In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain

Uçkun, Ezgi; Wolfstetter, Georg; Anthonydhason, Vimala; Sukumar, Sanjay Kumar; Umapathy, Ganesh; Molander, Linnea; Fuchs, Johannes; Palmer, Ruth

doi:10.1016/j.jmb.2021.167282

Cited by 23 publications

(15 citation statements)

References 64 publications

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“…Proteomics based on proximity-labeling strategies using biotin ligases (e.g. BioID, TurboID-NES) are being increasingly used in in vitro and in vivo experimental contexts and in model systems ranging from plants 17,[51][52][53] , yeast 54 , zebrafish 55,56 , Drosophila 57,58 , C. elegans 16,59 , to mouse models 18,21,22,60 . TurboID is one of the most efficient biotin ligases that can effectively label several proteins within a 10nm labeling radius in mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines

Sunna

Bowen

Zeng

et al. 2022

Preprint

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Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain are incompatible with the survival of adult murine neurons and contamination from non-target cell-types and experimental artifacts confound the data. To overcome these barriers, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes. LPS treatment induced >500 differentially expressed proteins in transduced BV2 proteomes, including an increase in canonical M1 proteins (IL1a, Irg1, Oasl1) and a decrease in canonical M2 proteins (Arg1, MGl2).

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Section: Discussionmentioning

confidence: 99%

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines

Sunna

Bowen

Zeng

et al. 2022

Preprint

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“…Furthermore, by combining the fly in vivo platform with biochemistry applications, such as TurboID and APEX, one could identify the components that play key roles during SD assembly. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, which covalently labels proximal proteins ( Uçkun et al, 2021 ; Zhang et al, 2021 ), while APEX is an engineered peroxidase that can convert exogenous biotin for unselective covalent labelling of proximal proteins ( Lobingier et al, 2017 ). Either biotinylated complex can then be enriched using Streptavidin beads for subsequent analysis by mass spectrometry to achieve live-cell proteomics.…”

Section: Challenges In Slit Diaphragm Research and Advantages Of Usin...mentioning

confidence: 99%

Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm

Leemput

Wen

Han

2022

Front. Cell Dev. Biol.

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The podocyte slit diaphragm (SD) is an essential component of the glomerular filtration barrier and its disruption is a common cause of proteinuria and many types of kidney disease. Therefore, better understanding of the pathways and proteins that play key roles in SD formation and maintenance has been of great interest. Podocyte and SD biology have been mainly studied using mouse and other vertebrate models. However, vertebrates are limited by inherent properties and technically challenging in vivo access to the podocytes. Drosophila is a relatively new alternative model system but it has already made great strides. Past the initial obvious differences, mammalian podocytes and fly nephrocytes are remarkably similar at the genetic, molecular and functional levels. This review discusses SD formation and maintenance, and their dependence on cell polarity, the cytoskeleton, and endo- and exocytosis, as learned from studies in fly nephrocytes and mammalian podocytes. In addition, it reflects on the remaining gaps in our knowledge, the physiological implications for glomerular diseases and how we can leverage the advantages Drosophila has to offer to further our understanding.

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“…Many studies simply enrich over endogenous biotinylation and bead background. [59][60][61][62] Other studies expressed forms of TurboID alone that were localized to the specific cellular compartment that the protein of interest resided, such as the cellular membrane. 48,49,51,52 As a non-TurboID-expressing wild-type control with a similar genetic background, we used a fly strain in which endogenous kdm5 is removed and HA-tagged KDM5 is expressed using its endogenous promoter from a transgene inserted at the same locus as the TurboID constructs (kdm5 140 ;gkdm5 WT ).…”

Section: Determining the Proper Controls To Identify The Kdm5 Proximi...mentioning

confidence: 99%

Proximity labeling reveals a newin vivonetwork of interactors for the histone demethylase KDM5

Yheskel

Secombe

2022

Preprint

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KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins. Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads, and generated a novel control for DNA-adjacent background in the form of dCas9:TurboID. We identified both known and novel candidate proteins, including members of the SWI/SNF and NURF chromatin remodeling complexes, the non-specific lethal (NSL) complex, Mediator, and several insulator proteins. Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

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In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain

Cited by 23 publications

References 64 publications

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines

Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm

Proximity labeling reveals a newin vivonetwork of interactors for the histone demethylase KDM5

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