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:10.1126/sciadv.1602426

Cited by 60 publications

(63 citation statements)

References 37 publications

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“…Faster acting peroxidase-based labeling (e.g. APEX) also present limitations for intact organisms because of H2O2 toxicity and limited accessibility of the necessary biotin phenol substrate (Reinke et al, 2017). We provided the first demonstration of biotin ligase-based PL in C. elegans using the newly-engineered PL enzyme mutant TurboID which presents faster labeling kinetics and retains catalytic activity at lower temperatures than all other existing biotin ligases (Branon et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Sanchez

Branon

Cote

et al. 2020

Preprint

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SummaryReorganization of microtubules from the centrosome to non-centrosomal subcellular sites is central to cell differentiation. To identify components of non-centrosomal microtubule organizing centers in differentiated cells of a living organism, we developed the biotin ligase-based proximity labeling approach TurboID for use in C. elegans. We identified proteins proximal to the non-centrosomal microtubule minus end protein PTRN-1/Patronin at the apical membrane of epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. This regulation occurs downstream of cell polarity and in conjunction with actin. Our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes and uncover novel functions of spectraplakins and WDR62 family proteins.

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Section: Discussionmentioning

confidence: 99%

Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Sanchez

Branon

Cote

et al. 2020

Preprint

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“…Three studies reported APEX-mediated labeling in live tissue. Two of these studies were performed in C. elegans in a mutant background that compromised cuticle integrity, which allowed for successful delivery of the biotin-phenol substrate into the worm tissue (Reinke, Balla, et al, 2017;Reinke, Mak, et al, 2017). The third labeled the mitochondrial proteome in dissected Drosophila imaginal discs, salivary glands, and larval muscles (Chen, Hu, et al, 2015).…”

Section: Apex-mediated Proximity Labeling Can Be Used To Identify Promentioning

confidence: 99%

Proximity labeling reveals novel interactomes in live Drosophila tissue

Mannix

Starble

Kaufman

et al. 2019

Preprint

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Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10µm. While multiple proteins have been identified as components of ring canals (RCs), we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. We optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology. Summary StatementHere, we optimized a procedure for enzyme-catalyzed proximity labeling in live Drosophila ovary tissue to interrogate the Drosophila ring canal protein interactome.

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“…, 2017 ; Reinke et al. , 2017a , b ). Here we utilized both BioID and APEX techniques in addition to standard affinity-capture approaches toward the identification of interacting proteins for cytoplasmic localized ubiquitin pathway components.…”

Section: Discussionmentioning

confidence: 99%

Mapping the mammalian ribosome quality control complex interactome using proximity labeling approaches

Zuzow

Ghosh

Leonard

et al. 2018

MBoC

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Previous genetic and biochemical studies from Saccharomyces cerevisiae have identified a critical ribosome-associated quality control complex (RQC) that facilitates resolution of stalled ribosomal complexes. While components of the mammalian RQC have been examined in vitro, a systematic characterization of RQC protein interactions in mammalian cells has yet to be described. Here we utilize both proximity-labeling proteomic approaches, BioID and APEX, and traditional affinity-based strategies to both identify interacting proteins of mammalian RQC members and putative substrates for the RQC resident E3 ligase, Ltn1. Surprisingly, validation studies revealed that a subset of substrates are ubiquitylated by Ltn1 in a regulatory manner that does not result in subsequent substrate degradation. We demonstrate that Ltn1 catalyzes the regulatory ubiquitylation of ribosomal protein S6 kinase 1 and 2 (RPS6KA1, RPS6KA3). Further, loss of Ltn1 function results in hyperactivation of RSK1/2 signaling without impacting RSK1/2 protein turnover. These results suggest that Ltn1-mediated RSK1/2 ubiquitylation is inhibitory and establishes a new role for Ltn1 in regulating mitogen-activated kinase signaling via regulatory RSK1/2 ubiquitylation. Taken together, our results suggest that mammalian RQC interactions are difficult to observe and may be more transient than the homologous complex in S. cerevisiae and that Ltn1 has RQC-independent functions.

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In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans

Abstract: New approach enables unbiased determination of tissue- and subcellular-specific protein location within a live animal.

Cited by 60 publications

References 37 publications

Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Proximity labeling reveals novel interactomes in live Drosophila tissue

Mapping the mammalian ribosome quality control complex interactome using proximity labeling approaches

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