Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators

Li, Jiefu; Han, Shuo; Li, Hongjie; Udeshi, Namrata D.; Svinkina, Tanya; Mani, D. R.; Xu, Chuanyun; Guajardo, Ricardo; Xie, Qijing; Li, Tongchao; Luginbuhl, David J.; Wu, Bing; McLaughlin, Colleen N.; Xie, Anthony; Kaewsapsak, Pornchai; Quake, Stephen R.; Carr, Steven A.; Ting, Alice Y.; Luo, Liqun

doi:10.1016/j.cell.2019.12.029

Cited by 128 publications

(154 citation statements)

References 103 publications

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“…Dissecting the molecular composition of specific connections remains a major challenge. Sorting of growth cones 69 and specific synapse types (Pfeffer et al 70 , this study), imaging-based approaches [71][72][73] , affinity purification 74 , and proximity biotinylation [75][76][77] each have their advantages and disadvantages 78 . Here, we combined biochemical enrichment, antibody labeling of a synapse type-enriched surface marker, and fluorescent sorting to isolate MF synapses.…”

Section: Discussionmentioning

confidence: 99%

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

et al. 2020

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Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.

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Section: Discussionmentioning

confidence: 99%

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

et al. 2020

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“…All six samples were then digested with 0.1 μg Lys-C for 2 hours, followed by a reduction of the urea concentration to <2 M and continued digestion with 0.5 μg trypsin overnight. Reactions were quenched with formic acid at a final concentration of 5% and then desalted by reverse phase C18 stage tips as described previously 87 and dried down. Peptides were then resuspended in 50 μL of 50 mM HEPES buffer and isobarically labeled using 400 μg of Tandem Mass Tag 6-plex (TMT6) isobaric labeling reagent (Thermo Fisher Scientific).…”

Section: Rap-ms Protein Digestion and Tmt Labelingmentioning

confidence: 99%

“…The labeling reactions were then quenched with 4 μL of 5% hydroxylamine, samples were mixed together, and dried. The sample was fractionated by SCX stage tip strategy using three pH cuts at 5.15, 8.25, and 10.3 as described previously 87 .…”

Section: Rap-ms Protein Digestion and Tmt Labelingmentioning

confidence: 99%

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

Schmidt

Lareau

Keshishian

et al. 2020

Preprint

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SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.

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“…APEX, a modified ascorbate peroxidase derived from soybean, is a simple to use highly versatile marker for EM detection. The wide applicability of its use is demonstrated by studies utilizing APEX and its derivative, APEX2, for detection of fusion proteins in cells, Drosophila, zebrafish, and mice (Ariotti et al, 2015;Hirabayashi et al, 2018;Li et al, 2020;Meiring et al, 2019). APEX has also been combined with nanobody-based detection systems for rapid localization of GFP-and mCherry-tagged proteins (Ariotti et al, 2015;Ariotti et al, 2018), and used to detect protein interactions using split GFP and nanobodies or using a novel split-APEX system (Ariotti et al, 2018;Han et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

Rae

Ferguson

Ariotti

et al. 2020

Preprint

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Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

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Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators

Cited by 128 publications

References 103 publications

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

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