Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Adult rat hippocampal CA1 pyramidal cells were immunostained with antibodies for GM130 (green) and NR2A/B C-termini (red). NR2 clusters co-localized with GM130 (yellow arrows), consistent with native receptor clustering early in the secretory pathway (scale bar 10 µm). (B) Full-length myc-tagged NR2B was transfected for 3.5 hours, and maintained at 20°C for 2.5 additional hours to block progress of myc-NR2B-NR1 beyond the TGN. Cycloheximide (100 µM) was added for the last 1.5 hours to reduce ER staining from recently synthesized myc-NR2B. The results shown above consist of a pulse of myc-NR2B-NR1 heteromeric receptors limited to between the ER and the TGN. Antibody staining for myc (left panel) and SAP102 (middle panel) demonstrated some clustering and co-localization of myc-NR2B with SAP102. Yellow arrows indicate co-localized puncta in the Golgi region, and green arrows indicate diffuse staining consistent with ER (scale bar 10 µm). (C) Immunogold labeling of intracellular NR2A/B (5 nm gold) and SAP102 (10 nm gold) along microtubules in the pyramidal cell body layer of hippocampal CA1 indicated co-localization of NR2A/B and SAP102, which was consistent with NR2A/B and SAP102 association early in the secretory pathway (scale bar is 100 nm). (D) VE-2B was transfected and the following day incubated for 24 hours at 40°C. Full-length myc-NR2B was serially transfected as described in (B) while neurons were incubated at 40°C. After 3 hours at 40°C, neurons were shifted to 20°C incubation for an additional 2.5 hours in the presence of Cycloheximide (100 µM) followed by 30 minutes at 32°C to allow both VE-2B and myc-NR2B to exit the TGN. The top panel shows VE-2B in a proximal dendrite targeted similarly to myc-NR2B (middle panel; scale bar 5 µm) in the same dendrite (bottom panel, merge).

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Transfection, Blocking Assay, Staining, Synthesized, Labeling, Incubation

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) At 10-minutes after ER exit VE-2B clearly co-localized with endogenous SAP102 (top panels, VE-2B EGFP fluorescence in green, and endogenous immunostaining with antibody to SAP102 in red; boxes depicts the enlargements; scale bars are 10 µm). At 45 minutes after ER exit (panels second from the top), a time point at which the leading edge of the pulse of VE-2B cargo is arriving at the cell surface (see Fig. 1C), endogenous SAP102 continued to exhibit strong colocalization. VE-2A demonstrated indistinguishable patterns of colocalization with SAP102 along the secretory pathway (summarized in ). Colocalization of VE-2B with SAP102 is dependent on the distal C-terminal seven amino acid residues that contain the PDZ-binding domain of NR2B (bottom panels), as VE-2BΔ7 exhibited little colocalization with SAP102. Note that VE-2BΔ7 still exhibited apparent clustering (left panel at bottom; for quantification see Fig. 6E) and that endogenous SAP102 did not concentrate in the perinuclear Golgi region in the absence of bound receptor (bottom red panel). (B) Neurons transfected with VE, or VE-2B were incubated overnight at 40°C, then switched to media incubated at 15°C for 1 hour to block transport to the Golgi apparatus, and limit secretory cargo transport to no further than the IC. Neurons were then immunostained for endogenous SAP102. Concentration of VE was observed along the lengths of dendrites that was consistent with budding and protrusion from ER exits sites and initial transport to the Golgi (top panels; characterized previously ). However, concentrations of VE were not well colocalized with SAP102. Alternatively, concentrations of VE-2B demonstrated the beginnings of co-localization with SAP102 at the IC (bottom panels; scale bars 5 µm). (C) Colocalization was quantified as described in Experimental Methods with 5–15 images of neuronal soma from at least 3 separate transfection experiments for a minimum of 15 and a maximum of 26 measures. To assess whether concentrated clusters of VE-2B were co-localized with concentrated SAP102 clusters, the threshold of SAP102 was held constant at a threshold that included concentrated SAP102 clusters (104-255 gray level, inclusive), and the threshold of inclusion for VE-2B (green threshold) was varied from 52-255 to 182-255 (x-axis). The percent colocalization was averaged within transfection to reduce random error (the number of transfections equals N), and then within-group to obtain the means ± SEM. By one-way Anova with repeated measures, there was a significant group effect (P<0.01), and post hoc pairwise comparisons using the Tukey method that were significant are indicated by asterisks (p<0.05). E-2B was co-localized with SAP102 at both 10 minutes (filled circles), and 45 minutes (open squares) after ER exit. At 10 minutes after ER exit, VE-2B was also predominantly co-localized with GM130, a marker of cis- media - Golgi (filled squares). VE-2BΔ7 (filled triangles) showed incidental background levels of colocalization with SAP102. Similar results were obtained holding the green threshold at a high constant value and varying the red threshold.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Fluorescence, Immunostaining, Binding Assay, Transfection, Incubation, Blocking Assay, Concentration Assay, Marker

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Western Blot, Infection, Immunoprecipitation, Transfection

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p<0.05). VE-2B colocalization with SAP102 was significantly greater across all green thresholds using an Anova with repeated measures compared to both VE and VE-2BΔ7 (p<0.01), while the percent overlap of VE-2B with PSD-95 by Anova with repeated measures was significantly different than VE-2BΔ7 (p<0.01). (B) The distances between GFP, SAP102, and synaptophysin were obtained at 3 hours after release from the ER by defining a 2 µm region of interest around GFP puncta, and calculating the centroid for each color within the region of interest above 2X background using Metamorph software. Lengths are binned as <0.3 µm (unresolvable) to >1 µm in 100 nm increments as shown above. Results are shown as a fraction of the total puncta in all bins for 3 separate transfections with >50 GFP puncta per transfection ±SEM. A significantly greater percentage of VE-2B puncta are within an unresolvable distance to SAP102 compared to VE-2BΔ7 (B, left graph; p<0.05; one-way Anova with repeated measures), whereas there is no significant difference in the unresolvable groups of VE-2B, VE-2BΔ7, and synaptophysin (B, right graph). (C) Synaptic association of VE-2B is associated with an increase in SAP102 intensity. The mean intensity was calculated for all SAP102 and synaptophysin puncta within the 2 µm region of interest from the data pool in B. The resulting measure included both synaptic and non-synaptic VE-2B/SAP102. VE-2B-associated SAP102 staining showed a significantly higher mean intensity than VE-2BΔ7-associated puncta (C, left histogram; * p<0.05, Student’s t-test), while the mean synaptophysin intensity remained unchanged (C, left histogram). The mean intensities for the entire images from the data pool were the same. The arrow indicates the threshold at which SAP102 was included (104–255) for measurement of VE-2B/high-intensity MAGUK pixel overlap in A. To assess the effect of VE-2B on synaptic SAP102 (right histogram), the mean intensity of SAP102 puncta co-localized with both VE-2B and synaptophysin (<0.3 µm from each other) was extracted from a larger data pool (N = 4 transfections; total synaptic puncta, 289) and compared to the mean intensity of synaptic SAP102 in the same image pool and in the absence of VE-2B (total synaptic puncta, 699). Synaptic VE-2B significantly enhanced synaptic SAP102 intensity (*p<0.05, Student’s t-test; illustrated in D). (D) A typical example of how VE-2B (right picture, green) concentrated both synaptic and non-synaptic endogenous SAP102 (left picture, SAP102 in red; synaptophysin in blue) 3 hours after ER release (scale bar, 5 µm). Note that those SAP102 puncta that are co-localized with VE-2B (all arrows) are brighter than SAP102 puncta in the rest of the field. Yellow arrows indicate non-synaptic VE-2B/SAP102 puncta. White arrows indicate synaptic VE-2B/SAP102/synaptophysin puncta.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Software, Transfection, Staining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Expressing, Immunostaining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Adult rat hippocampal CA1 pyramidal cells were immunostained with antibodies for GM130 (green) and NR2A/B C-termini (red). NR2 clusters co-localized with GM130 (yellow arrows), consistent with native receptor clustering early in the secretory pathway (scale bar 10 µm). (B) Full-length myc-tagged NR2B was transfected for 3.5 hours, and maintained at 20°C for 2.5 additional hours to block progress of myc-NR2B-NR1 beyond the TGN. Cycloheximide (100 µM) was added for the last 1.5 hours to reduce ER staining from recently synthesized myc-NR2B. The results shown above consist of a pulse of myc-NR2B-NR1 heteromeric receptors limited to between the ER and the TGN. Antibody staining for myc (left panel) and SAP102 (middle panel) demonstrated some clustering and co-localization of myc-NR2B with SAP102. Yellow arrows indicate co-localized puncta in the Golgi region, and green arrows indicate diffuse staining consistent with ER (scale bar 10 µm). (C) Immunogold labeling of intracellular NR2A/B (5 nm gold) and SAP102 (10 nm gold) along microtubules in the pyramidal cell body layer of hippocampal CA1 indicated co-localization of NR2A/B and SAP102, which was consistent with NR2A/B and SAP102 association early in the secretory pathway (scale bar is 100 nm). (D) VE-2B was transfected and the following day incubated for 24 hours at 40°C. Full-length myc-NR2B was serially transfected as described in (B) while neurons were incubated at 40°C. After 3 hours at 40°C, neurons were shifted to 20°C incubation for an additional 2.5 hours in the presence of Cycloheximide (100 µM) followed by 30 minutes at 32°C to allow both VE-2B and myc-NR2B to exit the TGN. The top panel shows VE-2B in a proximal dendrite targeted similarly to myc-NR2B (middle panel; scale bar 5 µm) in the same dendrite (bottom panel, merge).

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Transfection, Blocking Assay, Staining, Synthesized, Labeling, Incubation

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) At 10-minutes after ER exit VE-2B clearly co-localized with endogenous SAP102 (top panels, VE-2B EGFP fluorescence in green, and endogenous immunostaining with antibody to SAP102 in red; boxes depicts the enlargements; scale bars are 10 µm). At 45 minutes after ER exit (panels second from the top), a time point at which the leading edge of the pulse of VE-2B cargo is arriving at the cell surface (see Fig. 1C), endogenous SAP102 continued to exhibit strong colocalization. VE-2A demonstrated indistinguishable patterns of colocalization with SAP102 along the secretory pathway (summarized in ). Colocalization of VE-2B with SAP102 is dependent on the distal C-terminal seven amino acid residues that contain the PDZ-binding domain of NR2B (bottom panels), as VE-2BΔ7 exhibited little colocalization with SAP102. Note that VE-2BΔ7 still exhibited apparent clustering (left panel at bottom; for quantification see Fig. 6E) and that endogenous SAP102 did not concentrate in the perinuclear Golgi region in the absence of bound receptor (bottom red panel). (B) Neurons transfected with VE, or VE-2B were incubated overnight at 40°C, then switched to media incubated at 15°C for 1 hour to block transport to the Golgi apparatus, and limit secretory cargo transport to no further than the IC. Neurons were then immunostained for endogenous SAP102. Concentration of VE was observed along the lengths of dendrites that was consistent with budding and protrusion from ER exits sites and initial transport to the Golgi (top panels; characterized previously ). However, concentrations of VE were not well colocalized with SAP102. Alternatively, concentrations of VE-2B demonstrated the beginnings of co-localization with SAP102 at the IC (bottom panels; scale bars 5 µm). (C) Colocalization was quantified as described in Experimental Methods with 5–15 images of neuronal soma from at least 3 separate transfection experiments for a minimum of 15 and a maximum of 26 measures. To assess whether concentrated clusters of VE-2B were co-localized with concentrated SAP102 clusters, the threshold of SAP102 was held constant at a threshold that included concentrated SAP102 clusters (104-255 gray level, inclusive), and the threshold of inclusion for VE-2B (green threshold) was varied from 52-255 to 182-255 (x-axis). The percent colocalization was averaged within transfection to reduce random error (the number of transfections equals N), and then within-group to obtain the means ± SEM. By one-way Anova with repeated measures, there was a significant group effect (P<0.01), and post hoc pairwise comparisons using the Tukey method that were significant are indicated by asterisks (p<0.05). E-2B was co-localized with SAP102 at both 10 minutes (filled circles), and 45 minutes (open squares) after ER exit. At 10 minutes after ER exit, VE-2B was also predominantly co-localized with GM130, a marker of cis- media - Golgi (filled squares). VE-2BΔ7 (filled triangles) showed incidental background levels of colocalization with SAP102. Similar results were obtained holding the green threshold at a high constant value and varying the red threshold.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Fluorescence, Immunostaining, Binding Assay, Transfection, Incubation, Blocking Assay, Concentration Assay, Marker

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Western Blot, Infection, Immunoprecipitation, Transfection

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p<0.05). VE-2B colocalization with SAP102 was significantly greater across all green thresholds using an Anova with repeated measures compared to both VE and VE-2BΔ7 (p<0.01), while the percent overlap of VE-2B with PSD-95 by Anova with repeated measures was significantly different than VE-2BΔ7 (p<0.01). (B) The distances between GFP, SAP102, and synaptophysin were obtained at 3 hours after release from the ER by defining a 2 µm region of interest around GFP puncta, and calculating the centroid for each color within the region of interest above 2X background using Metamorph software. Lengths are binned as <0.3 µm (unresolvable) to >1 µm in 100 nm increments as shown above. Results are shown as a fraction of the total puncta in all bins for 3 separate transfections with >50 GFP puncta per transfection ±SEM. A significantly greater percentage of VE-2B puncta are within an unresolvable distance to SAP102 compared to VE-2BΔ7 (B, left graph; p<0.05; one-way Anova with repeated measures), whereas there is no significant difference in the unresolvable groups of VE-2B, VE-2BΔ7, and synaptophysin (B, right graph). (C) Synaptic association of VE-2B is associated with an increase in SAP102 intensity. The mean intensity was calculated for all SAP102 and synaptophysin puncta within the 2 µm region of interest from the data pool in B. The resulting measure included both synaptic and non-synaptic VE-2B/SAP102. VE-2B-associated SAP102 staining showed a significantly higher mean intensity than VE-2BΔ7-associated puncta (C, left histogram; * p<0.05, Student’s t-test), while the mean synaptophysin intensity remained unchanged (C, left histogram). The mean intensities for the entire images from the data pool were the same. The arrow indicates the threshold at which SAP102 was included (104–255) for measurement of VE-2B/high-intensity MAGUK pixel overlap in A. To assess the effect of VE-2B on synaptic SAP102 (right histogram), the mean intensity of SAP102 puncta co-localized with both VE-2B and synaptophysin (<0.3 µm from each other) was extracted from a larger data pool (N = 4 transfections; total synaptic puncta, 289) and compared to the mean intensity of synaptic SAP102 in the same image pool and in the absence of VE-2B (total synaptic puncta, 699). Synaptic VE-2B significantly enhanced synaptic SAP102 intensity (*p<0.05, Student’s t-test; illustrated in D). (D) A typical example of how VE-2B (right picture, green) concentrated both synaptic and non-synaptic endogenous SAP102 (left picture, SAP102 in red; synaptophysin in blue) 3 hours after ER release (scale bar, 5 µm). Note that those SAP102 puncta that are co-localized with VE-2B (all arrows) are brighter than SAP102 puncta in the rest of the field. Yellow arrows indicate non-synaptic VE-2B/SAP102 puncta. White arrows indicate synaptic VE-2B/SAP102/synaptophysin puncta.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Software, Transfection, Staining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

Techniques: Expressing, Immunostaining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Adult rat hippocampal CA1 pyramidal cells were immunostained with antibodies for GM130 (green) and NR2A/B C-termini (red). NR2 clusters co-localized with GM130 (yellow arrows), consistent with native receptor clustering early in the secretory pathway (scale bar 10 µm). (B) Full-length myc-tagged NR2B was transfected for 3.5 hours, and maintained at 20°C for 2.5 additional hours to block progress of myc-NR2B-NR1 beyond the TGN. Cycloheximide (100 µM) was added for the last 1.5 hours to reduce ER staining from recently synthesized myc-NR2B. The results shown above consist of a pulse of myc-NR2B-NR1 heteromeric receptors limited to between the ER and the TGN. Antibody staining for myc (left panel) and SAP102 (middle panel) demonstrated some clustering and co-localization of myc-NR2B with SAP102. Yellow arrows indicate co-localized puncta in the Golgi region, and green arrows indicate diffuse staining consistent with ER (scale bar 10 µm). (C) Immunogold labeling of intracellular NR2A/B (5 nm gold) and SAP102 (10 nm gold) along microtubules in the pyramidal cell body layer of hippocampal CA1 indicated co-localization of NR2A/B and SAP102, which was consistent with NR2A/B and SAP102 association early in the secretory pathway (scale bar is 100 nm). (D) VE-2B was transfected and the following day incubated for 24 hours at 40°C. Full-length myc-NR2B was serially transfected as described in (B) while neurons were incubated at 40°C. After 3 hours at 40°C, neurons were shifted to 20°C incubation for an additional 2.5 hours in the presence of Cycloheximide (100 µM) followed by 30 minutes at 32°C to allow both VE-2B and myc-NR2B to exit the TGN. The top panel shows VE-2B in a proximal dendrite targeted similarly to myc-NR2B (middle panel; scale bar 5 µm) in the same dendrite (bottom panel, merge).

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Transfection, Blocking Assay, Staining, Synthesized, Labeling, Incubation

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) At 10-minutes after ER exit VE-2B clearly co-localized with endogenous SAP102 (top panels, VE-2B EGFP fluorescence in green, and endogenous immunostaining with antibody to SAP102 in red; boxes depicts the enlargements; scale bars are 10 µm). At 45 minutes after ER exit (panels second from the top), a time point at which the leading edge of the pulse of VE-2B cargo is arriving at the cell surface (see Fig. 1C), endogenous SAP102 continued to exhibit strong colocalization. VE-2A demonstrated indistinguishable patterns of colocalization with SAP102 along the secretory pathway (summarized in ). Colocalization of VE-2B with SAP102 is dependent on the distal C-terminal seven amino acid residues that contain the PDZ-binding domain of NR2B (bottom panels), as VE-2BΔ7 exhibited little colocalization with SAP102. Note that VE-2BΔ7 still exhibited apparent clustering (left panel at bottom; for quantification see Fig. 6E) and that endogenous SAP102 did not concentrate in the perinuclear Golgi region in the absence of bound receptor (bottom red panel). (B) Neurons transfected with VE, or VE-2B were incubated overnight at 40°C, then switched to media incubated at 15°C for 1 hour to block transport to the Golgi apparatus, and limit secretory cargo transport to no further than the IC. Neurons were then immunostained for endogenous SAP102. Concentration of VE was observed along the lengths of dendrites that was consistent with budding and protrusion from ER exits sites and initial transport to the Golgi (top panels; characterized previously ). However, concentrations of VE were not well colocalized with SAP102. Alternatively, concentrations of VE-2B demonstrated the beginnings of co-localization with SAP102 at the IC (bottom panels; scale bars 5 µm). (C) Colocalization was quantified as described in Experimental Methods with 5–15 images of neuronal soma from at least 3 separate transfection experiments for a minimum of 15 and a maximum of 26 measures. To assess whether concentrated clusters of VE-2B were co-localized with concentrated SAP102 clusters, the threshold of SAP102 was held constant at a threshold that included concentrated SAP102 clusters (104-255 gray level, inclusive), and the threshold of inclusion for VE-2B (green threshold) was varied from 52-255 to 182-255 (x-axis). The percent colocalization was averaged within transfection to reduce random error (the number of transfections equals N), and then within-group to obtain the means ± SEM. By one-way Anova with repeated measures, there was a significant group effect (P<0.01), and post hoc pairwise comparisons using the Tukey method that were significant are indicated by asterisks (p<0.05). E-2B was co-localized with SAP102 at both 10 minutes (filled circles), and 45 minutes (open squares) after ER exit. At 10 minutes after ER exit, VE-2B was also predominantly co-localized with GM130, a marker of cis- media - Golgi (filled squares). VE-2BΔ7 (filled triangles) showed incidental background levels of colocalization with SAP102. Similar results were obtained holding the green threshold at a high constant value and varying the red threshold.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Fluorescence, Immunostaining, Binding Assay, Transfection, Incubation, Blocking Assay, Concentration Assay, Marker

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Western Blot, Infection, Immunoprecipitation, Transfection

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p<0.05). VE-2B colocalization with SAP102 was significantly greater across all green thresholds using an Anova with repeated measures compared to both VE and VE-2BΔ7 (p<0.01), while the percent overlap of VE-2B with PSD-95 by Anova with repeated measures was significantly different than VE-2BΔ7 (p<0.01). (B) The distances between GFP, SAP102, and synaptophysin were obtained at 3 hours after release from the ER by defining a 2 µm region of interest around GFP puncta, and calculating the centroid for each color within the region of interest above 2X background using Metamorph software. Lengths are binned as <0.3 µm (unresolvable) to >1 µm in 100 nm increments as shown above. Results are shown as a fraction of the total puncta in all bins for 3 separate transfections with >50 GFP puncta per transfection ±SEM. A significantly greater percentage of VE-2B puncta are within an unresolvable distance to SAP102 compared to VE-2BΔ7 (B, left graph; p<0.05; one-way Anova with repeated measures), whereas there is no significant difference in the unresolvable groups of VE-2B, VE-2BΔ7, and synaptophysin (B, right graph). (C) Synaptic association of VE-2B is associated with an increase in SAP102 intensity. The mean intensity was calculated for all SAP102 and synaptophysin puncta within the 2 µm region of interest from the data pool in B. The resulting measure included both synaptic and non-synaptic VE-2B/SAP102. VE-2B-associated SAP102 staining showed a significantly higher mean intensity than VE-2BΔ7-associated puncta (C, left histogram; * p<0.05, Student’s t-test), while the mean synaptophysin intensity remained unchanged (C, left histogram). The mean intensities for the entire images from the data pool were the same. The arrow indicates the threshold at which SAP102 was included (104–255) for measurement of VE-2B/high-intensity MAGUK pixel overlap in A. To assess the effect of VE-2B on synaptic SAP102 (right histogram), the mean intensity of SAP102 puncta co-localized with both VE-2B and synaptophysin (<0.3 µm from each other) was extracted from a larger data pool (N = 4 transfections; total synaptic puncta, 289) and compared to the mean intensity of synaptic SAP102 in the same image pool and in the absence of VE-2B (total synaptic puncta, 699). Synaptic VE-2B significantly enhanced synaptic SAP102 intensity (*p<0.05, Student’s t-test; illustrated in D). (D) A typical example of how VE-2B (right picture, green) concentrated both synaptic and non-synaptic endogenous SAP102 (left picture, SAP102 in red; synaptophysin in blue) 3 hours after ER release (scale bar, 5 µm). Note that those SAP102 puncta that are co-localized with VE-2B (all arrows) are brighter than SAP102 puncta in the rest of the field. Yellow arrows indicate non-synaptic VE-2B/SAP102 puncta. White arrows indicate synaptic VE-2B/SAP102/synaptophysin puncta.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Software, Transfection, Staining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Expressing, Immunostaining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Adult rat hippocampal CA1 pyramidal cells were immunostained with antibodies for GM130 (green) and NR2A/B C-termini (red). NR2 clusters co-localized with GM130 (yellow arrows), consistent with native receptor clustering early in the secretory pathway (scale bar 10 µm). (B) Full-length myc-tagged NR2B was transfected for 3.5 hours, and maintained at 20°C for 2.5 additional hours to block progress of myc-NR2B-NR1 beyond the TGN. Cycloheximide (100 µM) was added for the last 1.5 hours to reduce ER staining from recently synthesized myc-NR2B. The results shown above consist of a pulse of myc-NR2B-NR1 heteromeric receptors limited to between the ER and the TGN. Antibody staining for myc (left panel) and SAP102 (middle panel) demonstrated some clustering and co-localization of myc-NR2B with SAP102. Yellow arrows indicate co-localized puncta in the Golgi region, and green arrows indicate diffuse staining consistent with ER (scale bar 10 µm). (C) Immunogold labeling of intracellular NR2A/B (5 nm gold) and SAP102 (10 nm gold) along microtubules in the pyramidal cell body layer of hippocampal CA1 indicated co-localization of NR2A/B and SAP102, which was consistent with NR2A/B and SAP102 association early in the secretory pathway (scale bar is 100 nm). (D) VE-2B was transfected and the following day incubated for 24 hours at 40°C. Full-length myc-NR2B was serially transfected as described in (B) while neurons were incubated at 40°C. After 3 hours at 40°C, neurons were shifted to 20°C incubation for an additional 2.5 hours in the presence of Cycloheximide (100 µM) followed by 30 minutes at 32°C to allow both VE-2B and myc-NR2B to exit the TGN. The top panel shows VE-2B in a proximal dendrite targeted similarly to myc-NR2B (middle panel; scale bar 5 µm) in the same dendrite (bottom panel, merge).

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Transfection, Blocking Assay, Staining, Synthesized, Labeling, Incubation

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) At 10-minutes after ER exit VE-2B clearly co-localized with endogenous SAP102 (top panels, VE-2B EGFP fluorescence in green, and endogenous immunostaining with antibody to SAP102 in red; boxes depicts the enlargements; scale bars are 10 µm). At 45 minutes after ER exit (panels second from the top), a time point at which the leading edge of the pulse of VE-2B cargo is arriving at the cell surface (see Fig. 1C), endogenous SAP102 continued to exhibit strong colocalization. VE-2A demonstrated indistinguishable patterns of colocalization with SAP102 along the secretory pathway (summarized in ). Colocalization of VE-2B with SAP102 is dependent on the distal C-terminal seven amino acid residues that contain the PDZ-binding domain of NR2B (bottom panels), as VE-2BΔ7 exhibited little colocalization with SAP102. Note that VE-2BΔ7 still exhibited apparent clustering (left panel at bottom; for quantification see Fig. 6E) and that endogenous SAP102 did not concentrate in the perinuclear Golgi region in the absence of bound receptor (bottom red panel). (B) Neurons transfected with VE, or VE-2B were incubated overnight at 40°C, then switched to media incubated at 15°C for 1 hour to block transport to the Golgi apparatus, and limit secretory cargo transport to no further than the IC. Neurons were then immunostained for endogenous SAP102. Concentration of VE was observed along the lengths of dendrites that was consistent with budding and protrusion from ER exits sites and initial transport to the Golgi (top panels; characterized previously ). However, concentrations of VE were not well colocalized with SAP102. Alternatively, concentrations of VE-2B demonstrated the beginnings of co-localization with SAP102 at the IC (bottom panels; scale bars 5 µm). (C) Colocalization was quantified as described in Experimental Methods with 5–15 images of neuronal soma from at least 3 separate transfection experiments for a minimum of 15 and a maximum of 26 measures. To assess whether concentrated clusters of VE-2B were co-localized with concentrated SAP102 clusters, the threshold of SAP102 was held constant at a threshold that included concentrated SAP102 clusters (104-255 gray level, inclusive), and the threshold of inclusion for VE-2B (green threshold) was varied from 52-255 to 182-255 (x-axis). The percent colocalization was averaged within transfection to reduce random error (the number of transfections equals N), and then within-group to obtain the means ± SEM. By one-way Anova with repeated measures, there was a significant group effect (P<0.01), and post hoc pairwise comparisons using the Tukey method that were significant are indicated by asterisks (p<0.05). E-2B was co-localized with SAP102 at both 10 minutes (filled circles), and 45 minutes (open squares) after ER exit. At 10 minutes after ER exit, VE-2B was also predominantly co-localized with GM130, a marker of cis- media - Golgi (filled squares). VE-2BΔ7 (filled triangles) showed incidental background levels of colocalization with SAP102. Similar results were obtained holding the green threshold at a high constant value and varying the red threshold.

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Fluorescence, Immunostaining, Binding Assay, Transfection, Incubation, Blocking Assay, Concentration Assay, Marker

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Western Blot, Infection, Immunoprecipitation, Transfection

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p<0.05). VE-2B colocalization with SAP102 was significantly greater across all green thresholds using an Anova with repeated measures compared to both VE and VE-2BΔ7 (p<0.01), while the percent overlap of VE-2B with PSD-95 by Anova with repeated measures was significantly different than VE-2BΔ7 (p<0.01). (B) The distances between GFP, SAP102, and synaptophysin were obtained at 3 hours after release from the ER by defining a 2 µm region of interest around GFP puncta, and calculating the centroid for each color within the region of interest above 2X background using Metamorph software. Lengths are binned as <0.3 µm (unresolvable) to >1 µm in 100 nm increments as shown above. Results are shown as a fraction of the total puncta in all bins for 3 separate transfections with >50 GFP puncta per transfection ±SEM. A significantly greater percentage of VE-2B puncta are within an unresolvable distance to SAP102 compared to VE-2BΔ7 (B, left graph; p<0.05; one-way Anova with repeated measures), whereas there is no significant difference in the unresolvable groups of VE-2B, VE-2BΔ7, and synaptophysin (B, right graph). (C) Synaptic association of VE-2B is associated with an increase in SAP102 intensity. The mean intensity was calculated for all SAP102 and synaptophysin puncta within the 2 µm region of interest from the data pool in B. The resulting measure included both synaptic and non-synaptic VE-2B/SAP102. VE-2B-associated SAP102 staining showed a significantly higher mean intensity than VE-2BΔ7-associated puncta (C, left histogram; * p<0.05, Student’s t-test), while the mean synaptophysin intensity remained unchanged (C, left histogram). The mean intensities for the entire images from the data pool were the same. The arrow indicates the threshold at which SAP102 was included (104–255) for measurement of VE-2B/high-intensity MAGUK pixel overlap in A. To assess the effect of VE-2B on synaptic SAP102 (right histogram), the mean intensity of SAP102 puncta co-localized with both VE-2B and synaptophysin (<0.3 µm from each other) was extracted from a larger data pool (N = 4 transfections; total synaptic puncta, 289) and compared to the mean intensity of synaptic SAP102 in the same image pool and in the absence of VE-2B (total synaptic puncta, 699). Synaptic VE-2B significantly enhanced synaptic SAP102 intensity (*p<0.05, Student’s t-test; illustrated in D). (D) A typical example of how VE-2B (right picture, green) concentrated both synaptic and non-synaptic endogenous SAP102 (left picture, SAP102 in red; synaptophysin in blue) 3 hours after ER release (scale bar, 5 µm). Note that those SAP102 puncta that are co-localized with VE-2B (all arrows) are brighter than SAP102 puncta in the rest of the field. Yellow arrows indicate non-synaptic VE-2B/SAP102 puncta. White arrows indicate synaptic VE-2B/SAP102/synaptophysin puncta.

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Software, Transfection, Staining

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: These results were confirmed with a second SAP102 specific antibody (Alomone Labs, data not shown).

Techniques: Expressing, Immunostaining