@article {69534, title = {Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching.}, journal = {Cell}, year = {2024}, month = {2024 Jul 03}, abstract = {

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins{\textquoteright} functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.06.022}, author = {Xu, Chuanyun and Li, Zhuoran and Lyu, Cheng and Hu, Yixin and McLaughlin, Colleen N and Wong, Kenneth Kin Lam and Xie, Qijing and Luginbuhl, David J and Li, Hongjie and Udeshi, Namrata D and Svinkina, Tanya and Mani, D R and Han, Shuo and Li, Tongchao and Li, Yang and Guajardo, Ricardo and Ting, Alice Y and Carr, Steven A and Li, Jiefu and Luo, Liqun} } @article {68418, title = {In situ cell-type-specific cell-surface proteomic profiling in mice.}, journal = {Neuron}, year = {2022}, month = {10/2022}, abstract = {

Cell-surface proteins (CSPs) mediate intercellular communication throughout the lives of multicellular organisms. However, there are no generalizable methods for quantitative CSP profiling in specific cell types in vertebrate tissues. Here, we present in situ cell-surface proteome extraction by extracellular labeling (iPEEL), a proximity labeling method in mice that enables spatiotemporally precise labeling of cell-surface proteomes in a cell-type-specific environment in native tissues for discovery proteomics. Applying iPEEL to developing and mature cerebellar Purkinje cells revealed differential enrichment in CSPs with post-translational protein processing and synaptic functions in the developing and mature cell-surface proteomes, respectively. A proteome-instructed in\ vivo loss-of-function screen identified a critical, multifaceted role for Armh4 in Purkinje cell dendrite morphogenesis. Armh4 overexpression also disrupts dendrite morphogenesis; this effect requires its conserved cytoplasmic domain and is augmented by disrupting its endocytosis. Our results highlight the utility of CSP profiling in native mammalian tissues for identifying regulators of cell-surface signaling.

}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.09.025}, author = {Shuster, S Andrew and Li, Jiefu and Chon, URee and Sinantha-Hu, Miley C and Luginbuhl, David J and Udeshi, Namrata D and Carey, Dominique Kiki and Takeo, Yukari H and Xie, Qijing and Xu, Chuanyun and Mani, D R and Han, Shuo and Ting, Alice Y and Carr, Steven A and Luo, Liqun} } @article {68419, title = {Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code.}, journal = {Neuron}, volume = {110}, year = {2022}, month = {07/2022}, pages = {2299-2314.e8}, abstract = {

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains, and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combined expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.

}, keywords = {Animals, Dendrites, Drosophila, Drosophila Proteins, Ion Channels, Membrane Proteins, Nerve Tissue Proteins, Olfactory Pathways, Olfactory Receptor Neurons, POU Domain Factors, Proteomics, Transcription Factors}, issn = {1097-4199}, doi = {10.1016/j.neuron.2022.04.026}, author = {Xie, Qijing and Li, Jiefu and Li, Hongjie and Udeshi, Namrata D and Svinkina, Tanya and Orlin, Daniel and Kohani, Sayeh and Guajardo, Ricardo and Mani, D R and Xu, Chuanyun and Li, Tongchao and Han, Shuo and Wei, Wei and Shuster, S Andrew and Luginbuhl, David J and Quake, Stephen R and Murthy, Swetha E and Ting, Alice Y and Carr, Steven A and Luo, Liqun} } @article {68429, title = {Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.}, journal = {Cell}, volume = {180}, year = {2020}, month = {01/2020}, pages = {373-386.e15}, abstract = {

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in\ vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

}, keywords = {Animals, Axons, Brain, Dendrites, Drosophila melanogaster, Drosophila Proteins, Gene Expression Profiling, Gene Expression Regulation, Developmental, Membrane Proteins, Neurogenesis, Olfactory Nerve, Olfactory Pathways, Olfactory Receptor Neurons, Proteomics, Receptors, Lipoprotein, Smell}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.12.029}, author = {Li, Jiefu and Han, Shuo and Li, Hongjie and Udeshi, Namrata D and Svinkina, Tanya and Mani, D R and Xu, Chuanyun and Guajardo, Ricardo and Xie, Qijing and Li, Tongchao and Luginbuhl, David J and Wu, Bing and McLaughlin, Colleen N and Xie, Anthony and Kaewsapsak, Pornchai and Quake, Stephen R and Carr, Steven A and Ting, Alice Y and Luo, Liqun} } @article {68434, title = {Proximity labeling: spatially resolved proteomic mapping for neurobiology.}, journal = {Curr Opin Neurobiol}, volume = {50}, year = {2018}, month = {06/2018}, pages = {17-23}, abstract = {

Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.

}, keywords = {Animals, Biotinylation, Humans, Neurobiology, Neuroimaging, Proteome, Proteomics}, issn = {1873-6882}, doi = {10.1016/j.conb.2017.10.015}, author = {Han, Shuo and Li, Jiefu and Ting, Alice Y} } @article {65893, title = {Antibodies to biotin enable large-scale detection of biotinylation sites on proteins}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {Jan-12-2017}, pages = {1167 - 1170}, abstract = {

Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.

}, issn = {1548-7091}, doi = {10.1038/nmeth.4465}, url = {http://www.nature.com/articles/nmeth.4465http://www.nature.com/doifinder/10.1038/nmeth.4465http://www.nature.com/articles/nmeth.4465.pdfhttp://www.nature.com/articles/nmeth.4465.pdfhttp://www.nature.com/articles/nmeth.4465}, author = {Udeshi, Namrata D and Pedram, Kayvon and Svinkina, Tanya and Fereshetian, Shaunt and Myers, Samuel A and Aygun, Ozan and Krug, Karsten and Clauser, Karl and Ryan, Dominic and Ast, Tslil and Mootha, Vamsi K and Ting, Alice Y and Carr, Steven A} } @article {65891, title = {Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2}, journal = {Nature Protocols}, volume = {11}, year = {2016}, month = {Jan-03-2016}, pages = {456 - 475}, abstract = {

This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a {\textquoteright}ratiometric{\textquoteright} three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5{\textendash}7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2{\textendash}5 d and analysis of the data to obtain the final proteomic list takes 1 week.

}, issn = {1754-2189}, doi = {10.1038/nprot.2016.018}, url = {http://www.nature.com/articles/nprot.2016.018http://www.nature.com/articles/nprot.2016.018.pdfhttp://www.nature.com/articles/nprot.2016.018.pdfhttp://www.nature.com/articles/nprot.2016.018}, author = {Hung, Victoria and Udeshi, Namrata D and Lam, Stephanie S and Loh, Ken H and Cox, Kurt J and Pedram, Kayvon and Carr, Steven A and Ting, Alice Y} } @article {46410, title = {The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release.}, journal = {Journal of Cell Science}, volume = {123}, year = {2010}, month = {2010 Jul 1}, pages = {2308-18}, abstract = {

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

}, keywords = {Amino Acid Sequence, Amphiregulin, Animals, Cell Communication, Cell Proliferation, Cercopithecus aethiops, COS Cells, EGF Family of Proteins, Glycoproteins, Heparan Sulfate Proteoglycans, Heparin, Heparin-binding EGF-like Growth Factor, Intercellular Junctions, Intercellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Mutation, Peptides, Protein Precursors, Protein Structure, Tertiary}, issn = {1477-9137}, doi = {10.1242/jcs.058321}, author = {Prince, Robin N and Schreiter, Eric R and Zou, Peng and Wiley, H Steven and Ting, Alice Y and Lee, Richard T and Lauffenburger, Douglas A} }