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Quantitative Multiplex Immunoprecipitation (QMI) analysis of protein interaction network rearrangements during homeostatic plasticity

Citation

Heavner, Whitney; Smith, Stephen (2021), Quantitative Multiplex Immunoprecipitation (QMI) analysis of protein interaction network rearrangements during homeostatic plasticity, Dryad, Dataset, https://doi.org/10.5061/dryad.pg4f4qrp5

Abstract

Neurons maintain constant levels of excitability using homeostatic synaptic scaling, which adjusts the strength of all of a neuron’s postsynaptic inputs to compensate for large changes in overall activity. Here, we asked how prolonged changes in activity affect network-level protein interactions at the synapse. We assessed a glutamatergic synapse protein interaction network (PIN) composed of 380 binary associations among 21 protein members to identify protein complexes altered by activity manipulation in vitro or sensory deprivation in vivo. In cultured cortical neurons, we observed widespread bidirectional PIN alterations during up- and downscaling that reflected rapid rearrangements of glutamate receptor co-associations via synaptic scaffold remodeling. Sensory deprivation of the barrel cortex caused unique PIN rearrangements, including changes in co-associations between the glutamate receptor mGluR5 and the kinase Fyn, consistent with emerging models of experience dependent plasticity involving multiple types of homeostatic responses. Mice lacking Homer1 or Shank3B did not undergo normal PIN rearrangements, suggesting that these Autism Spectrum Disorder (ASD)-linked proteins serve as structural hubs for synaptic homeostasis. This dataset contains the raw XML and PBX (bioplex protocol) files necessary for performing adaptive non-parametric analysis with an emprical alpha cutoff (ANC) to identify high-confidence IP_Probe pairs, here called "PiSCES" for proteins in shared complexes detected by surface epitopes, that are signifianctly differently associated between two conditions.

Methods

Quantitative multiplex immunoprecipitation: QMI was performed as described previously using previously validated antibodies (PMCID: PMC6150823). Briefly, a master mix containing equal numbers of each antibody-coupled Luminex bead was prepared and distributed to lysates containing equal amounts of protein and incubated overnight on a rotator at 4°C. The next day, each sample was washed twice in cold Fly-P buffer (50mM tris pH7.4, 100mM NaCl, 1% bovine serum albumin, and 0.02% sodium azide) and distributed into twice as many wells of a 96-well plate as there were probe antibodies (for technical duplicates). Biotinylated detection (probe) antibodies were added to the appropriate wells and incubated at 4°C with gentle agitation for 1 hour. The resulting bead-probe complexes were washed 3 times with Fly-P buffer, incubated for 30 minutes with streptavidin-PE on ice, washed another 3 times, resuspended in 125ml ice cold Fly-P buffer, and processed for fluorescence using a customized refrigerated Bio-Plex 200. Antibody clone names, catalog numbers, and lot numbers are the same as in (PMCID: PMC6150823).

Usage Notes

The XML files serve as input for ANC. The PBX files, which are readable by the Bio-Plex Manager Software, contain information pertaining to the layout of each 96-well plate used in the study, including the order of the samples, the IP bead regions, and the order of the probes on each plate. This information is necessary metadata for performing ANC.  


For experiments with 2 plates (_P1 and _P2), the order of the probes on plate P1 are Fyn, PSD95, NMDAR1, NMDAR2A, NMDAR2B, mGluR5, Shank3, Homer1, Homer1a, and PI3K; the order of the probes on plate P2 are GluA1, GluA2, Syngap, Sap97, NL3, Ube3a, CaMKIIa, SAPAP, panShank, Shank1, and PIKE. The exception is the set of HomeostaticScaling_48hr_ samples #5-8, which have Fyb at the end of plate P1.


For experiments with 3 plates (_P1, _P2, and _P3), the order of the probes on plate P1 are Fyn, PSD95, NMDAR1, NMDAR2A, NMDAR2B, mGluR5, Shank3; the order of the probes on plate P2 are Homer1, Homer1a, and PI3K, GluA1, GluA2, Syngap, Sap97; the order of the probes on plate P3 are NL3, Ube3a, CaMKIIa, SAPAP, panShank, Shank1, and PIKE.

Funding

National Institute of Mental Health, Award: MH102244

National Institute of Mental Health, Award: MH113545

National Institute of Mental Health, Award: NS31224