Journal: Nature Communications

Article Title: Surfaceome dynamics reveal proteostasis-independent reorganization of neuronal surface proteins during development and synaptic plasticity

doi: 10.1038/s41467-020-18494-6

Figure Lengend Snippet: a cLTP paradigm b The plot shows averaged fast EPSC amplitudes for each cell (triangles) and their averages (mean ± s.e.m) for control (untreated, n = 45) and cLTP (glycine treated, n = 38) cells. Mann–Whitney test. c – f Boxplots of distributions of log2 surface abundances for replicates of cLTP ( n = 12) and control ( n = 12). Boxes indicate median and percentiles (25th and 75th). g Volcano plot of statistical significance ( y -axis) and surface abundance change ( x -axis). Horizontal line is located at an adjusted p -value of 0.05. h Proteins with significantly different surface abundances (fold-change > 1.5 and p < 0.05) after cLTP grouped into categories. Shape size indicates scaled −log10 adjusted p -value. Border color indicates fold-change directionality (green > 0, purple < 0). Edges represent string confidence > 0.7. i Representative images of analysed primary dendrites before and after cLTP induction. Bars 5 µm. j Quantification of mean fluorescent intensity of antibody signal on the primary dendrite surface using antibodies against GluA: n = 63, Bai1: n = 42, Adcy3: n = 28 (CTRL) n = 22 (cLTP) cells/group. Means + 95% CI presented. Two-way T -test (Adcy3 + Bai1) or Mann–Whitney test (GluA). * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Antibodies targeting the extracellular domains of ADGRB1 (ABR-021, Alomone), AMPARs (182 411, Synaptic Systems), and AC3 (AAR-043, Alomone) were diluted 1:100 in blocking solution containing 10% normal goat serum (NGS) and incubated with the cells for 60 min.

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Determinants of affinity, specificity, and phase separation in a supramodule from Post-synaptic density protein 95

doi: 10.1016/j.isci.2022.105069

Figure Lengend Snippet: Phase transition of PSD-95 PSG:FL CC ADGRB1 complex (A) Schematic representation of a synapse highlighting the postsynaptic neuron were ADGRB1 receptors are located in the postsynaptic membrane. The C-terminal part of ADGRB1 (pink) is intracellular and located next to the PSD. The PSG supramodule of PSD-95 is highlighted to illustrate the possible interaction between PSG and full-length coiled-coil C-terminal part of ADGRB1. (B) Fluorescence polarization-based measurement of the PSG:ADGRB1 interaction. Data were obtained by titration of FITC-14 AA ADGRB1 with PSG or PSG G322A G335A , respectively. The experiments were performed at room temperature in technical triplicates and biological duplicates. (C) Analysis of FL CC ADGRB1 by circular dichroism between 200 and 260 nm suggests it is a disordered protein. The spectrum is an average of five scans measured at 25°C in 100-mM NaCl, 50-mM Tris pH 7.8, 1-mM TCEP. (D and E) ITC experiments of the interaction between PSG and FL CC ADGRB1 (D) and PSG G322A G335A and FL CC ADGRB1 (E). (F) LLPS formation measured as turbidity over time by monitoring absorbance at 350 nm at 25°C. PSG G322A G335A at a constant concentration of 15 μM was mixed with different concentrations of FL CC ADGRB1. LLPS formation correlates with the increasing FL CC ADGRB1 concentration. (G) SDS-PAGE of DSG-mediated cross-linking shows that 14 AA ADGRB1 cannot induce dimerization of PSG G322A G335A . 15 AA SynGap is a positive control and 15 AA CRIPT and water are negative controls. (H) LLPS of PSG and FL CC ADGRB1. Isolated PSG and FL CC ADGRB1 are soluble and homogeneous at 200 μM, as judged by light microscopy at room temperature. Mixing of PSG and FL CC ADGRB1 in a 1:3 ratio resulted in the formation of small liquid droplets that merged over time and formed larger droplets. The large liquid droplets formed by PSG and FL CC ADGRB1 immediately started to disappear upon the addition of 15 AA CRIPT. (I) SDS-PAGE sedimentation assay where PSD-95, Homer, Shank, and GKAP (each 20 μM) were incubated with increasing concentration of FL CC ADGRB1. Higher concentration of ADGRB1 clearly correlates with higher inclusion of PSD-95 in the pellets representing LLPS, whereas a correlation of lower significance was observed with Homer, Shank, and GKAP. Data were quantified by Lab software from BioRad. The significance (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001) was evaluated by two-way ANOVA with Tukey test using Prism 9.0 software (GraphPad).

Article Snippet: Interestingly, ADGRB1, similarly to SynGap, has a high content of coiled coil (CC) and a type I PBM in its C-terminus, and interaction with PSG resulted in LLPS.

Techniques: Sublimation, Membrane, Fluorescence, Titration, Circular Dichroism, Concentration Assay, SDS Page, Positive Control, Isolation, Light Microscopy, Sedimentation, Incubation, Software

Journal: iScience

Article Title: Determinants of affinity, specificity, and phase separation in a supramodule from Post-synaptic density protein 95

doi: 10.1016/j.isci.2022.105069

Figure Lengend Snippet: PSG and ADGRB1 form liquid-liquid phase separation in living cells Representative fluorescence microscopy images showing co-localization of PSG and ADGRB1 proteins in HeLa cells. DAPI-stained nucleus, mCherry-stained red cells, and EGFP-stained green cells along with a merge of the images are shown. (A–C) Expression of EGFP-FL CC Syngap, mCherry-PSG and EGFP-FL CC ADGRB1, respectively. (D and E) Co-localization of (D) EGFP-FL CC Syngap and (E) EGFP-FL CC ADGRB1 with mCherry-PSG. (F) Co-localization of EGFP-FL CC ADGRB1 with mCherry-PDZ1-2.

Article Snippet: Interestingly, ADGRB1, similarly to SynGap, has a high content of coiled coil (CC) and a type I PBM in its C-terminus, and interaction with PSG resulted in LLPS.

Techniques: Fluorescence, Microscopy, Staining, Expressing

Journal: iScience

Article Title: Determinants of affinity, specificity, and phase separation in a supramodule from Post-synaptic density protein 95

doi: 10.1016/j.isci.2022.105069

Figure Lengend Snippet:

Article Snippet: Interestingly, ADGRB1, similarly to SynGap, has a high content of coiled coil (CC) and a type I PBM in its C-terminus, and interaction with PSG resulted in LLPS.

Techniques: Virus, Recombinant, Software, Spectrophotometry, Microscopy, Mass Spectrometry, Inverted Microscopy