In vivo mapping of tissue- and subcellular-specific proteomes in<i>Caenorhabditis elegans</i>

Reinke, Aaron W.; Mak, Raymond; Troemel, Emily R.; Bennett, Eric J.

doi:10.1101/066134

Cited by 13 publications

(20 citation statements)

References 28 publications

(2 reference statements)

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“…We expect that APEX-seq will be broadly applicable to many organisms and cell types, just as APEX proteomics has been extended to flies (Chen et al, 2015a), worms (Reinke et al, 2017), yeast (Hwang and Espenshade, 2016), and neurons . APEX-seq could be fruitfully applied to polarized cells, cells with long extensions (e.g.…”

Section: Discussionmentioning

confidence: 99%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.(ERM), which we previously attempted but failed to map using APEX2-ERM and APEX-RIP (Kaewsapsak et al., 2017). When we performed a comparison of APEX-seq and APEX-RIP using HEK 239T cells expressing APEX on the ER membrane (facing the cytosol), we found that APEX-seq could clearly enrich secretory mRNAs on the ER membrane over cytosolic mRNAs, whereas APEX-RIP could not ( Figure 1D). Given the very close proximity between ER membrane-associated RNAs and cytosolic RNAs that are immediately adjacent to the ER, these results suggest that APEX-seq has a labeling radius of only a few nanometers. Validation of APEX-seqEncouraged by the results above, we expanded APEX-seq to nine distinct subcellular locations ( Figure 1E). Correct localization of each APEX2 fusion protein (domain structures shown in Figure S2A) in HEK-293T cells was confirmed by imaging with organelle markers ( Figure 1F). We prepared two replicates for each location ( Figure S2B and S2C, correlation coefficient r between replicates ranged from 0.97 to 1) as well as negative controls in which H2O2 was omitted from the labeling reaction. After labeling for 1 minute and cell lysis, we enriched biotinylated RNAs and prepared APEX-seq libraries with polyA+ selection. Transcripts shorter than 100 nt were excluded during purification. We used DESeq2 (Love et al., 2014) for data analysis and used a qvalue (FDR-adjusted p-value) < 0.05 and a log2fold-change > 0.75 to select for significantly enriched transcripts.Consistent with the RT-qPCR results for the mitochondrial matrix shown in Figure 1C, our sequencing-based analysis showed that all 13 mRNAs and 2 rRNAs encoded by mtDNA are strongly enriched by MITO-APEX2 labeling (Figure 2A, S2D and S2E, mean enrichment > 11fold), whereas no mRNAs encoded by the nuclear genome are enriched. Figure 2B shows good correlation (r > 0.9) between technical replicates.To assess APEX-seq in an "open" subcellular region (not enclosed by a membrane), we focused again on the ER membrane (ERM). The ERM is particularly valuable for methodology validation because there are well-established "true positives" (mRNAs encoding secreted proteins that are known to be translated at the ERM) and well-established "false positive...

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Section: Discussionmentioning

confidence: 99%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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“…BioID has only been used in the mouse brain for mapping of the inhibitory post-synapse, where biotin was supplied by IP injection for 7 days 22 , and in a xenograft model, where biotin was supplied by IP injection for 2 days 33 . APEX peroxidase has been used in three in vivo studies, but in each case, genetic modification to compromise cuticle integrity 35,36 or manual dissection of tissue had to be performed 37 to deliver APEX chemical substrates to the relevant cells. APEX also relies on H2O2 which is toxic.…”

Section: Discussionmentioning

confidence: 99%

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Branon

2017

Preprint

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Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs -BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans. Main textProximity labeling (PL) has emerged as an alternative to immunoprecipitation and biochemical fractionation for the proteomic analysis of macromolecular complexes, organelles, and protein interaction networks 1 . In PL, a promiscuous labeling enzyme is targeted by genetic fusion to a specific protein or subcellular region. Addition of a small molecule substrate, such as biotin, initiates covalent tagging of endogenous proteins within a few nanometers of the promiscuous enzyme (Figure 1a). Subsequently, the biotinylated proteins are harvested using streptavidin-coated beads and identified by mass spectrometry (MS).Two enzymes are commonly used for PL: APEX2, an engineered variant of soybean ascorbate peroxidase 2,3 , and BirA-R118G (here, referred to as "BioID"), a point mutant of E. coli biotin ligase 4,5 . The main advantage of APEX2 is its speed: proximal proteins can be tagged in 1 minute or less, enabling dynamic analysis of protein interaction networks 6,7 . However, APEX labeling requires the use of H2O2, which is toxic to cells and difficult to deliver into live organisms without causing severe tissue damage. By contrast, BioID is attractive because of the simplicity of its labeling protocol and non-toxic labeling conditions -only biotin needs to be added to initiate tagging. These attributes have resulted in over 100 applications of BioID over the past 5 years, in cultured mammalian cells 5,8,9 , plant protoplasts 10 , parasites [11][12][13][14][15][16][17][18][19] , slime mold 20,21 , and mouse 22 . BioID has been used, for example, to map the protein composition of the centrosome-cilium interface 8 and the inhibitory postsynaptic region 22 , each with nanometer spatial specificity. The major disadvantage of BioID, however, is its slow kinetics, which necessitates labeling with biotin for 18-24 hours, and sometimes much longer 22 , to accumulate sufficient quantities of biotinylated material for proteomic analysis. This precludes the use of BioID for studying dynamic processes that occur on the timescale of...

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“…The complete neuron morphologies and synaptic connections of key memory centres 74,75 or even entire nervous systems 72,76,77 have been reconstructed using electron microscopy for Drosophila , C. elegans and Platynereis . In addition, methods for transcriptome profiling of single cells and cell types have been applied to chart the complement of neuromodulators across neurons in learning circuits 78‐81 . This allows these neuromodulatory connections to be mapped onto the synaptic connectome, which has highlighted the notion that neuromodulators mainly signal extrasynaptically at the same time as forming a tractable framework for comprehensive analysis of neuromodulator action within learning circuits 70‐71,82 …”

Section: Neuromodulatory Pathways In Invertebrate Learning Circuitsmentioning

confidence: 99%

Neuromodulatory pathways in learning and memory: Lessons from invertebrates

Damme

Fruyt

Watteyne

et al. 2020

J Neuroendocrinology

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In an ever‐changing environment, animals have to continuously adapt their behaviour. The ability to learn from experience is crucial for animals to increase their chances of survival. It is therefore not surprising that learning and memory evolved early in evolution and are mediated by conserved molecular mechanisms. A broad range of neuromodulators, in particular monoamines and neuropeptides, have been found to influence learning and memory, although our knowledge on their modulatory functions in learning circuits remains fragmentary. Many neuromodulatory systems are evolutionarily ancient and well‐conserved between vertebrates and invertebrates. Here, we highlight general principles and mechanistic insights concerning the actions of monoamines and neuropeptides in learning circuits that have emerged from invertebrate studies. Diverse neuromodulators have been shown to influence learning and memory in invertebrates, which can have divergent or convergent actions at different spatiotemporal scales. In addition, neuromodulators can regulate learning dependent on internal and external states, such as food and social context. The strong conservation of neuromodulatory systems, the extensive toolkit and the compact learning circuits in invertebrate models make these powerful systems to further deepen our understanding of neuromodulatory pathways involved in learning and memory.

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In vivo mapping of tissue- and subcellular-specific proteomes inCaenorhabditis elegans

Cited by 13 publications

References 28 publications

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Neuromodulatory pathways in learning and memory: Lessons from invertebrates

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