Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts

Loh, Ken H.; Stawski, Philipp; Draycott, Austin S.; Udeshi, Namrata D.; Lehrman, Emily K.; Wilton, Daniel K.; Svinkina, Tanya; Deerinck, Thomas J.; Ellisman, Mark H.; Stevens, Beth; Carr, Steven A.; Ting, Alice Y.

doi:10.1016/j.cell.2016.07.041

Cited by 342 publications

(412 citation statements)

References 41 publications

(49 reference statements)

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“…Both the Celsr3 KO and cKO showed no change in the number of inhibitory synapses, and the Celsr3 cKO displayed no change in the activity of inhibitory synapses. The remaining excitatory synapses after the loss of Celsr3 may be formed by other Celsr isoforms, such as Celsr2 expressed in an overlapping pattern in the hippocampus (31,32), or a separate signaling pathway. These synaptic defects manifested in hippocampus-dependent behavioral deficits in adult Celsr3 cKOs.…”

Section: Discussion Opposing Roles Of Celsr3 and Vangl2 In Glutamatermentioning

confidence: 99%

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation

Thakar

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The signaling mechanisms that choreograph the assembly of the highly asymmetric pre-and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.Celsr3 | Vangl2 | glutamatergic | synapse formation G lutamatergic synapses, the predominant excitatory synapses in the brain, are asymmetric cell-cell junctions formed from distinct pre-and postsynaptic components involving highly organized complexes of hundreds of proteins across the 20-nm synaptic cleft (1, 2). The signaling pathway that directly assembles these asymmetric protein complexes has not been well understood. Understanding mechanisms of glutamatergic synapse formation will provide important insights into the function and plasticity as well as dysfunction of glutamatergic synapses, which underlie numerous nervous system disorders.Many epithelial tissues show planar cell polarity, the global asymmetry of cellular and tissue morphology and/or structure along the tissue plane (3, 4). The conserved core planar cell polarity (PCP) components, Frizzled, Dishevelled, Diego, Prickle, Vang(l), and Flamingo (Fmi)/Celsr, form asymmetric complexes at the cadherin-mediated adherens junctions that connect neighboring epithelial cells (3, 4). Recent studies suggest that mutations of some components of the PCP signaling pathway, Celsr3/Fmi and Vangl2, affect GABAergic circuit development in zebrafish retina, GABAergic motoneuron synapse development in Caenorhabditis elegans, and hippocampal/cortical glutamatergic and GABAergic synapse formation (5-11). PCP components are critical regulators of neuronal migration and axon guidance, which take p...

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Section: Discussion Opposing Roles Of Celsr3 and Vangl2 In Glutamatermentioning

confidence: 99%

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation

Thakar

Wang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…MDGA1 binds specifically to NLGN2 with nanomolar affinity, but not to NLGN1 or NLGN3, forming a side-by-side ( in-cis ) complex involving the extracellular domains; by contrast, MDGA2 prefers to interact with NLGN1 (Connor et al, 2016; Lee et al, 2013; Pettem et al, 2013). The interaction of MDGA1 with NLGN2 blocks the ability of NLGN2 to form a trans-synaptic bridge with neurexins, thus downregulating the ability of NLGN2 to promote inhibitory synapse development; on the other hand, there is consensus that MDGA2 downregulates excitatory synapse formation (Connor et al, 2016; Lee et al, 2013; Loh et al, 2016; Pettem et al, 2013). …”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of MDGA1: Regulation of Neuroligin 2:Neurexin Trans-synaptic Bridges

Gangwar

Zhong

Seshadrinathan

et al. 2017

Neuron

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Summary Neuroligins and neurexins promote synapse development and validation by forming trans-synaptic bridges spanning the synaptic cleft. Select pairs promote excitatory and inhibitory synapses, respectively, with neuroligin 2 (NLGN2) limited to inhibitory synapses and neuroligin 1 (NLGN1) dominating at excitatory synapses. The cell surface molecules, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1) and 2 (MDGA2), regulate trans-synaptic adhesion between neurexins and neuroligins, impacting NLGN2 and NLGN1, respectively. We have determined the molecular mechanism of MDGA action. MDGA1 Ig1–Ig2 is sufficient to bind NLGN2 with nanomolar affinity; its crystal structure reveals an unusual locked rod-shaped array. In the crystal structure of the complex, two MDGA1 Ig1–Ig2 molecules each span the entire NLGN2 dimer. Site-directed mutagenesis confirms the observed interaction interface. Strikingly, Ig1 from MDGA1 binds to the same region on NLGN2 as neurexins do. Thus, MDGAs regulate the formation of neuroligin-neurexin trans-synaptic bridges by sterically blocking access of neurexins to neuroligins.

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“…12) and wild-type ascorbate peroxidase (APX) because of its monomericity, superior catalytic activity, resistance to H 2 O 2 -induced inactivation, and efficiency of heme incorporation within cells 15 . For applications in the eukaryotic secretory pathway and on the cell surface, we recommend testing both HRP 52,53 and APEX2. HRP (Addgene, cat.…”

Section: Introductionmentioning

confidence: 99%

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

et al. 2017

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Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

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Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts

Cited by 342 publications

References 41 publications

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation

Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation

Molecular Mechanism of MDGA1: Regulation of Neuroligin 2:Neurexin Trans-synaptic Bridges

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

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