Cell-specific mitochondria affinity purification (CS-MAP) from Caenorhabditis elegans.
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ABSTRACT:
SUBMITTER: Ahier A
PROVIDER: S-EPMC8605396 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell type-specific mRNA profiles of any genetically defined cell type, and it has been used in organisms ranging from Drosophila melanogaster to mice and human cultured cells. Unlike other methodologies that rely on microdissection, cell panning or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and the potential artifacts that these treatments introduce) and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implement the TRAP methodology, which takes 2 d to complete once all materials are in hand.
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Project description:The innexin family of gap junction proteins has 25 members in Caenorhabditis elegans. Here, we describe the first high-resolution expression map of all members through analysis of live worms transformed with green fluorescent protein under the control of entire promoter regions. Our analyses show that innexins have dynamic expression patterns throughout development and are found in virtually all cell types and tissues. Complex tissues, such as the pharynx, intestine, gonad, as well as scaffolding tissues and guidepost cells express a variety of innexins in overlapping or complementary patterns, suggesting they may form heteromeric and heterotypic channels. Innexin expression occurs in several types of cells that are not known to form gap junctions as well as in a pair of migrating cells, suggesting they may have hemichannel function. Therefore, innexins likely play roles in almost all body functions, including embryonic development, cell fate determination, oogenesis, egg laying, pharyngeal pumping, excretion, and locomotion.
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Project description:Post-transcriptional regulation including mRNA binding to ribosomes plays an important role in determining cell-type-specific gene expression patterns. Here, we applied an approach that profiles cell-type-specific mRNAs. The Translating Ribosome Affinity Purification method (TRAP; Heiman et al., Cell, 2008 and Doyle et al., Cell, 2008) was developed in mice and has been combined with the UAS/Gal4 system in Drosophila (Thomas et al., PLoS ONE, 2012). TRAP is a powerful method to find cell-type-specific differences at the level of the 'translatome' (Dougherty, Schmidt, Nakajima, & Heintz, Nucleic Acids Research, 2010). In parallel to published efforts, we developed and implemented the method for the fly and compared distinct head cell types and identified cell-type-specific transcript classes with neuronal (e.g. receptor-, neuropeptide- or hormone activity) or glial function (e.g. transporter activity). Neuronal TRAP genes are over-represented in the brain, larval CNS and thoracico-abdominal ganglion (Chintapalli, Wang, & Dow, Nature Genetics, 2007). Using cell-type-to-cell-type comparisons (e.g. neurons vs. glia), instead of a given cell population to the total (e.g. neurons vs. head), the differences could be identified with greater resolution. TRAP uncovered more neuronal genes compared to neuronal RNA polymerase II ChIP-seq data (Schauer et al., Cell Reports, 2013). Thus, TRAP data confirm the importance of post-transcriptional regulation in defining cell identity. TRAP is one of the best methods to reveal differential "omics" data among distinct cell types by profiling ribosome-bound mRNAs. TRAP is a promising tool to reveal cell-type-specific transcriptional and translational changes in a perturbed environment.
2017-02-01 | GSE89993 | GEO
Project description:BackgroundAffinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide.ResultsWe generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules.ConclusionsThe method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.
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Project description:High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5' and 3' ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans.
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