Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: Heat map showing 22 putative interactors of the Munc18‐1/CASK/Mint1/Lin7b presynaptic complex identified in a proteomics screen using CASK, Mint1, or Lin7b as bait. Detection of the putative interactors using SALM1 or GluR2 (control) as bait is also shown. Bar values indicate average log10 LFQ intensity of three replicates. Gray indicates no detection in the IP. Partial interactome of the putative SALM1/Munc18‐1/CASK/Mint1/Lin7b complex identified by IP‐MS analysis in (A). Blue lines indicate interactions identified in the IP‐MS screen using CASK, Mint1, or Lin7b as bait. Red lines indicate interactions identified by reverse IP‐MS with SALM1. Black lines indicate previously established interactions with the Munc18‐1/CASK/Mint1/Lin7b complex (Butz et al , ; Tabuchi et al , ). Co‐immunoprecipitation of V5‐tagged cytoplasmic SALM1, SALM1ΔPDZ, or an empty vector with CASK in HEK cells. The co‐IP was repeated 3 times.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Control, Protein-Protein interactions, Immunoprecipitation, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: Sandwich‐cultured mouse hippocampal neurons stained at DIV16 for endogenous SALM1 (green), dendritic marker MAP2 (blue), and synapse markers Homer or VGluT1 (red), or the axonal marker SMI‐312 (red). Boxes indicate area of zoom. Arrows indicate overlap between SALM1 and synapse markers. Bars = 10 μm in full neuron images. Bars = 5 μm in zoomed images. Average overlap ± SEM of SALM1 clusters with VGluT1 or Homer puncta. Numbers in bars indicate number of zoomed images/total number of neurons in two independent experiments. Example images showing differential overlap of SALM1 with Homer or VGluT1. Bars = 0.5 μm. Electron micrographs showing subsynaptic localization of endogenous SALM1 in mouse hippocampal brain slices. SALM1 immunogold particles are detected in presynapses (orange and magenta arrows) and postsynapses (cyan and blue arrows). Bars = 100 nm. Mean percentage of gold particles ± SEM detected in pre‐ versus postsynapses of mouse hippocampal slices stained for SALM1. Percentages are based on detected gold particles in 32 synapses in hippocampal brain slices of three different animals (Mann–Whitney U ‐tests with Bonferroni correction, ns = not significant, * P < 0.025 after Bonferroni correction). Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Cell Culture, Staining, Marker, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A Collapsed z ‐stack image of a calcium phosphate transfected HEK cell showing surface expression (red) and intracellular expression (green) of SALM1‐pHl. B Single z ‐slice from the z ‐stack image given in (A). C, D Example images of calcium phosphate transfected HEK cells expressing GFP, SALM1‐pHl, Nrxn1β‐pHl, or HA‐Nlg1 (green) co‐cultured with DIV9‐10 sandwich‐cultured mouse hippocampal neurons. Dendrites were stained for MAP2 (blue), postsynapses for Homer (C), and presynapses for VGluT1 (D) (red). E, F Average number of postsynapses ± SEM (E) or presynapses ± SEM (F) per HEK cell. Numbers in bars indicate total number of cells/total number of independent cultures. Kruskal–Wallis tests with post hoc pairwise comparisons were used for (E) ( P < 0.001) and (F) ( P < 0.001). ns = not significant and *** P < 0.001. Data information: Scale bars (A–D) = 5 μm. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Transfection, Expressing, Cell Culture, Staining

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A–J (A and F) Schematic representation of SALM1‐depleted neurons forming postsynapses on Neurexin expressing HEK cells (A) or forming presynapses on Neuroligin expressing HEK cells (F). Example images showing postsynapses positive for postsynaptic marker Homer (red) formed on calcium phosphate transfected HEK cells expressing Nrxn1β‐pHl (B) or showing presynapses positive for presynaptic vesicle marker VGluT1 (red) formed on calcium phosphate transfected HEK cells expressing HA‐Nlg1 (G). HEK cells were co‐cultured for 24 h with sandwich‐cultured mouse hippocampal neurons infected with scrambled or SALM1 knockdown (shRNA#2) lentivirus (DIV3→10). Bars = 5 μm. Average number of Homer (C) or VGluT1 (H) particles ± SEM detected per HEK cell. Average size of the Homer (D) or VGluT1 (I) particles ± SEM detected per HEK cell. Average intensity ± SEM of Homer (E) and VGluT1 (J) puncta detected per HEK cell. For all graphs, the n is indicated in the bars and represents the total number of cells/total number of independent cultures. Kruskal–Wallis tests with post hoc paired comparisons were used for (C and H) ( P = 0.001), (D) ( P = 0.065), (E) ( P = 0.327), (I) ( P = 0.06), and (J) ( P = 0.003). ns = not significant, * P < 0.05 and ** P < 0.01. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Expressing, Marker, Transfection, Cell Culture, Infection, Knockdown, shRNA

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: Examples of expression patterns of Nrxn1β‐FLAG or HA‐Nlg1 at the surface of calcium phosphate transfected HEK cells. Example of surface expression patterns of SALM1‐pHl (red) and Nrxn1β‐FLAG (green) when co‐expressed in calcium phosphate transfected HEK cells. Example of surface expression patterns of SALM1‐pHl (red) and HA‐Nlg1 (green) when co‐expressed in calcium phosphate transfected HEK cells. Data information: Images represent collapsed z ‐stacks. Blue boxes indicate area of zoom; white arrows indicate examples of SALM1 puncta in close proximity of Nlg1 or Nrxn1β puncta. Bars = 5 μm in full HEK cell images. Bars = 2 μm in zoomed images.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Expressing, Transfection

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A–I (A, C) Single z ‐slice images through calcium phosphate transfected HEK cells co‐expressing SALM1‐pHl and Nrxn1β‐FLAG treated with DMSO (A) or Latrunculin A (C) and stained for surface GFP (blue), surface FLAG (green), and Phalloidin (red). White boxes indicate area of zoom; zoomed images are depicted below the full image for each channel. White lines in merged zoomed images represent cross sections used for intensity plots in (B, D). Bars = 5 μm in full HEK cell images. Bars = 3 μm in zoomed images. (B, D) Representative fluorescence intensity plots of cross sections depicted by white lines in the zoomed merge image in (A, C). SALM1 puncta are highlighted in yellow. (E) Partial amino acid sequence of the transmembrane and intracellular juxtamembrane domain of SALM1 and mutant SALM1 RAKA . The point mutations R556A and K558A are indicated by * and highlighted in red. Single z‐slice images through HEK cells co‐expressing calcium phosphate transfected SALM1‐pHl (F) or SALM1 RAKA (H) (blue, surface staining) with Nrxn1β‐FLAG (green, surface staining) and lentivirally expressed PLC‐PH‐mCherry (red). White boxes indicate area of zoom; zoomed images are depicted below the full image for each channel. White lines in merged zoomed images represent cross sections used for intensity plots in (B, D). Bars = 5 μm in full HEK cell images. Bars = 3 μm in zoomed images. (G, I) Fluorescence intensity plots of cross sections depicted by white lines in the zoomed merge image in (F, H). SALM1 puncta are highlighted in yellow. J Average number of surface Nrxn1β‐FLAG clusters ± SEM per HEK cell for HEK cells expressing the different indicated constructs. K Average Nrxn1β‐FLAG diffusion ratio (D‐ratio) ± SEM per HEK cell for HEK cells expressing the different indicated constructs. Data information: The n is indicated in the bars and represents the total number of cells/total number of independent cultures. S1 = SALM1‐pHl, Nrxn = Nrxn1β‐FLAG, and S1 RAKA = SALM1 RAKA ‐pHl. Kruskal–Wallis tests with post hoc paired comparisons were used on (J) ( P < 0.001) and (K) ( P < 0.001). *** P < 0.001. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Transfection, Expressing, Staining, Fluorescence, Sequencing, Mutagenesis, Construct, Diffusion-based Assay

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A Example images of neurites from DIV 10 sandwich‐cultured mouse excitatory hippocampal neurons lentivirally infected at DIV4 with Nrxn1β‐FLAG (green, surface staining) and scrambled, shRNA#2, shRNA#2+rSALM1, or shRNA#2+rSALM1 RAKA . Arrows indicate overlap between VGluT1 puncta and Nrxn1β‐FLAG clusters. Bars = 5μm B Average surface Nrxn1β‐FLAG intensity ± SEM detected in VGluT1 puncta per neuron. C Schematic representation of neurons expressing GFP (control), Nrxn1βFLAG (green puncta), or SALM1‐pHl with Nrxn1βFLAG forming presynapses (red puncta) on HA‐Nlg1 expressing HEK cells. D Example images showing presynapses positive for presynaptic marker VGLuT1 (red) formed on calcium phosphate transfected HEK cells expressing HA‐Nlg1 and stained for HEK cell filler GFP and surface Nrxn1β‐FLAG (green). HEK cells were co‐cultured for 24 h with sandwich‐cultured mouse hippocampal neurons infected with GFP (control), Nrxn1β‐FLAG, SALM1‐pHl, SALM1‐pHl+Nrxn1β‐FLAG, SALM1 RAKA ‐pHl+Nrxn1β‐FLAG, or SALM1ΔPDZ‐pHl+Nrxn1β‐FLAG lentivirus (DIV3→10). Bars = 5 μm in full HEK cell images. Bars = 1 μm in zoomed images. E–G Average number of VGluT1 puncta (E), puncta size (F), and puncta intensity (G) ± SEM detected per HEK cell. Data information: For all graphs, the n is indicated in the bars and represents the total number of cells/total number of independent cultures. Kruskal–Wallis tests with post hoc paired comparisons were used on data sets (B) ( P < 0.001), (E) ( P = 0.001), and (F) ( P = 0.775). A one‐way ANOVA test was used in (G) ( P = 0.772). ns = not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Cell Culture, Infection, Staining, shRNA, Expressing, Control, Marker, Transfection

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A Example images of neurites from DIV 11 sandwich‐cultured mouse excitatory hippocampal neurons lentivirally infected at DIV4 with Nrxn1β‐FLAG, SALM1‐pHl, or SALM1 RAKA ‐pHl. Intracellular and surface stainings are shown for each construct. Bars = 5 μm. B Example images of neurites from DIV 11 sandwich‐cultured mouse excitatory hippocampal neurons lentivirally co‐infected at DIV4 with Nrxn1β‐FLAG and SALM1‐pHl or Nrxn1β‐FLAG and SALM1 RAKA ‐pHl. Surface stainings are shown for each construct. Arrows indicate examples of overlap between SALM1‐pHl and Nrxn1β‐FLAG clusters. Bars = 5 μm. C–E Average number (C), size (D), and intensity (E) ± SEM of surface Nrxn1β‐FLAG clusters. F Average ratio ± SEM of surface over total staining intensity for SALM1‐pHl and SALM1 RAKA ‐pHl. G Example images of neurites from DIV 11 sandwich‐cultured mouse excitatory hippocampal neurons lentivirally co‐infected at DIV4 with Nrxn1β‐FLAG and SALM1‐pHl or Nrxn1β‐FLAG and SALM1 RAKA ‐pHl. Surface stainings are shown for SALM1‐pHl and SALM1 RAKA ‐pHl. Total staining is shown for Nrxn1β‐FLAG. Bars = 5 μm. H–J Average number (H), size (I), and intensity (J) ± SEM of total (intracellular and surface) Nrxn1β‐FLAG clusters. Data information: For all graphs, the n is indicated in the bars and represents the total number of cells/total number of independent cultures. A one‐way ANOVA test with post hoc Bonferroni test was used for (C) ( P < 0.001), (F) ( P < 0.001), and (H) ( P = 0.987). Kruskal–Wallis tests with post hoc paired comparisons were used for (D) ( P < 0.001), (E) ( P < 0.001), (I) ( P = 0.06), and (J) ( P = 0.192). ns = not significant, ** P < 0.01, and *** P < 0.001. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Cell Culture, Infection, Construct, Staining

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: A Example images of neurites from single isolated (autaptic) mouse excitatory hippocampal neurons stained for GFP and VGluT1. Cells were lentivirally infected with scrambled or SALM1 shRNA constructs at different time points (DIV7 or DIV9) and analyzed 7 days later (DIV14 or DIV16). At DIV7, cells were lentivirally infected with shRNA#2 + rSALM1, shRNA#2 + rSALM1 RAKA or with shRNA#2 + rSALM1ΔPDZ. Bars = 5 μm. B, C Average number of VGluT1 puncta per μm neurite ± SEM per neuron for DIV7→14 (B) and DIV9→16 (C). D, E Average intensity ± SEM of VGluT1 puncta per neuron for DIV7→14 (D) and DIV9→16 (E). F Electron micrographs of synapses from DIV14 autaptic hippocampal neurons lentivirally infected at DIV7 with scrambled, shRNA#1, or shRNA#2 (black, red and blue boxed images, respectively). Synaptic clefts are indicated by green arrows. Presynapses are localized above the synaptic cleft showing distinct vesicular structures; postsynapses are localized below the synaptic cleft. Bars = 100 nm. G Average number of synaptic vesicles per synapse ± SEM in electron micrographs. H Average size of the synaptic vesicle cluster per synapse ± SEM. I, J Average length of the presynaptic active zone (K) and the postsynaptic density (L) ± SEM per synapse. K Average number of membrane proximate vesicles ± SEM per synapse. Data information: For all graphs, numbers in bars indicate total number of neurons/total number of independent cultures. Kruskal–Wallis tests with post hoc paired comparisons were used for all data sets, P < 0.001 for (B, D, and G), P = 0.004 for (H), P = 0.349 for (C), P = 0.031 for (E), P = 0.879 for (I), P = 0.368 for (J), and P = 0.463 for (K). ns = not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Isolation, Staining, Infection, shRNA, Construct, Membrane

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: Patch‐clamp analysis (A–K) on single isolated mouse hippocampal neurons lentivirally infected with the indicated constructs at DIV7 and analyzed at DIV14‐15. A Example traces of EPSCs in control (black), shRNA#1 (red), shRNA#2 (blue), shRNA#2+rSALM1 (green), or shRNA#2+rSALM1 RAKA (orange) expressing cells. B Average evoked EPSC amplitudes ± SEM. Kruskal–Wallis ( P < 0.001). These experiments could not be performed with visual confirmation of lentiviral infection of rescue constructs, which typically is around 80%. Together with the high variability in evoked synaptic responses, this uncertain factor may explain that the average EPSC amplitudes are incompletely restored after SALM1 knockdown and that the RAKA mutant is not significantly different from the WT. C Example traces of mEPSCs from control (black), shRNA#1 (red), or shRNA#2 (blue) expressing cells. D, E Average mEPSC amplitudes ± SEM (D) or average frequencies ± SEM (E). Kruskal–Wallis ( P < 0.001 for E and P = 0.879 for D). F Average paired‐pulse ratios obtained using different intervals ± SEM. n = 20, n = 9, and n = 12 for scrambled, shRNA#1, and shRNA#2, respectively. G Average normalized EPSC response ± SEM upon 10 Hz train stimulation. n = 20, n = 9, and n = 13 for scrambled, shRNA#1, and shRNA#2, respectively. Tau ± SEM is indicated in the graph for each condition and was not significant (Kruskal–Wallis, P = 0.084). H Average normalized EPSC response ± SEM upon 40 Hz train stimulation. n = 20, n = 9, and n = 13 for scrambled, shRNA#1, and shRNA#2, respectively. Tau ± SEM is indicated in the graph for each condition and was not significant (Kruskal–Wallis, P = 0.897). I Cumulative EPSC responses to 40 Hz train stimulation. Green lines represent extrapolation used to determine RRP size. J Average RRP size ± SEM determined by back extrapolation of cumulative EPSCs in I (one‐way ANOVA with post hoc Games‐Howell, P = 0.007). K Average initial release probability (Pr) ± SEM per neuron calculated as the ratio of EPSC 0 /RRP size (Kruskal–Wallis, P = 0.347). M Average trace of the fluorescence intensity of SypHy in calcium phosphate transfected isolated hippocampal neurons expressing scrambled shRNA (black) or shRNA#2 (blue). The timing of the 300 action potentials at 10 Hz stimulus and the exposure to NH 4 Cl are indicated in the graph. N Average SypHy intensity trace normalized to the maximum intensity upon NH 4 Cl superfusion for each of the given conditions. N, O Average SypHy intensity ± SEM upon NH 4 Cl exposure (N) or upon stimulation with 300 action potentials (O) (Mann–Whitney U ‐test, P = 0.02 for M and P = 0.03 for N). P Normalized average SypHy intensity ± SEM upon 300 action potentials stimulation (Mann–Whitney U ‐test, P = 0.899). Q Average percentage of silent synapses ± SEM (Mann–Whitney U ‐test, P = 0.652). R Average trace of the fluorescence intensity of SypHy in lentivirally infected isolated hippocampal neurons expressing shRNA#2 (blue), shRNA#2+rSALM1 (green), shRNA#2+SALM1 RAKA (orange), or shRNA#2+SALM1ΔPDZ (gray). The timing of the 300 action potentials at 10 Hz stimulus and the exposure to NH 4 Cl are indicated in the graph. S Average SypHy intensity trace normalized to the maximum intensity upon NH 4 Cl superfusion for each of the given conditions in (Q). T, U Average percentage ± SEM rescue compared to rSALM1 upon NH 4 Cl exposure (U) or upon stimulation with 300 action potentials (T) (Kruskal–Wallis, P < 0.001 for T and U). V Normalized average SypHy intensity ± SEM upon 300 action potentials stimulation (Kruskal–Wallis, P = 0.06). Data information: For all graphs, numbers in bars indicate total number of cells/total number of independent experiments. ns = not significant, * P < 0.05, ** P < 0.01 and *** P < 0.001. Source data are available online for this figure.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: Patch Clamp, Isolation, Infection, Construct, Control, shRNA, Expressing, Knockdown, Mutagenesis, Fluorescence, Transfection, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: SALM 1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

doi: 10.15252/embj.2018101289

Figure Lengend Snippet: Schematic model illustrating clustering of Neurexin by SALM1 in four steps. (1) SALM1 self‐clusters on cell membranes through direct homomeric cis ‐interactions. (2) SALM1 clusters recruit negatively charged PIP2 via electrostatic interactions with SALM1's polybasic domain. (3) PIP2 microdomains formed by SALM1 induce local F‐actin network formation. (4) Neurexin is recruited to SALM1 microdomains via F‐actin and PIP2. Together, the data in this study suggest a role for the Neurexin/SALM1/PIP2/F‐actin complex in synapse development and synaptic vesicle recruitment.

Article Snippet: For immunoEM, we used a different antibody (ProSci Cat#5067, RRID:AB_10906317) that recognized SALM1 with high specificity ( ).

Techniques: