10.1074/mcp.m115.053975
ISSN1535-9484
AutoresJia‐Xuan Chen, Patricia G. Cipriani, Desirea Mecenas, Jolanta Polanowska, Fabio Piano, Kristin C. Gunsalus, Matthias Selbach,
Tópico(s)Bioinformatics and Genomic Networks
ResumoStudying protein interactions in whole organisms is fundamental to understanding development. Here, we combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development. Our network reflects known biology and is highly enriched in functionally relevant interactions. To demonstrate the utility of the map, we looked for new regulators of P granule dynamics and found that GEI-12, a novel binding partner of the DYRK family kinase MBK-2, is a key regulator of P granule formation and germline maintenance. Our data corroborate a recently proposed model in which the phosphorylation state of GEI-12 controls P granule dynamics. In addition, we find that GEI-12 also induces granule formation in mammalian cells, suggesting a common regulatory mechanism in worms and humans. Our results show that in vivo interaction proteomics provides unique insights into animal development. Studying protein interactions in whole organisms is fundamental to understanding development. Here, we combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development. Our network reflects known biology and is highly enriched in functionally relevant interactions. To demonstrate the utility of the map, we looked for new regulators of P granule dynamics and found that GEI-12, a novel binding partner of the DYRK family kinase MBK-2, is a key regulator of P granule formation and germline maintenance. Our data corroborate a recently proposed model in which the phosphorylation state of GEI-12 controls P granule dynamics. In addition, we find that GEI-12 also induces granule formation in mammalian cells, suggesting a common regulatory mechanism in worms and humans. Our results show that in vivo interaction proteomics provides unique insights into animal development. Protein-protein interactions (PPIs) 1The abbreviations used are:PPIprotein-protein interactionAP-MSaffinity purification and mass spectrometryDYRKdual-specificity tyrosine-regulated kinaseGOgene ontologyIPimmunoprecipitationIVIin vivo interactomeLClow complexityLCIliterature-curated interactionsLC-MS/MSliquid chromatography mass spectrometryLFQlabel-free quantificationRNPribonucleoproteinRNAiRNA interferenceSILACstable isotope labelling by amino acids in cell cultureWI8worm interactome 8. are central to virtually all aspects of life. The systematic characterization of all PPIs is therefore a major goal and challenge in the post genomic era. Large scale in vitro screens using cell lines or the yeast two-hybrid system have generated protein interaction maps that can help to better understand the functional organization of the proteome (1Malovannaya A. Lanz R.B. Jung S.Y. Bulynko Y. Le N.T. Chan D.W. Ding C. Shi Y. Yucer N. Krenciute G. Kim B.J. Li C. Chen R. Li W. Wang Y. O'Malley B.W. 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Similarly, many PPIs are regulated by condition-specific post-translational modifications, which may not be adequately represented in yeast or cell-based assay systems. These limitations provide compelling reasons to develop approaches that can capture the endogenous interaction partners of proteins within a living organism. protein-protein interaction affinity purification and mass spectrometry dual-specificity tyrosine-regulated kinase gene ontology immunoprecipitation in vivo interactome low complexity literature-curated interactions liquid chromatography mass spectrometry label-free quantification ribonucleoprotein RNA interference stable isotope labelling by amino acids in cell culture worm interactome 8. During embryogenesis, PPIs play key roles in directing and coordinating essential developmental processes. 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Briefly, inoculated RNAi bacterial culture was grown in LB media containing 50 μg/ml ampicillin for ∼7 h, followed by IPTG (1 mm) induction for 1 h. The bacterial culture was then seeded onto NGM plates containing 50 μg/ml ampicillin and 1 mm IPTG. Seeded plates were let dry, protected from light and incubated at room temperature overnight. Synchronized L1 larvae were then added to the seeded plates and were incubated at 25 °C for ∼48 h until F1 embryos could be harvested for further examinations. For trans-generational feeding and sterility assays, L1 larvae were subjected to gei-12(RNAi) continuously through adulthood on solid medium at 15 °C or 25 °C; multiple L1 progeny were individually transferred to new feeding plates and the process was repeated through two filial generations. Adults from each generation were examined individually and scored as sterile if no embryos were visible in the uterus. Embryos were fixed by the freeze-cracking method in liquid nitrogen, followed by methanol/acetone fixation and rehydration in a descending acetone series modified from Takeda et al. (39Takeda K. Watanabe C. Qadota H. Hanazawa M. Sugimoto A. Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans embryos using an antigen subtraction method.Genes Cells. 2008; 13: 653-665Crossref PubMed Scopus (11) Google Scholar). P granules were stained by monoclonal K76 antibody (1:5 dilution) (40Strome S. Wood W.B. Generation of asymmetry and segregation of germ-line granules in early C. elegans embryos.Cell. 1983; 35: 15-25Abstract Full Text PDF PubMed Scopus (419) Google Scholar), followed by FITC-conjugated anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA). Slides were mounted using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Transfected HEK293T cells were fixed by 4% paraformaldehyde, washed and briefly stained with DAPI. Slides were mounted using VECTASHIELD HardSet mounting medium. Live and fixed embryo fluorescence imaging and time-lapse microscopy were carried out using a Leica DM RA2 microscope equipped with a Hamamatsu C9100–12 EM-CCD camera. Fixed mammalian cell images were acquired with a Zeiss Axio Imager M2 system. Images were processed in Volocity (PerkinElmer, Waltham, MA) and ImageJ software (41Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat. Methods. 2012; 9: 671-675Crossref PubMed Scopus (34981) Google Scholar). Embryos (∼2 million per replicate) were freshly harvested in biological triplicate by bleaching young gravid hermaphrodites and sonicated on ice (cycle: 0.5 s, amplitude: 40–45%, 5 strokes/session, 5 sessions, interval between sessions: 30 s; UP200S ultrasonic processor (Hielscher Ultrasonics GmbH)) in lysis buffer (total volume: ∼600 μl; 50 mm Tris-HCl, pH 7.4, 100 mm KCl, 1 mm MgCl2, 1 mm EGTA, 1 mm DTT, 10% glycerol, protease inhibitor mixture (Roche), 0.1% Nonidet P-40 Substitute (Sigma)). After sonication, Nonidet P-40 Substitute was added up to 1% and the lysates were incubated with head over tail rotation at 4 °C for 30 min, followed by centrifugation at 20,000 × g for 20 min at 4 °C. Cleared lysate was then aspirated without disturbing the upper lipid layer and split by half into either the anti-GFP agarose beads or the blocked control beads (40–50 μl, Chromotek) (Fig. 1A). After head over tail rotation at 4 °C for 60–90 min, the beads were washed once with lysis buffer containing 0.1% Nonidet P-40 Substitute, followed by two times of washing in either buffer I (25 mm Tris-HCl, pH 7.4, 300 mm NaCl, 1 mm MgCl2) or buffer II (1 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl2) or both. For GFP::MBK-2 pull-downs, two separate experiments were performed using different washing conditions. Proteins were eluted by orbital shaking in 50 μl of 6 m urea/2 M thiourea at room temperature. For the MBK-1::GFP pull-down experiments, proteins were eluted twice by shaking in 50 μl of 8 m guanidinium chloride at 90 °C, followed by ethanol precipitation. Eluted protein samples were then digested in-solution as previously reported (19Paul F.E. Hosp F. Selbach M. Analyzing protein-protein interactions by quantitative mass spectrometry.Methods. 2011; 54: 387-395Crossref PubMed Scopus (55) Google Scholar). For checking the impact of post-lysis interactions, SILAC-labeled "light" BS1080 (GLD-1::GFP) young adults were mixed with "heavy" N2 worms, either before lysis or only at the last washing step before elution (Fig. 2A). For the samples mixed before the pull-down, lysates were incubated with anti-GFP agarose beads for 60 min and the bound proteins were eluted in 6 m urea/2 M thiourea. For the samples mixed after the pull-down, the incubation time was 30 min and the elution was performed in 100 mm glycine-HCl, pH 2.5. For the label-swap SILAC pull-down experiments using HEK293T cells, the EGFP::GEI-12 expressing cells and the control EGFP-only expressing cells were lysed separately in lysis buffer (25 mm Tris-HCl, pH 7.4, 125 mm KCl, 1 mm MgCl2, 1 mm EGTA, 1 mm DTT, 5% glycerol, protease inhibitor mixture, 1% Triton X-100). Cleared lysates were incubated with anti-GFP agarose beads at 4 °C for 90 min, followed by three sequential washes in the following buffers: I (25 mm Tris-HCl, pH 7.4, 125 mm KCl, 1 mm MgCl2, 1 mm EGTA, 0.1% Triton X-100), II (25 mm Tris-HCl, pH 7.4, 125 mm KCl, 1 mm MgCl2, 1 mm EGTA), III (1 mm Tris-HCl, pH 7.4, 150 mm KCl, 1 mm MgCl2). Beads of the two SILAC states were combined before the final wash. Proteins were eluted using 8 m guanidinium chloride, ethanol precipitated and further processed as mentioned above. To check whether GEI-12 interactions were partially mediated by RNA, pull-down experiments against EGFP::GEI-12 were performed using cell lysates pre-treated with or without nuclease (250 U, Pierce Universal Nuclease, Thermo Scientific) for 20 min. To simulate the detection of label-free pull-down enrichment in a complex nonspecific background, two samples of predefined composition were prepared: sample 1: 1× E. coli lysate, 1× UPS2 human standard (Sigma Aldrich), recombinant CDC42 (gift from Florian Paul); sample 2: 1× E. coli lysate, 4× UPS2 human standard, recombinant RAC1, RHOA, and GFP. These two samples were measured in succession in triplicate. Peptide mixtures were separated by reversed phase chromatography using the Eksigent NanoLC Ultra system or the EASY-nLC system (Th
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