Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of synaptic stability by AMPA receptor reverse signaling

doi: 10.1073/pnas.1015163108

Figure Lengend Snippet: Stable presynaptic puncta are associated with postsynaptic AMPA receptors. Time-lapse imaging revealed that Syn-GFP puncta preferentially stabilized on dendrites and are associated with postsynaptic AMPA receptors the majority of the time. (A) Example of a location in which Syn-GFP–expressing axons (green) contacted mCherry-expressing dendrites (red). The white boxes depict areas that are magnified in B and C. In each imaging experiment, we confirmed by DIC imaging that no untransfected dendrites were in the field of view. (Scale bar, 5 μm.) (B and C) Micrographs of locations identified in A at 0, 20, 40, and 60 min. (B) A dendrite segment receiving stable Syn-GFP inputs (white arrows). (C) Syn-GFP puncta in an area without dendrites. Note the lack of stable puncta in this field of view, suggesting that dendrite contact is necessary for presynaptic input stabilization. (D) Quantification of the dynamics of Syn-GFP puncta at and away from sites of dendritic contact. ***P < 0.001. (E–G) Stable and eliminated Syn-GFP puncta with retrospective staining for AMPA and NMDA receptors. Arrowheads indicate the position of Syn-GFP puncta at the time point indicated. (E) A stable Syn-GFP punctum. Live-labeling at the 6-h mark revealed the presence of a surface-expressed GluR2 punctum that colocalized with the Syn-GFP punctum. (F) A Syn-GFP punctum that was present at 0 and 3 h but was eliminated by 6 h. No staining for surface GluR2 was seen at the elimination site. (G) Another example of an eliminated Syn-GFP punctum. Note the presence of an NR1 punctum at the elimination site. (Scale bar, 1 μm.) (H) Quantification of the fraction of stable and eliminated Syn-GFP puncta that colocalized with total NR1 or surface-expressed GluR1 or GluR2. *P < 0.05.

Article Snippet: Untagged and GFP-tagged GluR1 and GluR2 were provided by Richard Huganir (Johns Hopkins University, Baltimore, MD).

Techniques: Imaging, Expressing, Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of synaptic stability by AMPA receptor reverse signaling

doi: 10.1073/pnas.1015163108

Figure Lengend Snippet: AMPA receptor down-regulation decreases the number and stability of presynaptic inputs. (A and B) A control neuron cotransfected with mCherry (red) and CFP (green) (A) and a neuron cotransfected with mCherry and CFP:GluR1 and -2 C-tails (B). C-tails were expressed robustly and targeted to dendrites. (Scale bar, 5 μm.) (C) Quantification of the number of surface GluR1 puncta per length of dendrite (normalized to controls). ***P < 0.0001. (D) Quantification of the number of surface GluR2 puncta per length of dendrite (normalized to controls). *P = 0.03. (E) Quantification of the number of PSD-95 puncta per length of dendrite (normalized to controls). (F and G) Endogenous VGLUT-positive inputs onto control (F) or C-tail–expressing neurons (G) at 14 DIV. (Upper) VGLUT-positive inputs (green) onto mCherry-filled dendrites (red). (Lower) The same VGLUT inputs are shown in white with the dendrite outlined in yellow, allowing better visualization of inputs. (Scale bar, 2 μm.) (H) Quantification of the number of excitatory, VGLUT-positive inputs per length of dendrite (normalized to controls). ***P < 0.0001. (I) Quantification of the number of inhibitory, GAD6-positive inputs per length of dendrite (normalized to controls). NS, not significant. (J and K) Examples from imaging experiments in which Syn-GFP–expressing axons (green) contacted mCherry expressing dendrites (red) that also were expressing CFP or CFP:GluR1 and two C-tails. (J) Micrographs from an experiment in which Syn-GFP puncta contacted an mCherry-expressing dendrite that also was expressing CFP (control). The panels show time points at 0, 30, and 60 min. Several Syn-GFP puncta were stabilized on the control neuron for the duration of 1 h, whereas other puncta either appeared or disappeared. (K) Micrographs from the imaging experiment shown in J, but in this case Syn-GFP puncta contacted an mCherry-expressing dendrite that also was expressing GluR C-tails. Although several Syn-GFP puncta contacted the dendrite expressing C-tails, no puncta were stabilized across the hour. (Scale bar, 2 μm.) (L) Quantification of the fraction of stable Syn-GFP inputs contacting control dendrites or dendrites expressing C-tails. There was a significant decrease in stable inputs onto neurons expressing C-tails relative to controls. ***P = 0.0003.

Article Snippet: Untagged and GFP-tagged GluR1 and GluR2 were provided by Richard Huganir (Johns Hopkins University, Baltimore, MD).

Techniques: Control, Expressing, Imaging

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of synaptic stability by AMPA receptor reverse signaling

doi: 10.1073/pnas.1015163108

Figure Lengend Snippet: Overexpression of AMPA receptors increases presynaptic input number and stability. (A–C) Representative images of VGLUT-positive inputs (green) onto 14-DIV neurons transfected with vector (A), STG (B), or STG plus GFP-GluR1 and -2 (C), along with mCherry for visualization of dendrites (red). Lower panels show the same image but with the VGLUT-positive inputs in white and the dendrite outlined in yellow, allowing better visualization of inputs. (Scale bar, 1 μm.) (D–F) Representative images of GAD6-positive inputs (blue) onto 14-DIV neurons transfected with vector (D), STG (E), or STG plus GFP-GluR1 and -2 (F), along with mCherry for visualization of dendrites (red). Lower panels show the same image, but with the GAD6-positive inputs in white and the dendrite outlined in yellow. (G) Quantification of the number of excitatory (VGLUT-positive) inputs per length of dendrite, normalized to control values. Overall P = 0.0004. Intergroup comparisons: ***P < 0.001; **P < 0.01. (H) Quantification of the number of inhibitory (GAD6-positive) inputs per length of dendrite, normalized to controls. (I) Quantification of Syn-GFP stabilization onto control and AMPA receptor-expressing neurons. *P = 0.03.

Article Snippet: Untagged and GFP-tagged GluR1 and GluR2 were provided by Richard Huganir (Johns Hopkins University, Baltimore, MD).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Regulation of synaptic stability by AMPA receptor reverse signaling

doi: 10.1073/pnas.1015163108

Figure Lengend Snippet: The NTD of GluR2 in concert with NLG-1 is sufficient to induce presynaptic input stability. (A–C) Live-imaged 293T cells (red) expressing vector (A), NLG-1 plus GluR1 and -2 (B), or NLG-1 plus GluR2 NTD (G2NTD) (C) contacted by Syn-GFP–expressing axons (green) from cocultured neurons. The white rectangles in the left panels of A–C demarcate an area of a Syn-GFP–expressing axon that is magnified in the panels on the right. The panels on the right correspond to various time points during the imaging period (0, 10, 20, and 60 min). A large fraction of Syn-GFP puncta contacting control 293T cells were trafficking (present in only one imaging window) (A). In contrast, a large fraction of Syn-GFP puncta contacting NLG-1 plus GluR2 NTD-expressing 293T cells were stable across 60 min (C). (Scale bar, 5 μm.) (D) Confirmation that NLG-1 induces presynaptic inputs in our imaging paradigm. The 293T cells expressing NLG-1 received a significantly greater number of Syn-GFP inputs than control cells expressing pRK5 vector at the beginning of the imaging period. ***P = 0.001. (E) A stability index, defined as the ratio of stable puncta to total (stable plus trafficking) puncta, for axons contacting 293T cells expressing the indicated constructs. Overall P < 0.0001. Intergroup comparisons: ***P < 0.001; **P < 0.01; *P < 0.05. n = 24–43 cells for each condition. (F The 293T cells expressing NLG-1 received a significantly greater number of Syn-GFP inputs than control (pDisplay-expressing) cells at the beginning of the imaging period. *P = 0.013. (G) Quantification of stability indexes for 293T cells expressing indicated constructs shows that the first 109 amino acids of the NTD of GluR2 are necessary for presynaptic input stabilization. **P < 0.01; *P < 0.05. (H) Model of the proposed role of the NTD of GluR2 in regulating presynaptic stability. AMPAR, AMPA receptor; NMDAR, NMDA receptor.

Article Snippet: Untagged and GFP-tagged GluR1 and GluR2 were provided by Richard Huganir (Johns Hopkins University, Baltimore, MD).

Techniques: Expressing, Plasmid Preparation, Imaging, Control, Construct

Journal: eNeuro

Article Title: Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing

doi: 10.1523/ENEURO.0056-22.2022

Figure Lengend Snippet: Vectors

Article Snippet: These fragments were cloned into the AfeI site of pOC2 PSD95-Halo (Addgene #183449) and pOC1 GluA1-GFP (Addgene #183430), respectively, using HiFi assembly.

Techniques: Cloning, Plasmid Preparation, Knock-In, Expressing

Journal: eNeuro

Article Title: Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing

doi: 10.1523/ENEURO.0056-22.2022

Figure Lengend Snippet: Primers

Article Snippet: These fragments were cloned into the AfeI site of pOC2 PSD95-Halo (Addgene #183449) and pOC1 GluA1-GFP (Addgene #183430), respectively, using HiFi assembly.

Techniques: Sequencing

Journal: Cell reports

Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

doi: 10.1016/j.celrep.2021.109396

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GFP-GluA1 (C terminus) , Addgene , 34857.

Techniques: Recombinant, Modification, Control, DC Protein Assay, Western Blot, Lysis, Isolation, Plasmid Preparation, In Situ, Mutagenesis, Cloning, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum

doi: 10.3389/fnmol.2023.1107355

Figure Lengend Snippet: Key reagents and resources used in the present study.

Article Snippet: C-Myc-GRIA1 , Sino Biological , HG15792-CM.

Techniques: Recombinant, Protein Extraction, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum

doi: 10.3389/fnmol.2023.1107355

Figure Lengend Snippet: CYLD stabilizes AMPARs. (A) Representative immunoblots of total protein isolated from the DLS of Cyld +/+ and Cyld −/− littermates. (B) Quantification of immunoblots in (A) reveals that total protein expression levels remain unchanged between genotypes (GluA1, GluA2 and mGluR5: n = 4 per group; CaMKIIα, CaMKIIβ, pCaMKIIα and pCaMKIIβ: n = 6 per group). (C) Representative immunoblots of surface protein isolated from the DLS of Cyld +/+ and Cyld −/− littermates. (D) Quantification of immunoblots in (C) reveals a significant decrease in surface GluA1 and GluA2 protein levels in Cyld −/− mice (GluA1: n = 5 per group; GluA2: n = 8 per group; NMDAR1 and mGluR5: n = 3 per group; NMDAR2B: n = 11 per group). β-actin and Na, K-ATPase were used as loading controls. Data are presented as the mean ± SEM; *** p < 0.001.

Article Snippet: C-Myc-GRIA1 , Sino Biological , HG15792-CM.

Techniques: Western Blot, Isolation, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum

doi: 10.3389/fnmol.2023.1107355

Figure Lengend Snippet: CYLD regulates GluA1 and GluA2 K63-ubiquitination. (A) HEK293 cells were cotransfected with CYLD and Myc-GluA1 or Myc-GluA2 expression vectors. The cell lysates were immunoprecipitated using anti-Myc and anti-CYLD antibodies and immunoblotted with anti-CYLD and anti-GluA2 antibodies. (B) Interaction between endogenous CYLD and GluA1 and GluA2 in the mouse brain, analyzed by immunoprecipitation. Rabbit IgG was used as a negative control in immunoprecipitation experiments. (C) HEK293 cells were cotransfected with expression vectors for various combinations of Myc-GluA1 (left) or Myc-GluA2 (right), HA-K63Ub and CYLD, followed by immunoprecipitation with anti-Myc and immunoblotting with anti-K63Ub. (D) Quantification of immunoblots in (C) shows a reduction in K63Ub conjugated to GluA1 (left) and GluA2 (right) by CYLD in HEK293 cells. (E) DLS lysates from Cyld +/+ and Cyld −/− littermates were immunoprecipitated with anti-GluA1 (left) or anti-GluA2 (right) antibodies and immunoblotted with anti-K63Ub. Rabbit IgG was used as a negative control in immunoprecipitation experiments. (F) Quantification of immunoblots in (E) reveals increased GluA1 and GluA2 K63 ubiquitination in Cyld −/− mice ( n = 4 Cyld +/+ mice, n = 4 Cyld −/− mice). (G) In vitro deubiquitination assays showing that CYLD removes GluA1 (left) and GluA2 (right) K63Ub chains. Data are presented as the mean ± SEM; * p <0.05.

Article Snippet: C-Myc-GRIA1 , Sino Biological , HG15792-CM.

Techniques: Expressing, Immunoprecipitation, Negative Control, Western Blot, In Vitro

Journal: Frontiers in Molecular Neuroscience

Article Title: Neural mechanism underlies CYLD modulation of morphology and synaptic function of medium spiny neurons in dorsolateral striatum

doi: 10.3389/fnmol.2023.1107355

Figure Lengend Snippet: CYLD deficiency impairs both DHPG-and HFS-induced LTD in the DLS. (A) Representative confocal images showing surface GluA1 (red) and DAPI (blue) in acute striatal slices treated with or without DHPG (Ctrl) in Cyld +/+ and Cyld −/− mice. Scale bar: 20 μm. (B) Normalized surface GluA1 in DHPG-treated slices compared to Ctrl ( n = 3 Cyld +/+ mice, n = 3 Cyld −/− mice). (C) Absence of DHPG-induced LTD in whole-cell recordings in Cyld −/− mice. (D) Representative traces of AMPAR-mediated EPSCs evoked in a MSN showing the baseline (gray) and 1–30 min after addition of DHPG in Cyld +/+ (black) and Cyld −/− (blue) mice. (E) The graph shows the mean AMPAR-mediated EPSC amplitude at 1–30 min after addition of DHPG taken from (C) ( n = 9 neurons from 7 Cyld +/+ mice, n = 8 neurons from 4 Cyld −/− mice). (F) Absence of LTD induced by four trains of HFS (100 Hz, 1 s, with 10 s inter-train intervals) in field potential recordings in Cyld −/− mice. After HFS of the corticostriatal pathway, PS amplitude values in Cyld −/− mice were significantly higher than those in Cyld +/+ mice between 1 and 60 min. (G) Representative traces showing the baseline (gray) and 1–60 min after LTD induction in Cyld +/+ (black) and Cyld −/− (blue) mice. (H) Mean PS amplitude at 1–60 min after HFS taken from (F) ( n = 16 slices from 10 Cyld +/+ mice, n = 16 slices from 10 Cyld −/− mice). Data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: C-Myc-GRIA1 , Sino Biological , HG15792-CM.

Techniques:

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: Data collection, phasing and refinement statistics

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Solvent

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: Crystal structure of Alphavirus nsP4 RdRp domain. ( A ) Schematic presentation of RRV and SINV nsP4. The crystallized regions of nsP4: SINV RdRp (aa 91–610) and RRV RdRp (aa 109–611) are annotated. Their flexible region (aa 135–184; denoted as ‘flex’ in smudge green) occupying the central tunnel, and (sub)domain distribution is shown as follows: N-terminal domain (NTD) in white and RdRp domain consisting of fingers with colored fingertips (index in green, middle in orange, ring in yellow and pinky in pink), palm in gray and thumb in purple. The mutations targeting disulfide-bond forming cysteine (C164S) in SINV RdRp and surface-entropy site (labeled as SER3 to represent surface-entropy mutations of Q192A and Q196A) in RRV RdRp are marked. The locations of the A-G motifs are shown in colors (colored as A: teal; B: marine; C: red; D: brown; E: yellow; F: black; G: forest green). (B, C) The annotated crystal structures of RRV RdRp (RdRp SER3 ) in ( B ) and SINV RdRp (wt) in ( C ) are colored according to (A) for displaying their subdomain spatial distribution from the front right-hand view (left panel) and their motifs in the 180° rear view (right panel). Motif C that comprises conserved GDD active-site residues is highlighted in red sticks. Polyalanine considered in the flex region of RRV RdRp SER3 and built residues in the SINV RdRp flex region are both represented as a smudge-green ribbon which is traced by backbone Cα residues as a sphere. Additional features in SINV RdRp in (C): the cysteines (C164 in flex, C323 in pinky) are labeled as black sticks (left panel) while magnesium cations are colored navy blue. (D, E) The contrast of RdRp crystallographic configurations for ( D ) monomeric RRV and ( E ) dimeric SINV is presented in both crystal structure (top view) and simplified block (non-hydrophobic-interacting part colored as gray and zig-zag part symbolizes flex region) to present the hydrophobic interactions between the index finger (green region) and thumb (magenta region) on the left panel respectively. The hydrophobic interface of RRV and SINV between the N-terminal index finger and C-terminal thumb region is highlighted in the respective zoom-in box on the right panel of both (D) and (E). In these zoom-in boxes, all hydrophobic residues (hydrophobic interaction distance within 4Å) are shown as sticks and listed accordingly to the color of the index finger and thumb. The top list is the common residues shared between RdRp of RRV and SINV while the bottom list is SINV-RdRp-unique residues at the hydrophobic interface.

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Labeling, Blocking Assay

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: Structure dynamics of RRV nsP4 in solution. ( A ) The HDX profiles of the recombinant constructs of both RRV and SINV: nsP4FL (blue), RdRp (red), and NTD (green), in solution. On the graph, the deuteration incorporation levels expressed in percentage ( D %) are plotted against the nsP4 residue numbers (RRV: aa 1–611, SINV: aa 1–610). The residues missing in the resolved structure are shaded as gray areas, and their positions are indicated. The longest unresolved region in RRV RdRp (residues 135–184) is designated as ‘flex’. (B, C) The color-coded ternary structures of Robetta homology models for RRV ( B ) nsP4FL, ( C ) RdRp and NTD, and for SINV nsP4FL and RdRp in ( D ), according to the representation of D% in rainbow heatmap to visualize structural flexibilities. These models were generated via Robetta to fill the missing residues not covered in the current crystal structures, including the flexibly orientated NTD. The HDX-protection profiles of RRV nsP4 in (A) were color-mapped to these RRV homology models at single-amino-acid resolutions to guide the visual analyses and 3D comparisons between nsP4FL and RdRp and NTD. RRV nsP4FL is annotated with N/C-terminus, subdomains, motifs, and highlighted regions (Ring Motif F, Pinky, flex, Index-to-NTD linker, HLH substructure) as a reference to other models in (C) and (D). The missing residues are presented as Cα spheres in these homology models. The residues colored in black are those not covered/excluded by HDX data rendering. Rainbow bar as reference for (B),(C), and (D): D% is visualized in the rainbow heatmap spectrum, where the red end represents high D% (structured region) and the blue end represents low D% (disordered region).

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Recombinant, Construct, Residue, Generated

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: Comparison of the structure of alphavirus nsP4 with those of other viral RdRps. ( A ) The RRV RdRp SER3 (RRV, PDB 7F0S) and SINV RdRp (SINV, PDB 7VB4), and their shared top structural homologs, including Norwalk virus (NV, PDB 5TSN), enterovirus-71 (EV71, PDB 6KWQ), and classical swine fever virus (CSFV, PDB 5Y6R). The motifs of these PDB structures were structurally aligned for comparison. The summarized motif and subdomain arrangement at the center are colored the same as Figure . The conserved aspartate residues in motifs C and A are displayed as peptide carbon backbone Cα spheres, while the conserved phenylalanine residue in motif E is shown in stick format. ( B ) The conservation of RdRps of +RNA virus based on primary sequence and secondary structure presented through Espript*. The sequence and secondary structure of RRV nsP4 (genus Alphavirus , family Togaviridae ) were compared to those of different alphaviruses (RRV-to-SPDV) and members of the Caliviridae (NV) , Flaviviridae (CSFV-to-HCV), and Picornaviridae (PV-to-COXV) families. The secondary structure is labeled on top of the alignment and shows only α-helices (spring), β-strands (arrow), and β-turns (TT). Motifs A-G are boxed and colored according to (A). [*Espript coloring: The red-boxed columns are the most conserved, while the blue-boxed columns classify amino acid conservation based on their similar functional groups. *Espript label: Togaviridae - Ross River virus (RRV), Getah virus (GETV), Sindbis virus (SINV), chikungunya virus (CHIKV), o’nyong’nyong virus (ONNV), Venezuela equine encephalitis virus (VEEV), Barmah forest virus (BFV), and salmon pancreas disease virus (SPDV); Caliviridae - Norwalk virus (NV); Flaviviridae - classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV); Picornaviridae - poliovirus (PV), encephalomyocarditis virus (EMCV), enterovirus-71 (EV71), and coxsackievirus (COXV).

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Comparison, Virus, Residue, Sequencing, Labeling, Functional Assay

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: RNA polymerase activities of nsP4FL, RdRp, and RdRp SER3 . ( A ) Schematic presentation of the discontinuous time point RNA polymerase reaction. Extension of fluorescently labeled (green star on the 5′ end) RNA template T1 by recombinant nsP4 protein (gray particle) results in ATP incorporations as A-residue (orange circle) into the RNA intermediates (RIs; a snapshot of one possible RI; right panel) and RNA product (RP; right panel), both of which are shown below with expected band migration with each band corresponding to the number of A-residue added to T1 on a urea-PAGE gel. ( B , C ) The biochemical characterization of recombinant nsP4 proteins using discontinuous time-point gel-based polymerase assay was performed for 0–24 h. The denaturing urea-PAGE gels are annotated with T1, time-point (t) in hour unit (h), products made by dengue virus RdRp used as a positive control (named as D), and the tested recombinant nsP4 proteins from RRV (nsP4FL, polymerase weakened mutant of nsP4FL GNN, RdRp, RdRp SER3 , and NTD) and SINV (nsPFL and RdRp, in purple text). For the polymerase reactions between RRV RdRp or RdRp SER3 and NTD, the stoichiometric molar ratio is shown in (C), that is marked with lane number for reference. The incorporation of each base is marked with a short white line shown between the last 2 time points.

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Labeling, Recombinant, Residue, Migration, Virus, Positive Control, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold

doi: 10.1093/nar/gkab1302

Figure Lengend Snippet: Effects of mutations in nsP4 on the activities of RRV and SINV trans -replicases. ( A ) The trans -replication system is composed of two plasmids (CMV-P123 and CMV-ubi-nsP4) for the expression of RRV and SINV replicase and a plasmid HSPolI-FG for the production of replication-competent RNA containing Fluc and Gluc reporters. In this assay, the increase in expression levels of Fluc and Gluc represented the production of full-length genomic RNA and SG RNA, respectively. HSPolI, a truncated promoter (residues −211 to −1) for human RNA polymerase I; 5′ UTR, full length 5′ UTR of an alphavirus; nsP1 N*, region encoding the N-terminal 77 (RRV) or 114 (SINV) amino acid residues of nsP1; SG, SG promoter spanning (with respect to termination codon of nsP4) from position –79 to the end of the intergenic region; 3′ UTR, truncated (last 110 residues) 3′ UTR of an alphavirus; HDV RZ, antisense strand ribozyme of hepatitis delta virus; MmTer, a terminator for RNA polymerase I in mice; CMV, an immediate early promoter of human cytomegalovirus; LI, leader sequence of the herpes simplex virus thymidine kinase gene with artificial intron; SV40Ter, simian virus 40 late polyadenylation region; Ubi, sequence encoding for human ubiquitin. Positions of mutations in nsP4 (residues numbered as in nsP4 of RRV) are indicated. ( B, C ) U2OS cells in 12-well plates were cotransfected with matching combinations of CMV-P123 and CMV-ubi-nsP4 or its mutant versions (in a 1:1 molar ratio) and the corresponding HSPolI-FG plasmids. As negative controls, CMV-ubi-nsP4 GAA , which encodes nsP4 lacking RNA polymerase activity, was used instead of CMV-ubi-nsP4. Cells were incubated at 37°C and lysed 18 h post-transfection. Fluc and Gluc activities produced by wt and mutant replicases were normalized to the nsP4 GAA controls. Values obtained for nsP4 GAA controls were taken as 1. Means + standard deviation of three independent experiments are shown. (B) Effects of introduced mutations on RRV replicase activity. ( C ) Effects of introduced mutations on SINV replicase activity.

Article Snippet: The sequences encoding SINV nsP4FL (aa 1–610) and its RdRp domain (aa 91–610) were obtained using PCR on a SINV cDNA plasmid template (pSIN ReP5-GFP-GluR1 from Addgene).

Techniques: Expressing, Plasmid Preparation, Virus, Sequencing, Ubiquitin Proteomics, Mutagenesis, Activity Assay, Incubation, Transfection, Produced, Standard Deviation

Journal: Biology Direct

Article Title: Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

doi: 10.1186/1745-6150-1-37

Figure Lengend Snippet: Immunofluorescence localization of NMDA zeta receptor subunit (NR1), Hsc70, and synaptotagmin VII to epithelial tight junctions . (A) Confocal images showing immunofluorescence localization of postsynaptic NMDA receptor zeta subunit co-localized with occludin in MDCK I cells (one week post-confluent). (B) Confocal images showing immunofluorescence localization of presynaptic Hsc70 localized to tight junction domains of C2bbE1 cells (4 weeks post-confluent). (C) Confocal images showing immunofluorescence localization of presynaptic synaptotagmin VII localized to tight junction domains of C2bbE1 cells (4 weeks post-confluent). Note the punctate patterns of NMDA zeta, Hsc70, and synaptotagmin VII.

Article Snippet: Antibodies to P/Q-type calcium CP alpha 1A (sc-28619), synaptotagmin VII (sc-15420), EAAT1 (sc-15316), piccolo (sc-18569), connexin 36 (sc-14904), GRIP1 (sc-28934), rabaptin-5 (sc-6162), Homer (sc-15321), NMDA zeta (sc-1467), GluR1 (sc-13152), Hsc70 (sc-7298) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence

Journal: Biology Direct

Article Title: Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

doi: 10.1186/1745-6150-1-37

Figure Lengend Snippet: Analysis of synaptic hits . (A) Categorization of 202 synaptic hits based on synaptic localizations. (B) Sub-categorization of 202 synaptic hits based on synaptic functions. (C) Confocal images showing immunofluorescence localization of presynaptic P/Q-type voltage-gated calcium channel subunit CPα1A and postsynaptic AMPA receptor subunit GluR1 to tight junctions of C2bbE1 intestinal epithelial cells (4 weeks post-confluent). Tight junction formation has been monitored by measurement of transepithelial electrical resistance before the cells are utilized for staining. Occludin is used as a marker for tight junction domain. Merged yellow images show overlapping of occludin and synaptic proteins.

Article Snippet: Antibodies to P/Q-type calcium CP alpha 1A (sc-28619), synaptotagmin VII (sc-15420), EAAT1 (sc-15316), piccolo (sc-18569), connexin 36 (sc-14904), GRIP1 (sc-28934), rabaptin-5 (sc-6162), Homer (sc-15321), NMDA zeta (sc-1467), GluR1 (sc-13152), Hsc70 (sc-7298) were purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Staining, Marker

Journal: Molecular Psychiatry

Article Title: The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking

doi: 10.1038/s41380-024-02701-7

Figure Lengend Snippet: A Model depicting effects of C4 overexpression on GluR1 recycling in dendritic spines. B Representative images (60X) showing a GFP-positive dendritic spine (white), GluR1 (green) and Rab11a (magenta) of P21–23 apical tufts in GFP-controls (blue frame). C Representative images (60X) showing a dendritic spine identified with GFP signal (white), GluR1 (green) and Rab11a (magenta) in C4-OE (red frame). B , C Yellow arrowhead, GluR1 or Rab11a clusters in spines. White-filled arrowhead, GluR1/Rab11a colocalization. White empty arrowhead, non-colocalized GluR1/Rab11a. Spine silhouette, white dotted line. Orthogonal views are shown (XY, YZ and XZ). Scale bar = 2 μm. D C4-OE caused a 47% decrease in the amount of GluR1 colocalized with Rab11a compared to GFP-control. E There was no change in the amount of Rab11a colocalized with GluR1 in C4-OE relative to GFP-control. F C4-OE increased the minimum distance between GluR1 and Rab11a clusters by 35% relative to GFP-control. Green circle: GluR1, Magenta circle: Rab11a. G C4-OE led to a 25% decrease in the overlapping volume between GluR1 and Rab11a relative to GFP-control. Green circle: GluR1, Magenta circle: Rab11a. H Schematic showing the effects of C4-OE on GluR1 degradation in dendritic spines. I Representative images (60X) showing a GFP-positive spine (white), GluR1 (green) and LAMP1 (magenta) in GFP-control (blue frame). J Representative images (60X) showing a GFP-positive spine (white), GluR1 (green) and LAMP1 (magenta) in C4-OE (red frame). I , J Yellow arrowhead, GluR1 or Rab11a clusters in spines. White empty arrowhead, non-colocalized GluR1/LAMP1. White-filled arrowhead, GluR1/LAMP1 colocalization. Spine silhouette, white dotted line. Orthogonal views are shown (XY, YZ and XZ). Scale bar = 2 μm. K C4-OE led to a 145% increase in the amount of GluR1 colocalized with LAMP1. L C4-OE caused a 103% increase in the amount of LAMP1 colocalized with GluR1. M C4-OE induced a 31% decrease in the minimum distance between GluR1 and LAMP1 clusters. Green circle: GluR1, Magenta circle: LAMP1. N Compared to GFP-control, C4-OE did not alter the overlapping volume between GluR1 and LAMP1. Green circle: GluR1, Magenta circle: LAMP1. D , E , K , L N = 8 dendrites, 3 animals for Con and C4-OE. F , G , M , N N = 7 dendrites, 3 animals, Con; and N = 8 dendrites, 3 animals, C4-OE. D – G , K – N t- test. * p < 0.05, ** p < 0.01, *** p < 0.001. All graphs, Mean ± SEM.

Article Snippet: For GluR1 expression in HEK293T cells, we used Gria1 (NM_008165 ) under the CMV promoter (pCMV6-Gria1, OriGene Technologies plasmid #MC203472).

Techniques: Over Expression, Control

Journal: Molecular Psychiatry

Article Title: The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking

doi: 10.1038/s41380-024-02701-7

Figure Lengend Snippet: A Left: Schematic of dissected GFP-positive tissue from IUE animals, used for isolating cytosolic and synaptosome fractions. Right: Western blot (WB) showing levels of GluR1 with C4 overexpression. GluR1 levels were detected in synaptosome and cytosolic fractions in control and C4-OE conditions. Since C4 was expressed at relatively low levels, it could only be detected when several brains were pooled. Therefore, each lane corresponds to pooled brain lysate from a single litter (6 mice). Protein molecular weights, kilodaltons (kDa, left column). “+” indicates the presence of C4 (top row). Numbers in the bottom row indicate fold change in GluR1 levels relative to control synaptosome and cytosolic fraction. B Left: Schematic of degradation assay in HEK293T cells. Right: WB quantified protein levels of GluR1 in GS and GSC cells with CHX and CHX + MG132 treatment. Protein molecular weights, kilodaltons (kDa, left column). “+” indicates the presence of C4, CHX or MG132. C Left: CHX treatment led to decreased GluR1 levels in GS cells, which was rescued upon application of MG132. Right: GluR1 levels in GSC cells were unaffected by CHX or CHX + MG132 treatments. Light gray circles: DMSO treated cells, Dark gray triangles: CHX treated cells, Dark gray squares: CHX + MG132 treated cells. N = 3 sample replicates per condition. One-way ANOVA. * p < 0.05, ** p < 0.01. All graphs, Mean ± SEM.

Article Snippet: For GluR1 expression in HEK293T cells, we used Gria1 (NM_008165 ) under the CMV promoter (pCMV6-Gria1, OriGene Technologies plasmid #MC203472).

Techniques: Western Blot, Over Expression, Control, Degradation Assay

Journal: Advanced Science

Article Title: Allele‐Specific Regulation of PAXIP1‐AS1 by SMC3/CEBPB at rs112651172 in Psychiatric Disorders Drives Synaptic and Behavioral Dysfunctions in Mice

doi: 10.1002/advs.202508259

Figure Lengend Snippet: Expression of AMPA and GABA(A) receptor subunits and dendritic spine analysis in the PFC of mice. A,B) qPCR validation of AMPA receptor subunit from Scr (n = 6), OE (n = 6) and RE (n = 7) groups (A) and GABA(A) receptor subunit from Scr (n = 6), OE (n = 4) and RE (n = 5) groups (B) expressions in the PFC of mice. C,D) Western blot validation of GluR1 (C) and Gabrb2 (D) expression in the PFC of mice. E) Representative images of dendritic spine analysis in cortical layer 5 neurons of Scr (n = 3), OE (n = 3), and RE (n = 3) mice (left), showing no significant changes in spine density or morphology among the three groups (right). Data are presented as mean ± SD obtained from at least three mice per group. p values were calculated using an unpaired two‐tailed Student's t ‐test for the indicated comparisons, with p < 0.05 considered statistically significant (ns, not significant).

Article Snippet: The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies: CNTNAP3 ( Ab252413 , Abcam), ZGPAT (SC‐515524, Santa Cruz Biotechnology), GluR1 (67642‐1‐Ig, Proteintech), GABRB2 (H681506024, HUABio), and GAPDH (60004‐1‐Ig, Proteintech).

Techniques: Expressing, Biomarker Discovery, Western Blot, Two Tailed Test