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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 <t>or</t> <t>myr-AIP</t> for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.
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( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 or myr-AIP for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.

Journal: bioRxiv

Article Title: Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

doi: 10.64898/2026.02.27.708433

Figure Lengend Snippet: ( A ) Schematic of XL-MS workflow. ( B ) XL-based interactome of synaptic ribosomes. Synaptic annotation is based on SynGO (dataset version 20231201). ( C ) Protein domain-level crosslink map between RPL35 and CaMKIIα. Domain classification based on . ( D ) Structural model of CaMKIIα binding to the ribosome. Most CaMKIIα–RPL35 crosslinks satisfy distance constraints (green), with remaining links accommodated by CaMKIIα linker flexibility (yellow). ( E ) Detection of newly-synthesized proteins in dendrites and synapses from control (untreated) neurons or neurons treated with 10 μM KN-93 or myr-AIP for 30 min. Nascent proteins were labeled with puromycin (5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (see Methods). Scale bar, 5 µm. ( F ) Quantification of puromycin signal in postsynaptic regions of the apical dendritic arbor from control and treated neurons. Error bars show mean ± SEM. **p<0.01; ****p<0.0001, Brown-Forsythe and Welch’s ANOVA, Dunnett’s multiple comparisons test; n=58-64 neurons from 3 independent cultures.

Article Snippet: DIV 18-19 hippocampal neurons were treated with either 10 μM KN-93 (Tocris, #5215) or 10 μM myr-AIP (Tocris, #5959) for 30 min at 37°C, or left untreated.

Techniques: Structural Proteomics, Binding Assay, Synthesized, Control, Labeling