Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: MS-based comparative enrichment analysis of VGluT3 immunoisolates’ proteome reveals age-dependent changes in the expression of synaptic and trafficking proteins . A , schematic representation of the immunoisolation approach to isolate IHC VGluT3-positive vesicular structures from immature (P8) and mature (P23) organs of Corti. S2 fraction was prepared as described in Figure 1 from ∼100 organs of Corti (per biological replicate) and used as starting material in VGluT3-and control IgG-specific immunoisolations. Two technical replicates (indicated as T1 and T2) from two independent immunoisolation procedures (biological replicates R1 and R2) were measured. B and C , approach used for the analysis of the MS data. B , protein enrichment was assessed by comparing VGluT3 immunoisolates with both control IgG immunoisolates and input S2 samples. The –log 10 adjusted p value was plotted against the log 2 iBAQ fold change of VGluT3 over control (IgG or input), with a significant t test FDR threshold of 5% and S 0 = 0. C , to visualize protein enrichment in VGluT3 immunoisolates as compared to both control IgG and input S2, log 2 iBAQ fold change VGluT3/Control IgG vs. log 2 iBAQ fold change VGluT3/Input was plotted; proteins in the upper right quadrant were enriched in VGluT3 immunoisolates – a threshold of log 2 iBAQ fold difference >0.7 was set to consider only proteins with at least 1.5-fold enrichment. D and E , Scatter plots showing differential enrichment of proteins in VGluT3 immunoisolates when compared to both control IgG and inputs at P8 ( D ) and P23 ( E ); displayed are IHC marker proteins VGluT3 and otoferlin, classical SV proteins, SNAREs, SNARE-binding proteins and other proteins. Numbers in parenthesis refer to the total number of significantly enriched proteins in VGluT3 immunoisolates over control IgG and Input (>1.5-fold enrichment). Gene names are displayed. Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community, but is still annotated in most databases as syntaxin-12). See also supplemental Fig. S3 for individual volcano plots. Source data are available for this figure ( supplemental Table S2 ). IHC, inner hair cell; MS, mass spectrometry; SV, synaptic vesicle.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Expressing, Protein Enrichment, Marker, Binding Assay, Mass Spectrometry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Comparative analysis of enriched proteins in P8 and P23 VGluT3 immunoisolates revealed a small overlap in protein identifications between ages and a different relative enrichment for shared proteins . A , Venn diagram showing the overlap between the significantly enriched proteins (>1.5-fold enrichment) at P8 and P23. B and C , ranking of significantly enriched proteins according to their relative enrichment in VGluT3 over Control IgG immunoisolates and input S2, before ( B ) and after ( C ) hearing onset. Relative Enrichment corresponds to the average of log 2 iBAQ (VGluT3/Input) and log 2 iBAQ (VGluT3/Control IgG). Displayed are only enriched proteins shown also in Figure 4 , D and E ( upper right quadrant and >1.5-fold enrichment). Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community, but is still annotated in most databases as syntaxin-12). Source data are available for this figure ( supplemental Table S3 ).

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Functional annotation of mature VGluT3-associated IHC proteome reveals a mixed SV-endosomal signature . A , sunburst diagram with functional annotation of the enriched proteins in VGluT3 immunoisolates at P23. Proteins were grouped for display according to their cellular component and their involvement in trafficking events. Annotations were done manually and based on information available in several databases; proteins were grouped according to cellular component and biological function. For detailed annotations see supplemental Table S4 . B–F , Scatter plots showing positively enriched proteins in VGluT3 immunoisolates at P23 when compared to both control IgG and input; log 2 iBAQ fold change VGluT3/Control IgG vs. log 2 iBAQ fold change VGluT3/Input was plotted. Displayed are proteins involved in trafficking events in different trafficking organelles (SV, endolysosomal, Golgi, and ER proteins) including SNAREs and resident proteins. Several Rab GTPase proteins, mostly of endolysosomal nature, were also enriched ( F ). Annotations were done manually and based on information available in several databases; proteins were grouped according to cellular compartment and biological function. For detailed annotation see supplemental Table S4 . In ( B – F ) gene names are displayed. Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community but is still annotated in most databases as syntaxin-12). For ranking of proteins according to their relative enrichment, see supplemental Fig. S5 . Source data are available for this figure ( supplemental Table S2 ). ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Functional Assay, Membrane

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Immunolocalization analysis of SNARE, SV, and kinase proteins in the adult organ of Corti . A–D , VAMP-7 ( A ), syntaxin-12/13 ( B ), syntaxin-8 ( C ), and syntaxin-7 ( D ), all highly enriched in our MS experiments, are expressed in IHCs. E–H , some classical neuronal SV proteins SCAMP1 ( E ), V-ATPase ( F ), and SV2B ( G ), and the kinase PKCα ( H ), all enriched in our MS experiments, are expressed in IHCs. Note that all proteins localize to the basolateral region of the IHCs, although in some cases the protein is also expressed in supporting cells and/or in afferent (postsynaptic) and efferent fibers of SGNs. Images correspond to high magnification views of representative P15–25 IHCs immunolabeled with antibodies against the candidate proteins ( red ), the ribbon marker CtBP2/RIBEYE ( green ), and the IHC marker otoferlin ( blue ). The upper panels show overviews of representative IHCs, displaying maximum intensity projections of 5 to 10 confocal optical sections through the longitudinal axis of the IHCs (scale bars: 5 μm). The bottom panels show a zoom into the synaptic area, displaying single confocal optical sections through the longitudinal axis of a single IHCs at the basal region (scale bars: 2 μm). IHC, inner hair cell; MS, mass spectrometry; SGN, spiral ganglion neuron.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Immunolabeling, Marker, Mass Spectrometry

Journal: Molecular Neurodegeneration

Article Title: Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase

doi: 10.1186/1750-1326-9-1

Figure Lengend Snippet: Localization and dynamic transport of BACE1 in recycling endosomes. (A) Colocalization of BACE1 with endogenous syntaxin 13 in dendrites and axons of cultured hippocampal neurons (DIV12). Insets show higher magnification of the boxed area. Images of the dendrites were acquired by confocal microscopy and those of the axon were generated by deconvolution of widefield z -stacks. Dendrites and axons were identified by the presence and absence of MAP2 staining, respectively. (B) Analysis of BACE1 colocalization with Rab11b in dendrites and axons. DIV12 neurons co-transfected with BACE1-YFP and mCherry-Rab11b were immunostained for MAP2. Insets show higher magnification of the area marked by a yellow arrowhead on a dendrite or the boxed area in an axon. Arrowheads indicate vesicles positive for BACE1 and Rab11b along the axon. Manders’ coefficient of colocalization (M Coeff) is indicated (n = 7 neurons). (C) Visualization of BACE1-YFP and mCherry-Rab11b dynamic co-transport by dual-color live cell imaging. Time-lapse images were acquired using the Dual-View two-channel simultaneous-imaging system at the rate of 1 frame/sec for 3 min (Additional file ). Kymograph analysis reveals bidirectional transport of BACE1 in Rab11b-positive endosomes (yellow tracks in the overlay panels) in both dendrites and axons.

Article Snippet: The coverslips were incubated for 1 h at room temperature with the primary antibodies diluted in PBS containing 3% BSA: MAP2 mAb (1:5,000; Sigma), EEA1 (1:200; Millipore), Syntaxin 13 (1:1000; Synaptic Systems); TfR mAb (C2F2, 1:250; Pharmingen), rat anti-LAMP1 (ID4B, 1:150; Developmental Studies Hybridoma Bank).

Techniques: Cell Culture, Confocal Microscopy, Generated, Staining, Transfection, Live Cell Imaging, Imaging