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  • 85
    Thermo Fisher rabbit anti rim1 polyclonal antibody
    Rabbit Anti Rim1 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore rim1 mvenus
    <t>RIM1-mEos3.1</t> PALM identifies NCs a. Neurons coexpressing <t>RIM1-mVenus</t> (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p
    Rim1 Mvenus, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rim1 2
    Distribution of synaptic proteins in postsynaptic density (PSD) and AZs. (A) Representative images of freeze-fracture replicas of PF-PC synapses labeled for GluA1–3, GluD2, <t>RIM1/2</t> and Ca V 2.1 (5-nm gold) with PF-PC markers (GluD2 for GluA1–3, VGluT1 for RIM1/2 and Ca V 2.1, 15-nm gold). PSD on the exoplasmic face (E-face), AZs on the protoplasmic face (P-face) and cross-fractured cytoplasm were indicated with red, blue and yellow, respectively. Scale bar = 200 nm. (B) Summary of gold particle density for GluA1–3, GluD2, RIM1/2, and Ca V 2.1 on PSD or AZs. Each scatter indicates the mean value obtained from an individual replica. Numerals in the plot indicate the numbers of analyzed replicas for each group. Asterisks indicate significant differences (* P
    Rim1 2, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rim 1 2 antibody
    Distribution of synaptic proteins in postsynaptic density (PSD) and AZs. (A) Representative images of freeze-fracture replicas of PF-PC synapses labeled for GluA1–3, GluD2, <t>RIM1/2</t> and Ca V 2.1 (5-nm gold) with PF-PC markers (GluD2 for GluA1–3, VGluT1 for RIM1/2 and Ca V 2.1, 15-nm gold). PSD on the exoplasmic face (E-face), AZs on the protoplasmic face (P-face) and cross-fractured cytoplasm were indicated with red, blue and yellow, respectively. Scale bar = 200 nm. (B) Summary of gold particle density for GluA1–3, GluD2, RIM1/2, and Ca V 2.1 on PSD or AZs. Each scatter indicates the mean value obtained from an individual replica. Numerals in the plot indicate the numbers of analyzed replicas for each group. Asterisks indicate significant differences (* P
    Rim 1 2 Antibody, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Becton Dickinson rim1
    Transcription is required for functional recovery from depolarization-induced muting. A. Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+act D) or without (recovered) 200 ng/ml actinomycin D. Actinomycin D was applied 0.5 h prior to and during recovery. B. Summary of EPSC amplitudes from neurons treated as in panel A ( n = 32 neurons). C. Quantification of <t>Rim1</t> immunostaining at vGluT-1-positive synapses (not shown; n = 15 fields). Integrated intensity values were normalized to the average control value for a given experiment. *p
    Rim1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rim1
    Mild impairment of sound encoding in <t>RIM1/2</t> cDKO and RIM2α SKO mice. ( A ) ABR waveforms of RIM1/2 cDKO mice (gray, n = 8) and RIM1/2 con (black, n = 12) in response to 80 dB (pe) click stimulation were generally preserved, but in RIM1/2 cDKO, the
    Rim1, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 89/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Syntaxin rim1
    Effect of the binding of CAST and <t>RIM1</t> on synaptic transmission. (A) Effects of the COOH-terminal regions of CAST on synaptic transmission. (a) Sequences of the CAST peptides. RID, RIM1-interacting domain; scb RID, scrambled RID. (b) Effects of the peptides (5 μM each) on the binding of HA-RIM1 to immobilized GST-CAST-4. The binding was inhibited by RID, but not by RIDΔIWA or scb RID. (c and d) Effects of the CAST peptides (1 mM each in the injection pipette) on synaptic transmission. Presynaptic neurons were stimulated every 20 s CAST peptides were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in c. Normalized and averaged EPSP amplitudes are plotted from five experiments with RID, RIDΔIWA, or scb RID peptide in d. (e) Effects of the COOH-terminal regions of CAST (5 μM each) on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST-CASTCΔIWA or GST-CASTC, followed by Western blotting using the anti-Myc and anti-HA Abs. GST-CASTC inhibited the binding but GST-CASTCΔIWA did not. (f and g) Effects of the recombinant CAST proteins (150 μM each in the injection) on synaptic transmission. Presynaptic neurons were stimulated every 20 s. Recombinant CAST proteins were introduced into presynaptic neurons at t = 0. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-CASTC or GST-CASTCΔIWA in f. (B) Effect of the PDZ domain of RIM1 on synaptic transmission. (a) Effect of the GST fusion protein containing the PDZ domain on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST alone or GST-RIM1 PDZ (5 μM each), followed by Western blotting using the anti-Myc and anti-HA Abs. GST-RIM1 PDZ inhibited the binding but GST alone did not. (b and c) Effect of GST-RIM1 PDZ on synaptic transmission. Presynaptic neurons were stimulated every 20 s GST alone or GST-RIM1 PDZ (170 μM each) were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in b. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-RIM1 PDZ or GST alone in c.
    Rim1, supplied by Syntaxin, used in various techniques. Bioz Stars score: 89/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems anti rim1 2
    Transsynaptic nanoscale alignment of AZ and PSD proteins a , Distributions of synaptic <t>RIM1/2</t> and PSD-95 pair as the original localizations (left) and with NCs highlighted (right), scale 200 nm. Filled arrows indicate aligned NCs, open arrows non-aligned NCs. b , Paired correlation function (PCF) of measured RIM1/2 and PSD-95 compared to PCF with either distribution randomized. c , PCF of simulated distributions with (cyan) and without (orange) shuffling NC positions. d , Cumulative distributions of cross-correlation index (n = 143 synapses). e , RIM1/2 protein enrichment as a function of distance from translated PSD-95 NC centers (top, filled points) and PSD-95 enrichment relative to RIM1/2 NCs (bottom, open points). Simulations with same randomizations as in d – e were performed for each synapse. f , Protein density profile for enriched vs non-enriched NCs, n = 119 PSD-95 NCs, 90 RIM1/2 NCs. g , Enrichment indices for RIM1/2, Munc13, and Bassoon relative to PSD-95 NCs (filled) and for the opposite direction (open), n > 260 NCs, *p
    Anti Rim1 2, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rabbit anti rim1
    Multi‐color imaging with X10 reveals synaptic ultrastructure in cell culture Three‐color imaging resolves synaptic vesicle clusters (identified by Synaptophysin), along with pre‐synaptic active zones (identified by Bassoon) and post‐synaptic densities (identified by Homer 1). The panel at the top right gives a schematic overview of the organization of a synapse, for orientation (colors as in the fluorescence images). The two panels on the bottom right provide a stereo view of the synapses. Expansion factor: 11.0×. Scale bars: 500 nm (both). Upper panels: higher magnification images show the alignment of pre‐synaptic active zones and post‐synaptic densities, as well as the distance between them, in side view. Expansion factor: 11.0×. Scale bar: 200 nm. Lower panels: a z ‐stack through an additional synapse, in face view. Expansion factor: 11.0×. Scale bar: 200 nm. Representative images of an immunostaining for pre‐synaptic <t>RIM1/2</t> and post‐synaptic PSD95, two markers known to be more closely associated than Bassoon/Homer 1 24 . Arrowheads indicate nanocolumns of aligned pre‐ and post‐synaptic proteins. Expansion factor: 10.4×. Scale bars: 500 nm (upper panel), 200 nm (lower panels). Line scans through Bassoon staining (green) in pre‐synaptic active zones and through Homer 1 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Line scans through RIM1/2 staining (green) in pre‐synaptic active zones and through PSD95 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Histogram showing the distribution of Bassoon to Homer 1 distances and RIM1/2 to PSD95 distances ( n = 15 neuronal areas, with the corresponding synapses, for Bassoon and Homer 1, n = 74 neuronal areas for RIM1/2 and PSD95).
    Rabbit Anti Rim1, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti rim1 2
    Multi‐color imaging with X10 reveals synaptic ultrastructure in cell culture Three‐color imaging resolves synaptic vesicle clusters (identified by Synaptophysin), along with pre‐synaptic active zones (identified by Bassoon) and post‐synaptic densities (identified by Homer 1). The panel at the top right gives a schematic overview of the organization of a synapse, for orientation (colors as in the fluorescence images). The two panels on the bottom right provide a stereo view of the synapses. Expansion factor: 11.0×. Scale bars: 500 nm (both). Upper panels: higher magnification images show the alignment of pre‐synaptic active zones and post‐synaptic densities, as well as the distance between them, in side view. Expansion factor: 11.0×. Scale bar: 200 nm. Lower panels: a z ‐stack through an additional synapse, in face view. Expansion factor: 11.0×. Scale bar: 200 nm. Representative images of an immunostaining for pre‐synaptic <t>RIM1/2</t> and post‐synaptic PSD95, two markers known to be more closely associated than Bassoon/Homer 1 24 . Arrowheads indicate nanocolumns of aligned pre‐ and post‐synaptic proteins. Expansion factor: 10.4×. Scale bars: 500 nm (upper panel), 200 nm (lower panels). Line scans through Bassoon staining (green) in pre‐synaptic active zones and through Homer 1 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Line scans through RIM1/2 staining (green) in pre‐synaptic active zones and through PSD95 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Histogram showing the distribution of Bassoon to Homer 1 distances and RIM1/2 to PSD95 distances ( n = 15 neuronal areas, with the corresponding synapses, for Bassoon and Homer 1, n = 74 neuronal areas for RIM1/2 and PSD95).
    Mouse Anti Rim1 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rim1 207 366
    Multi‐color imaging with X10 reveals synaptic ultrastructure in cell culture Three‐color imaging resolves synaptic vesicle clusters (identified by Synaptophysin), along with pre‐synaptic active zones (identified by Bassoon) and post‐synaptic densities (identified by Homer 1). The panel at the top right gives a schematic overview of the organization of a synapse, for orientation (colors as in the fluorescence images). The two panels on the bottom right provide a stereo view of the synapses. Expansion factor: 11.0×. Scale bars: 500 nm (both). Upper panels: higher magnification images show the alignment of pre‐synaptic active zones and post‐synaptic densities, as well as the distance between them, in side view. Expansion factor: 11.0×. Scale bar: 200 nm. Lower panels: a z ‐stack through an additional synapse, in face view. Expansion factor: 11.0×. Scale bar: 200 nm. Representative images of an immunostaining for pre‐synaptic <t>RIM1/2</t> and post‐synaptic PSD95, two markers known to be more closely associated than Bassoon/Homer 1 24 . Arrowheads indicate nanocolumns of aligned pre‐ and post‐synaptic proteins. Expansion factor: 10.4×. Scale bars: 500 nm (upper panel), 200 nm (lower panels). Line scans through Bassoon staining (green) in pre‐synaptic active zones and through Homer 1 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Line scans through RIM1/2 staining (green) in pre‐synaptic active zones and through PSD95 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Histogram showing the distribution of Bassoon to Homer 1 distances and RIM1/2 to PSD95 distances ( n = 15 neuronal areas, with the corresponding synapses, for Bassoon and Homer 1, n = 74 neuronal areas for RIM1/2 and PSD95).
    Rim1 207 366, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson rim1 monoclonal antibody
    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, <t>Rim1,</t> and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.
    Rim1 Monoclonal Antibody, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Synaptic Systems rabbit anti rim 1 2
    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, <t>Rim1,</t> and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.
    Rabbit Anti Rim 1 2, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rabbit polyclonal anti rim1
    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, <t>Rim1,</t> and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.
    Rabbit Polyclonal Anti Rim1, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti rim1 2 n 20
    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, <t>Rim1,</t> and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.
    Anti Rim1 2 N 20, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    P212121 seleno l methionine labeled rim1
    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, <t>Rim1,</t> and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.
    Seleno L Methionine Labeled Rim1, supplied by P212121, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    seleno l methionine labeled rim1 - by Bioz Stars, 2020-08
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    88
    Becton Dickinson anti rim1 mouse monoclonal
    Conditional knock-out of RIM2 in rod photoreceptors is under way as early as 3 weeks of age. A , Forms of <t>RIM1</t> and RIM2 found in the brain and retina of wild-type and transgenic RIM1ff/2ff mice. Expression of RIM 1α ≫ 1β in the retina, which is similar to the proportions in whole-brain lysates (probed with Ms monoclonal anti-RIM1). The floxed RIM2ff ) and therefore all of the RIM2 in the transgenic animal is RIM2-cfp, which is revealed as an upward shift in its migration compared with brain lysates from wild-type animals (the cfp tag is nonfluorescent in the retina). Relative RIM2 expression levels in the brain are RIM 2α > 2β ∼ 2α-splice variants (α-sp.) and, in the retina, 2α and 2α-sp are equally expressed, but 2β is not detected. The regional distribution of RIM1/2 was examined by immunostaining cryosections of retina ∼10 μm thick ( B – F ). B , Expression of RIM1 is largely confined to the IPL and this is demonstrated with a rabbit polyclonal anti-RIM1 from Synaptic Systems (left) and a mouse monoclonal against RIM1 from BD Transduction Laboratories (right). Scale bar, 25 μm. C , D ), demarcating the OPL. The section in D is from a LMOPcre + transgenic animal, which shows an intensely stained patch of Cre in the ONL that is designated as a Cre region (samples in C and D are from RIM1ff/2ff littermates, 3 weeks of age). Bottom overview cropped to the OPL is labeled “all probes” because the signals are derived from anti-RIM2 staining (rabbit polyclonal, Alexa Fluor488, shown in green), the cone marker PNA (linked to Alexa Fluor 568, shown in red), and anti-CtBP2 (Ms monoclonal, Alexa Fluor 648, shown in blue). E , F , Regions selected for presentation at higher resolution. The control condition presented in C and E and the “Cre-poor region” in F show that the RIM2 signal overlaps extensively with CtBP2/Ribeye (aqua). The “Cre-rich region” in F reveals a drop in RIM2 staining. G , H ) and Synaptotagmin1 (Syt1, ∼60 kDa) were used as presynaptic markers, whereas CtBP2 (∼45 kDa) in the context of Western analysis represents the transcription factor that is expressed throughout the retina. I , Graphical summary of the densitometry measurements from Western analysis. Only RIM2 was reduced (see text for values) and no apparent change in the intensity of the synaptic markers was witnessed at 3 weeks. Data are presented as mean ± SE.
    Anti Rim1 Mouse Monoclonal, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rim1 mouse monoclonal/product/Becton Dickinson
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rim1 mouse monoclonal - by Bioz Stars, 2020-08
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    Image Search Results


    RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Article Snippet: Neurons transfected with only RIM1-mVenus were immunostained with chicken anti-GFP (1:200, Chemicon) labeled with secondary anti-chicken-Alexa-488, rabbit anti-RIM1/2 labeled with secondary anti-rabbit-Cy-3, and mouse anti-Bassoon labeled with secondary anti-mouse-Alexa-647 to assess expression levels.

    Techniques: Expressing, Immunofluorescence, Transfection, Over Expression

    Purification and characterization of recombinant Rim1 protein and its C-terminal truncation variant. ( A ) Multiple sequence alignment of eukaryotic mitochondrial SSBs and bacterial SSBs using the ClustalW2 program to determine the C-terminal tail region of Rim1. The sequences for H. sapiens mtSSB ( Hs mtSSB) (GenBank™ accession: NP_003134), Xenopus laevis mtSSB ( Xl mtSSB) (GenBank™ accession: NP_001095241), Bombyx mori mtSSB ( Bm mtSSB) (GenBank™ accession: ABF51293), D. melanogaster mtSSB ( Dm mtSSB) (GenBank™ accession: AAF16936), E. coli SSB ( Ec SSB) (GenBank™ accession: YP_859663), Thermotoga maritima ( Tm SSB) (GenBank™ accession: Q9WZ73), Deinococcus radiodurans SSB ( Dr SSB) (GenBank™ accession: Q9RY51) and S. cerevisiae Rim1 ( Sc Rim1) (GenBank™ accession: AAB22978) are used for the alignment. The sequence alignment determined that the first 100 amino acids from the N-terminal end of Rim1 are involved in formation of the OB-fold domain, and the remaining 18 amino acids from the C-terminal end form the putative unstructured tail region. The amino acid sequences involved in the formation of the C-terminal tails of SSB proteins are highlighted in gray. The C-terminal tail of Rim1 contains five acidic amino acids that are indicated in bold. ( B ) Coomassie blue stained 15% SDS–PAGE gel to visualize purified Rim1 (lane 2) and Rim1ΔC18 (lane 2). The purified proteins were > 95% homogenous as assessed from the gel. ( C ) SEC-MALS detection reveals that the Rim1 and Rim1ΔC18 exist as a tetramer. The theoretical MM of monomeric Rim1 and Rim1ΔC18 is 13.29 and 11.43 kDa, respectively. The observed MM and hydrodynamic radius ( R h ) for Rim1 and Rim1ΔC18 proteins are as indicated. ( D ) Rim1 binding affinity for ssDNA was evaluated by fluorescence anisotropy. The anisotropy values for Rim1 binding to 1 nM 3′F-T 20 (open diamonds) and 3′F-T 70 (closed diamonds) were plotted as average values from three experiments with a standard deviation. Rim1 binding data to 3′F-T 20 were fit to the Hill equation resulting in a Hill coefficient of 2.5 and an apparent K d of 3.1 ± 0.1 nM (tetramer). Rim1 binding to 3′F-T 70 is stoichiometric under the conditions used here ( K d value

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: Purification and characterization of recombinant Rim1 protein and its C-terminal truncation variant. ( A ) Multiple sequence alignment of eukaryotic mitochondrial SSBs and bacterial SSBs using the ClustalW2 program to determine the C-terminal tail region of Rim1. The sequences for H. sapiens mtSSB ( Hs mtSSB) (GenBank™ accession: NP_003134), Xenopus laevis mtSSB ( Xl mtSSB) (GenBank™ accession: NP_001095241), Bombyx mori mtSSB ( Bm mtSSB) (GenBank™ accession: ABF51293), D. melanogaster mtSSB ( Dm mtSSB) (GenBank™ accession: AAF16936), E. coli SSB ( Ec SSB) (GenBank™ accession: YP_859663), Thermotoga maritima ( Tm SSB) (GenBank™ accession: Q9WZ73), Deinococcus radiodurans SSB ( Dr SSB) (GenBank™ accession: Q9RY51) and S. cerevisiae Rim1 ( Sc Rim1) (GenBank™ accession: AAB22978) are used for the alignment. The sequence alignment determined that the first 100 amino acids from the N-terminal end of Rim1 are involved in formation of the OB-fold domain, and the remaining 18 amino acids from the C-terminal end form the putative unstructured tail region. The amino acid sequences involved in the formation of the C-terminal tails of SSB proteins are highlighted in gray. The C-terminal tail of Rim1 contains five acidic amino acids that are indicated in bold. ( B ) Coomassie blue stained 15% SDS–PAGE gel to visualize purified Rim1 (lane 2) and Rim1ΔC18 (lane 2). The purified proteins were > 95% homogenous as assessed from the gel. ( C ) SEC-MALS detection reveals that the Rim1 and Rim1ΔC18 exist as a tetramer. The theoretical MM of monomeric Rim1 and Rim1ΔC18 is 13.29 and 11.43 kDa, respectively. The observed MM and hydrodynamic radius ( R h ) for Rim1 and Rim1ΔC18 proteins are as indicated. ( D ) Rim1 binding affinity for ssDNA was evaluated by fluorescence anisotropy. The anisotropy values for Rim1 binding to 1 nM 3′F-T 20 (open diamonds) and 3′F-T 70 (closed diamonds) were plotted as average values from three experiments with a standard deviation. Rim1 binding data to 3′F-T 20 were fit to the Hill equation resulting in a Hill coefficient of 2.5 and an apparent K d of 3.1 ± 0.1 nM (tetramer). Rim1 binding to 3′F-T 70 is stoichiometric under the conditions used here ( K d value

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Purification, Recombinant, Variant Assay, Sequencing, Staining, SDS Page, Size-exclusion Chromatography, Binding Assay, Fluorescence, Standard Deviation

    The N-terminal domain of Pif1 is essential for Rim1 mediated stimulation of helicase activity. ( A ) Schematic diagram of the Pif1 variants used: the N-terminal deletion mutant (Pif1ΔN) and the C-terminal deletion mutant (Pif1ΔC). ( B ) Results of Pif1ΔN-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1. The fraction of ssDNA formed over time for Pif1ΔN (closed squares) and Pif1ΔN+Rim1 (open diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1ΔN and Pif1ΔN+Rim1 were 0.52 ± 0.03 and 0.46 ± 0.03 per min, respectively. ( C ) Binding affinity of Pif1ΔN with FAM-Rim1. Fluorescence anisotropy of FAM-Rim1 was plotted as a function of increasing concentrations of Pif1ΔN. Data were fit to the equation for a hyperbola to obtain a K d value of 1.6 ± 0.2 µM.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: The N-terminal domain of Pif1 is essential for Rim1 mediated stimulation of helicase activity. ( A ) Schematic diagram of the Pif1 variants used: the N-terminal deletion mutant (Pif1ΔN) and the C-terminal deletion mutant (Pif1ΔC). ( B ) Results of Pif1ΔN-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1. The fraction of ssDNA formed over time for Pif1ΔN (closed squares) and Pif1ΔN+Rim1 (open diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1ΔN and Pif1ΔN+Rim1 were 0.52 ± 0.03 and 0.46 ± 0.03 per min, respectively. ( C ) Binding affinity of Pif1ΔN with FAM-Rim1. Fluorescence anisotropy of FAM-Rim1 was plotted as a function of increasing concentrations of Pif1ΔN. Data were fit to the equation for a hyperbola to obtain a K d value of 1.6 ± 0.2 µM.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Activity Assay, Mutagenesis, Binding Assay, Fluorescence

    Effect of heterologous SSBs on Pif1-catalyzed DNA helicase activity. ( A ) The fraction of ssDNA product formed under multiple turnover conditions with the 70T30bp substrate for Hs mtSSB+Pif1 (triangles), and gp32+Pif1 (diamonds) was plotted as the average value of three independent experiments along with Pif1 alone (circles) and Rim1+Pif1 (squares) which is replotted for comparison from Figure 5 B. The data were fit to a single exponential resulting in observed rate constants for product formation of 0.44 ± 0.04, 1.84 ± 0.09, 1.1 ± 0.1 and 0.58 ± 0.02 per min for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1, respectively. ( B ) Results of DNA strand separation experiments conducted with the 20T30bp substrate. The fraction of ssDNA formed over time for Pif1 alone (circles), Rim1+Pif1 (squares), Hs mtSSB+Pif1 (triangles) and gp32+Pif1 (diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01, 0.26 ± 0.02 and 0.25 ± 0.01 per min, respectively.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: Effect of heterologous SSBs on Pif1-catalyzed DNA helicase activity. ( A ) The fraction of ssDNA product formed under multiple turnover conditions with the 70T30bp substrate for Hs mtSSB+Pif1 (triangles), and gp32+Pif1 (diamonds) was plotted as the average value of three independent experiments along with Pif1 alone (circles) and Rim1+Pif1 (squares) which is replotted for comparison from Figure 5 B. The data were fit to a single exponential resulting in observed rate constants for product formation of 0.44 ± 0.04, 1.84 ± 0.09, 1.1 ± 0.1 and 0.58 ± 0.02 per min for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1, respectively. ( B ) Results of DNA strand separation experiments conducted with the 20T30bp substrate. The fraction of ssDNA formed over time for Pif1 alone (circles), Rim1+Pif1 (squares), Hs mtSSB+Pif1 (triangles) and gp32+Pif1 (diamonds) was plotted as the average of at least three independent experiments. The observed rate constants for Pif1 alone, Rim1+Pif1, Hs mtSSB+Pif1 and gp32+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01, 0.26 ± 0.02 and 0.25 ± 0.01 per min, respectively.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Activity Assay

    Rim1 and Rim1ΔC18 stimulate Pif1 DNA helicase activity. ( A ) Pif1-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( B ) Formation of ssDNA product over time was quantified and plotted as the average of at least three independent reactions with a standard deviation for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (A). The data were fit to a single exponential resulting in observed rate constants of 0.44 ± 0.04, 1.8 ± 0.1 and 0.90 ± 0.02 per min for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1, respectively. ( C ) Pif1-catalyzed separation of a partial duplex DNA substrate, 20T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( D ) The fraction of ssDNA product formed over time for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (C) was quantified and plotted as the average of at least three independent reactions with a standard deviation. The observed rate constants for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01 and 0.25 ± 0.01 per min, respectively.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: Rim1 and Rim1ΔC18 stimulate Pif1 DNA helicase activity. ( A ) Pif1-catalyzed separation of a partial duplex DNA substrate, 70T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( B ) Formation of ssDNA product over time was quantified and plotted as the average of at least three independent reactions with a standard deviation for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (A). The data were fit to a single exponential resulting in observed rate constants of 0.44 ± 0.04, 1.8 ± 0.1 and 0.90 ± 0.02 per min for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1, respectively. ( C ) Pif1-catalyzed separation of a partial duplex DNA substrate, 20T30bp, under multiple turnover conditions in the presence or absence of Rim1 or Rim1ΔC18 protein. ( D ) The fraction of ssDNA product formed over time for Pif1 alone (circles), Rim1+Pif1 (squares) and Rim1ΔC18+Pif1 (triangles) from (C) was quantified and plotted as the average of at least three independent reactions with a standard deviation. The observed rate constants for Pif1 alone, Rim1+Pif1 and Rim1ΔC18+Pif1 were 0.30 ± 0.01, 0.31 ± 0.01 and 0.25 ± 0.01 per min, respectively.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Activity Assay, Standard Deviation

    Rim1 has no effect on the k cat value for ATP hydrolysis catalyzed by Pif1. ( A ) DNA stimulated ATPase activity of Pif1 (100 nM) in the presence or absence of Rim1 (100 nM) at increasing concentrations of poly(dT). The ATPase activity of Pif1 was plotted as the average value from three independent experiments and data were fit to a hyperbola to obtain kinetic constants k cat and K eff . The observed k cat value for Pif1 was 94.7 ± 4.9 per s and it did not change in the presence of Rim1 (96.7 ± 1.8 per s). The measured K eff value for Pif1 was 1.07 ± 0.2 µM and it increased by 3-fold in the presence of Rim1 (3.4 ± 0.2 µM). ( B ) DNA-stimulated Pif1 (20 nM) ATPase activity at saturating concentrations of poly(dT) (20 µM) was measured with increasing concentrations of Rim1. The average Pif1 ATPase activity from three independent experiments was plotted. Titration with Rim1 had no effect on Pif1 ATPase activity.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: Rim1 has no effect on the k cat value for ATP hydrolysis catalyzed by Pif1. ( A ) DNA stimulated ATPase activity of Pif1 (100 nM) in the presence or absence of Rim1 (100 nM) at increasing concentrations of poly(dT). The ATPase activity of Pif1 was plotted as the average value from three independent experiments and data were fit to a hyperbola to obtain kinetic constants k cat and K eff . The observed k cat value for Pif1 was 94.7 ± 4.9 per s and it did not change in the presence of Rim1 (96.7 ± 1.8 per s). The measured K eff value for Pif1 was 1.07 ± 0.2 µM and it increased by 3-fold in the presence of Rim1 (3.4 ± 0.2 µM). ( B ) DNA-stimulated Pif1 (20 nM) ATPase activity at saturating concentrations of poly(dT) (20 µM) was measured with increasing concentrations of Rim1. The average Pif1 ATPase activity from three independent experiments was plotted. Titration with Rim1 had no effect on Pif1 ATPase activity.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Activity Assay, Titration

    The OB-fold domain and C-terminal tail of Rim1 form two independent Pif1 interaction sites. ( A ) Schematic diagram of the procedure used to measure the binding affinity between Pif1 and SSB protein. Rim1 or Rim1ΔC18 was labeled with the amine reactive fluorescein dye 5-FAM SE as described in ‘Materials and Methods’ section. The labeled proteins were used in binding assays to measure the change in fluorescence anisotropy as a function of protein binding. ( B ) FAM-labeled Rim1 protein binds to unlabeled Rim1 as indicated by increasing anisotropy; however, it did not bind to Hs mtSSB. FAM-labeled Rim1ΔC18 also did not bind to Hs mtSSB. ( C ) FAM-labeled Rim1 or FAM-labeled Rim1ΔC18 was titrated with Pif1 protein. The average anisotropy values from at least three independent experiments with a standard deviation were plotted using KaleidaGraph and fit to the equation for a hyperbola to obtain dissociation constants ( K d ) of 0.69 ± 0.03 and 2.5 ± 0.6 µM for Pif1 interaction with FAM-Rim1 and FAM-Rim1ΔC18, respectively.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: The OB-fold domain and C-terminal tail of Rim1 form two independent Pif1 interaction sites. ( A ) Schematic diagram of the procedure used to measure the binding affinity between Pif1 and SSB protein. Rim1 or Rim1ΔC18 was labeled with the amine reactive fluorescein dye 5-FAM SE as described in ‘Materials and Methods’ section. The labeled proteins were used in binding assays to measure the change in fluorescence anisotropy as a function of protein binding. ( B ) FAM-labeled Rim1 protein binds to unlabeled Rim1 as indicated by increasing anisotropy; however, it did not bind to Hs mtSSB. FAM-labeled Rim1ΔC18 also did not bind to Hs mtSSB. ( C ) FAM-labeled Rim1 or FAM-labeled Rim1ΔC18 was titrated with Pif1 protein. The average anisotropy values from at least three independent experiments with a standard deviation were plotted using KaleidaGraph and fit to the equation for a hyperbola to obtain dissociation constants ( K d ) of 0.69 ± 0.03 and 2.5 ± 0.6 µM for Pif1 interaction with FAM-Rim1 and FAM-Rim1ΔC18, respectively.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Binding Assay, Labeling, Fluorescence, Protein Binding, Standard Deviation

    Identification of specific protein complexes of Pif1 in S. cerevisiae using I-DIRT. ( A ) Schematic representation of the I-DIRT procedure. Pif1-TAP tag strains were cultured in a light isotopic media, whereas the parent strains were cultured in heavy isotopic media containing d4-lysine. Equal quantities of cell lysates were mixed and subjected to affinity capture for the Pif1-TAP protein complex followed by SDS–PAGE and MS analysis. ( B ) Immunoisolated Pif1-TAP and its associated proteins were resolved by SDS–PAGE on a 4–20% NuPAGE gel and visualized by Coomassie blue staining. ( C ) Representative mass spectra of peptides from the Pif1 I-DIRT experiment. A peptide from the Ssb2 protein containing a single lysine exhibits both isotopically light (1394.8 Da) and heavy (1398.8 Da) peptides. The Rim1 peptide has two lysines with the monoisotopic peak exhibiting only isotopically light peptides (1448.76 Da). As expected, the peptides of Pif1 protein exhibited only the monoisotopic peak for isotopically light peptides. ( D ) For each of the lysine containing peptides identified by MS, the peak area under the isotopically light peptides was compared with the peak area for the heavy peptides to obtain a ‘fraction light’. Twenty two proteins were identified as nonspecific interactors due to one or more peptides having a light to heavy ratio of ∼0.6. In contrast, the fraction of light to heavy peptides for Rim1 and Pif1 proteins was ∼1. All the identified proteins and their average ‘fraction light’ areas are listed in Supplementary Table S1 .

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: Identification of specific protein complexes of Pif1 in S. cerevisiae using I-DIRT. ( A ) Schematic representation of the I-DIRT procedure. Pif1-TAP tag strains were cultured in a light isotopic media, whereas the parent strains were cultured in heavy isotopic media containing d4-lysine. Equal quantities of cell lysates were mixed and subjected to affinity capture for the Pif1-TAP protein complex followed by SDS–PAGE and MS analysis. ( B ) Immunoisolated Pif1-TAP and its associated proteins were resolved by SDS–PAGE on a 4–20% NuPAGE gel and visualized by Coomassie blue staining. ( C ) Representative mass spectra of peptides from the Pif1 I-DIRT experiment. A peptide from the Ssb2 protein containing a single lysine exhibits both isotopically light (1394.8 Da) and heavy (1398.8 Da) peptides. The Rim1 peptide has two lysines with the monoisotopic peak exhibiting only isotopically light peptides (1448.76 Da). As expected, the peptides of Pif1 protein exhibited only the monoisotopic peak for isotopically light peptides. ( D ) For each of the lysine containing peptides identified by MS, the peak area under the isotopically light peptides was compared with the peak area for the heavy peptides to obtain a ‘fraction light’. Twenty two proteins were identified as nonspecific interactors due to one or more peptides having a light to heavy ratio of ∼0.6. In contrast, the fraction of light to heavy peptides for Rim1 and Pif1 proteins was ∼1. All the identified proteins and their average ‘fraction light’ areas are listed in Supplementary Table S1 .

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: Cell Culture, SDS Page, Mass Spectrometry, Staining

    In vitro co-precipitation experiments reveal a direct interaction between Rim1 and Pif1 proteins and two possible sites of interactions on Rim1. ( A ) Ammonium sulfate co-precipitation of Pif1 with Rim1 or Rim1ΔC18. The presence of Pif1, Rim1, Rim1ΔC18 and 270 g/l ammonium sulfate in the reaction are indicated by plus symbols. Both pellet and supernatant fractions were analyzed on a 15% SDS–PAGE gel. Rim1 alone (lane 3) or Rim1ΔC18 alone (lane 7) precipitate very little in the presence of ammonium sulfate; however, they co-precipitate completely with Pif1 under the same conditions (lane 5 and 9, respectively). ( B ) Co-precipitation of Pif1 protein with SSB-coated Dynabeads was performed as described in ‘Materials and Methods’ section. Purified Rim1, Rim1ΔC18 or Hs mtSSB protein was coated onto epoxy activated Dynabeads. As a negative control, Dynabeads were coated with glycine or BSA. SSB coated Dynabeads were incubated with equal amounts of purified Pif1. Dynabeads were captured with a magnet, washed and proteins were eluted using SDS–PAGE loading buffer followed by separation on a 4–20% resolving gel. Pif1 did not co-precipitate with glycine-coated (lane 2) or BSA-coated (lane 3) Dynabeads. Pif1 co-precipitated with Rim1-coated beads (lane 4) and its association was not affected in the presence of DNase I (lane 5). Pif1 was also observed to co-precipitate with Hs mtSSB-coated (lane 6) and Rim1ΔC18-coated Dynabeads (lane 7). ( C ) A semi-quantitative measurement of relative Pif1 protein association with different SSB-coated Dynabeads from (B). Pif1 protein co-precipitated with each SSB-coated Dynabead was quantified using ImageQuant software and normalized to the amount of SSB protein on the gel. Pif1 association with Rim1-coated beads was taken as 1 and the relative amount of Pif1 co-precipitated with Hs mtSSB or Rim1ΔC18-coated beads was 0.63 and 0.45, respectively.

    Journal: Nucleic Acids Research

    Article Title: Physical and functional interaction between yeast Pif1 helicase and Rim1 single-stranded DNA binding protein

    doi: 10.1093/nar/gks1088

    Figure Lengend Snippet: In vitro co-precipitation experiments reveal a direct interaction between Rim1 and Pif1 proteins and two possible sites of interactions on Rim1. ( A ) Ammonium sulfate co-precipitation of Pif1 with Rim1 or Rim1ΔC18. The presence of Pif1, Rim1, Rim1ΔC18 and 270 g/l ammonium sulfate in the reaction are indicated by plus symbols. Both pellet and supernatant fractions were analyzed on a 15% SDS–PAGE gel. Rim1 alone (lane 3) or Rim1ΔC18 alone (lane 7) precipitate very little in the presence of ammonium sulfate; however, they co-precipitate completely with Pif1 under the same conditions (lane 5 and 9, respectively). ( B ) Co-precipitation of Pif1 protein with SSB-coated Dynabeads was performed as described in ‘Materials and Methods’ section. Purified Rim1, Rim1ΔC18 or Hs mtSSB protein was coated onto epoxy activated Dynabeads. As a negative control, Dynabeads were coated with glycine or BSA. SSB coated Dynabeads were incubated with equal amounts of purified Pif1. Dynabeads were captured with a magnet, washed and proteins were eluted using SDS–PAGE loading buffer followed by separation on a 4–20% resolving gel. Pif1 did not co-precipitate with glycine-coated (lane 2) or BSA-coated (lane 3) Dynabeads. Pif1 co-precipitated with Rim1-coated beads (lane 4) and its association was not affected in the presence of DNase I (lane 5). Pif1 was also observed to co-precipitate with Hs mtSSB-coated (lane 6) and Rim1ΔC18-coated Dynabeads (lane 7). ( C ) A semi-quantitative measurement of relative Pif1 protein association with different SSB-coated Dynabeads from (B). Pif1 protein co-precipitated with each SSB-coated Dynabead was quantified using ImageQuant software and normalized to the amount of SSB protein on the gel. Pif1 association with Rim1-coated beads was taken as 1 and the relative amount of Pif1 co-precipitated with Hs mtSSB or Rim1ΔC18-coated beads was 0.63 and 0.45, respectively.

    Article Snippet: The fractions containing pure Rim1 protein were pooled and concentrated using centrifugal filter units (Millipore) and stored at −80°C in storage buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM BME, 0.1 mM EDTA and 30% glycerol).

    Techniques: In Vitro, SDS Page, Purification, Negative Control, Incubation, Software

    Syntaxin1, RIM1, SynaptotagminI, and mGluR7 are not SUMO1-conjugated in vivo. ( A ) SDS-PAGE (4–12%) followed by Western blot analysis using anti-RIM1 antibody of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of RIM1 in both WT and KI eluates indicates non-specific binding of RIM1 to the affinity matrix ( B ) Representative SDS-PAGE (8%) followed by Western blot analysis of input and eluate fractions of anti-RIM1 and anti-IgG immunopurifications in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-RIM1 confirms the enrichment of RIM1 in both WT and KI samples solely when anti-RIM1 antibody is used (upper panel). However, no SUMO1-RIM1 band is apparent (lower panel). ( C ) SDS-PAGE (4–12%) followed by anti-syntaxin1α Western blot analysis of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of syntaxin1a in both WT and KI eluates indicates non-specific binding of syntaxin1a to the affinity matrix ( D ) Representative SDS-PAGE (12%) followed by Western blot analysis of input and eluate fractions of anti-syntaxin1α and anti-IgG immunopurifications in the presence or absence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-syntaxin1a confirms the enrichment of syntaxin1a in both WT and KI samples solely when anti-syntaxin1a antibody is used (upper panel). However, no SUMO1-syntaxin1a band is apparent (lower panel). ( E ) SDS-PAGE (4–12%) followed by anti-synaptotagmin1 and mGluR7 Western blot of input and anti-HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of synaptotagmin1 in both WT and KI eluates indicates non-specific binding of synaptotagmin1 to the affinity matrix, while mGluR7 is not enriched in either case. Images are representatives of at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.26338.008

    Journal: eLife

    Article Title: Analysis of SUMO1-conjugation at synapses

    doi: 10.7554/eLife.26338

    Figure Lengend Snippet: Syntaxin1, RIM1, SynaptotagminI, and mGluR7 are not SUMO1-conjugated in vivo. ( A ) SDS-PAGE (4–12%) followed by Western blot analysis using anti-RIM1 antibody of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of RIM1 in both WT and KI eluates indicates non-specific binding of RIM1 to the affinity matrix ( B ) Representative SDS-PAGE (8%) followed by Western blot analysis of input and eluate fractions of anti-RIM1 and anti-IgG immunopurifications in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-RIM1 confirms the enrichment of RIM1 in both WT and KI samples solely when anti-RIM1 antibody is used (upper panel). However, no SUMO1-RIM1 band is apparent (lower panel). ( C ) SDS-PAGE (4–12%) followed by anti-syntaxin1α Western blot analysis of input and HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of syntaxin1a in both WT and KI eluates indicates non-specific binding of syntaxin1a to the affinity matrix ( D ) Representative SDS-PAGE (12%) followed by Western blot analysis of input and eluate fractions of anti-syntaxin1α and anti-IgG immunopurifications in the presence or absence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. Western blot analysis using anti-syntaxin1a confirms the enrichment of syntaxin1a in both WT and KI samples solely when anti-syntaxin1a antibody is used (upper panel). However, no SUMO1-syntaxin1a band is apparent (lower panel). ( E ) SDS-PAGE (4–12%) followed by anti-synaptotagmin1 and mGluR7 Western blot of input and anti-HA peptide eluate fractions from anti-HA immunoprecipitation in the presence of 20 mM NEM from WT and His 6 -HA-SUMO1 KI brains. The presence of synaptotagmin1 in both WT and KI eluates indicates non-specific binding of synaptotagmin1 to the affinity matrix, while mGluR7 is not enriched in either case. Images are representatives of at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.26338.008

    Article Snippet: Antibodies Primary antibodies used in IPs, Western blotting (WB) and immunocytochemistry (ICC) were as follows: mouse anti-SUMO1 21C7 (Hybridoma Bank, Iowa, WB: 1/1000, ICC: 1/50, RRID: AB_2198257 ), mouse anti-HA (Biolegend, 901515, WB: 1/1000, ICC: 1/100, RRID: AB_2565334 ) mouse anti-synapsin1 (Synaptic Systems, 106021, WB: 1/1000, RRID: AB_2617072 ), mouse anti-syntaxin1a (Synaptic Systems, 110111, WB: 1/1000, RRID: AB_887848 ), mouse anti-synaptotagmin (Synaptic System, 105001, WB: 1/1000, RRID: AB_887831 ), mouse anti-gephyrin (Synaptic Systems, 147111, WB: 1/1000, RRID: AB_887719 ), rabbit anti-RIM1 (Synapsin Systems, 140003, WB: 1/1000, RRID: AB_887774 ), rabbit anti-GluK2 (Millipore, 04–921, WB: 1/1000, RRID: AB_1587072 ), rabbit anti-mGlur7 (Upstate, 07–239, WB: 1/1000, RRID: AB_310459 ), mouse anti-GluN1 (Synaptic Systems, 114011, WB: 1/1000, RRID: AB_887750 ), mouse anti-synaptophysin (Synaptic Systems, 101011, WB: 1/1000, RRID: AB_887824 ), rabbit anti-synapsin1/2 (Synaptic Systems, 106002, ICC: 1/2000, RRID: AB_887804 ), rabbit anti-shank2 (Synaptic Systems, 162202, ICC: 1/1000, RRID: AB_2619860 ), chicken anti-Map2 (Novus, NB300213, ICC: 1/1000, RRID: AB_2138178 ), mouse anti-actin (Sigma-Aldrich, clone AC-40, WB: 1/5000, RRID: AB_476730 ).

    Techniques: In Vivo, SDS Page, Western Blot, Immunoprecipitation, Binding Assay

    Distribution of synaptic proteins in postsynaptic density (PSD) and AZs. (A) Representative images of freeze-fracture replicas of PF-PC synapses labeled for GluA1–3, GluD2, RIM1/2 and Ca V 2.1 (5-nm gold) with PF-PC markers (GluD2 for GluA1–3, VGluT1 for RIM1/2 and Ca V 2.1, 15-nm gold). PSD on the exoplasmic face (E-face), AZs on the protoplasmic face (P-face) and cross-fractured cytoplasm were indicated with red, blue and yellow, respectively. Scale bar = 200 nm. (B) Summary of gold particle density for GluA1–3, GluD2, RIM1/2, and Ca V 2.1 on PSD or AZs. Each scatter indicates the mean value obtained from an individual replica. Numerals in the plot indicate the numbers of analyzed replicas for each group. Asterisks indicate significant differences (* P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Advantages of Acute Brain Slices Prepared at Physiological Temperature in the Characterization of Synaptic Functions

    doi: 10.3389/fncel.2020.00063

    Figure Lengend Snippet: Distribution of synaptic proteins in postsynaptic density (PSD) and AZs. (A) Representative images of freeze-fracture replicas of PF-PC synapses labeled for GluA1–3, GluD2, RIM1/2 and Ca V 2.1 (5-nm gold) with PF-PC markers (GluD2 for GluA1–3, VGluT1 for RIM1/2 and Ca V 2.1, 15-nm gold). PSD on the exoplasmic face (E-face), AZs on the protoplasmic face (P-face) and cross-fractured cytoplasm were indicated with red, blue and yellow, respectively. Scale bar = 200 nm. (B) Summary of gold particle density for GluA1–3, GluD2, RIM1/2, and Ca V 2.1 on PSD or AZs. Each scatter indicates the mean value obtained from an individual replica. Numerals in the plot indicate the numbers of analyzed replicas for each group. Asterisks indicate significant differences (* P

    Article Snippet: These reports indicate a possibility that the amount and distribution of membrane-associated proteins at pre- and postsynaptic sites are altered through the brain slice preparation at CT. To investigate the two-dimensional distribution of synaptic proteins contributing synaptic transmission in the AZ and postsynaptic area of PF-PC synapses, we performed SDS-digested freeze-fracture replica labeling (SDS-FRL) for AMPAR (GluA1–3), GluD2, RIM1/2, and CaV 2.1 ( ).

    Techniques: Labeling

    Transcription is required for functional recovery from depolarization-induced muting. A. Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+act D) or without (recovered) 200 ng/ml actinomycin D. Actinomycin D was applied 0.5 h prior to and during recovery. B. Summary of EPSC amplitudes from neurons treated as in panel A ( n = 32 neurons). C. Quantification of Rim1 immunostaining at vGluT-1-positive synapses (not shown; n = 15 fields). Integrated intensity values were normalized to the average control value for a given experiment. *p

    Journal: PLoS ONE

    Article Title: Differential Requirement for Protein Synthesis in Presynaptic Unmuting and Muting in Hippocampal Glutamate Terminals

    doi: 10.1371/journal.pone.0051930

    Figure Lengend Snippet: Transcription is required for functional recovery from depolarization-induced muting. A. Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+act D) or without (recovered) 200 ng/ml actinomycin D. Actinomycin D was applied 0.5 h prior to and during recovery. B. Summary of EPSC amplitudes from neurons treated as in panel A ( n = 32 neurons). C. Quantification of Rim1 immunostaining at vGluT-1-positive synapses (not shown; n = 15 fields). Integrated intensity values were normalized to the average control value for a given experiment. *p

    Article Snippet: Primary antibodies [Munc13–1 (Synaptic Systems) at 1∶1000, Rim1 (BD Biosciences) at 1∶1000] and horseradish peroxidase-conjugated secondary antibodies were diluted in 1% NFDM in TTBS.

    Techniques: Functional Assay, Activated Clotting Time Assay, Immunostaining

    PKA signaling is required for recovery of Rim1 levels after depolarization-induced muting. A. Western blot analysis of whole-cell lysates from hippocampal mass cultures treated with 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+KT5720) or without (recovered) 2 µM KT5720. KT5720 was applied 0.5 h prior to and during recovery. B. Summary of Rim1 levels from Western blots as shown in A ( n = 3). Rim1 protein levels for each condition were normalized to SV2 and control treatment. *p

    Journal: PLoS ONE

    Article Title: Differential Requirement for Protein Synthesis in Presynaptic Unmuting and Muting in Hippocampal Glutamate Terminals

    doi: 10.1371/journal.pone.0051930

    Figure Lengend Snippet: PKA signaling is required for recovery of Rim1 levels after depolarization-induced muting. A. Western blot analysis of whole-cell lysates from hippocampal mass cultures treated with 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+KT5720) or without (recovered) 2 µM KT5720. KT5720 was applied 0.5 h prior to and during recovery. B. Summary of Rim1 levels from Western blots as shown in A ( n = 3). Rim1 protein levels for each condition were normalized to SV2 and control treatment. *p

    Article Snippet: Primary antibodies [Munc13–1 (Synaptic Systems) at 1∶1000, Rim1 (BD Biosciences) at 1∶1000] and horseradish peroxidase-conjugated secondary antibodies were diluted in 1% NFDM in TTBS.

    Techniques: Western Blot

    Synthesis is required for protein recovery from depolarization-induced muting. A. Western blot analysis of whole-cell lysates from hippocampal mass cultures treated with 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+cyh) or without (recovered) 1 µg/ml cycloheximide. Cycloheximide was applied 0.5 h prior to and during recovery. B. Summary of Rim1 levels from Western blots as shown in A ( n = 3). Rim1 protein levels for each condition were normalized to SV2 and control treatment. C. Summary of Munc13–1 levels from Western blots as shown in A ( n = 3). Munc13–1 protein levels for each condition were normalized to SV2 and control treatment. D. Representative images of Rim1 immunostaining in mass cultures after treatments as described in panel A. Scale bar represents 2 µm. E. Quantification of Rim1 immunostaining at vGluT-1-positive synapses (not shown; n = 15 fields). Integrated intensity values were normalized to the average control value for a given experiment. *p

    Journal: PLoS ONE

    Article Title: Differential Requirement for Protein Synthesis in Presynaptic Unmuting and Muting in Hippocampal Glutamate Terminals

    doi: 10.1371/journal.pone.0051930

    Figure Lengend Snippet: Synthesis is required for protein recovery from depolarization-induced muting. A. Western blot analysis of whole-cell lysates from hippocampal mass cultures treated with 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+cyh) or without (recovered) 1 µg/ml cycloheximide. Cycloheximide was applied 0.5 h prior to and during recovery. B. Summary of Rim1 levels from Western blots as shown in A ( n = 3). Rim1 protein levels for each condition were normalized to SV2 and control treatment. C. Summary of Munc13–1 levels from Western blots as shown in A ( n = 3). Munc13–1 protein levels for each condition were normalized to SV2 and control treatment. D. Representative images of Rim1 immunostaining in mass cultures after treatments as described in panel A. Scale bar represents 2 µm. E. Quantification of Rim1 immunostaining at vGluT-1-positive synapses (not shown; n = 15 fields). Integrated intensity values were normalized to the average control value for a given experiment. *p

    Article Snippet: Primary antibodies [Munc13–1 (Synaptic Systems) at 1∶1000, Rim1 (BD Biosciences) at 1∶1000] and horseradish peroxidase-conjugated secondary antibodies were diluted in 1% NFDM in TTBS.

    Techniques: Western Blot, Immunostaining

    Depolarization decreases Rim1 protein levels, which are rescued by MG-132 or forskolin. A , Representative images of vGluT-1 and Rim1α/β immunostaining in control neurons and after depolarization. B , Representative Western blot image after

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: A Role for the Ubiquitin-Proteasome System in Activity-Dependent Presynaptic Silencing

    doi: 10.1523/JNEUROSCI.4965-09.2010

    Figure Lengend Snippet: Depolarization decreases Rim1 protein levels, which are rescued by MG-132 or forskolin. A , Representative images of vGluT-1 and Rim1α/β immunostaining in control neurons and after depolarization. B , Representative Western blot image after

    Article Snippet: Rim1 primary antibody (BD Biosciences, San Jose, CA) was used at 1:500.

    Techniques: Immunostaining, Western Blot

    Synaptic Munc13-1 and Rim1 levels correlate with presynaptic function. A , Representative images of FM1-43FX uptake and Munc13-1 immunostaining at glutamate synapses under control conditions and after depolarization. Note that Munc13-1 levels are lower

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: A Role for the Ubiquitin-Proteasome System in Activity-Dependent Presynaptic Silencing

    doi: 10.1523/JNEUROSCI.4965-09.2010

    Figure Lengend Snippet: Synaptic Munc13-1 and Rim1 levels correlate with presynaptic function. A , Representative images of FM1-43FX uptake and Munc13-1 immunostaining at glutamate synapses under control conditions and after depolarization. Note that Munc13-1 levels are lower

    Article Snippet: Rim1 primary antibody (BD Biosciences, San Jose, CA) was used at 1:500.

    Techniques: Immunostaining

    Mild impairment of sound encoding in RIM1/2 cDKO and RIM2α SKO mice. ( A ) ABR waveforms of RIM1/2 cDKO mice (gray, n = 8) and RIM1/2 con (black, n = 12) in response to 80 dB (pe) click stimulation were generally preserved, but in RIM1/2 cDKO, the

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

    Article Title: Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

    doi: 10.1073/pnas.1417207112

    Figure Lengend Snippet: Mild impairment of sound encoding in RIM1/2 cDKO and RIM2α SKO mice. ( A ) ABR waveforms of RIM1/2 cDKO mice (gray, n = 8) and RIM1/2 con (black, n = 12) in response to 80 dB (pe) click stimulation were generally preserved, but in RIM1/2 cDKO, the

    Article Snippet: Primary antibodies were mouse anti-CtBP2 (1:200, BD Biosciences), rabbit anti-GluA2/3 (1:200, Chemicon), rabbit anti-CaV 1.3 (1: 50, Alomone Labs), mouse anti-GluA2 (1:75 Chemicon), RIM2 (#140103 recognizing the PSD-95/discs large/ZO-1 (PDZ) domain, 1:200; Synaptic Systems), RIM1 (1:200; Synaptic Systems), RIM3 (1:100) , and Vglut3 (1:500; Synaptic Systems).

    Techniques: Mouse Assay

    Conditional knock-out of RIM2 in rod photoreceptors is under way as early as 3 weeks of age. A , Forms of RIM1 and RIM2 found in the brain and retina of wild-type and transgenic RIM1ff/2ff mice. Expression of RIM 1α ≫ 1β in the retina, which is similar to the proportions in whole-brain lysates (probed with Ms monoclonal anti-RIM1). The floxed RIM2ff ) and therefore all of the RIM2 in the transgenic animal is RIM2-cfp, which is revealed as an upward shift in its migration compared with brain lysates from wild-type animals (the cfp tag is nonfluorescent in the retina). Relative RIM2 expression levels in the brain are RIM 2α > 2β ∼ 2α-splice variants (α-sp.) and, in the retina, 2α and 2α-sp are equally expressed, but 2β is not detected. The regional distribution of RIM1/2 was examined by immunostaining cryosections of retina ∼10 μm thick ( B – F ). B , Expression of RIM1 is largely confined to the IPL and this is demonstrated with a rabbit polyclonal anti-RIM1 from Synaptic Systems (left) and a mouse monoclonal against RIM1 from BD Transduction Laboratories (right). Scale bar, 25 μm. C , D ), demarcating the OPL. The section in D is from a LMOPcre + transgenic animal, which shows an intensely stained patch of Cre in the ONL that is designated as a Cre region (samples in C and D are from RIM1ff/2ff littermates, 3 weeks of age). Bottom overview cropped to the OPL is labeled “all probes” because the signals are derived from anti-RIM2 staining (rabbit polyclonal, Alexa Fluor488, shown in green), the cone marker PNA (linked to Alexa Fluor 568, shown in red), and anti-CtBP2 (Ms monoclonal, Alexa Fluor 648, shown in blue). E , F , Regions selected for presentation at higher resolution. The control condition presented in C and E and the “Cre-poor region” in F show that the RIM2 signal overlaps extensively with CtBP2/Ribeye (aqua). The “Cre-rich region” in F reveals a drop in RIM2 staining. G , H ) and Synaptotagmin1 (Syt1, ∼60 kDa) were used as presynaptic markers, whereas CtBP2 (∼45 kDa) in the context of Western analysis represents the transcription factor that is expressed throughout the retina. I , Graphical summary of the densitometry measurements from Western analysis. Only RIM2 was reduced (see text for values) and no apparent change in the intensity of the synaptic markers was witnessed at 3 weeks. Data are presented as mean ± SE.

    Journal: The Journal of Neuroscience

    Article Title: RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors

    doi: 10.1523/JNEUROSCI.0658-15.2015

    Figure Lengend Snippet: Conditional knock-out of RIM2 in rod photoreceptors is under way as early as 3 weeks of age. A , Forms of RIM1 and RIM2 found in the brain and retina of wild-type and transgenic RIM1ff/2ff mice. Expression of RIM 1α ≫ 1β in the retina, which is similar to the proportions in whole-brain lysates (probed with Ms monoclonal anti-RIM1). The floxed RIM2ff ) and therefore all of the RIM2 in the transgenic animal is RIM2-cfp, which is revealed as an upward shift in its migration compared with brain lysates from wild-type animals (the cfp tag is nonfluorescent in the retina). Relative RIM2 expression levels in the brain are RIM 2α > 2β ∼ 2α-splice variants (α-sp.) and, in the retina, 2α and 2α-sp are equally expressed, but 2β is not detected. The regional distribution of RIM1/2 was examined by immunostaining cryosections of retina ∼10 μm thick ( B – F ). B , Expression of RIM1 is largely confined to the IPL and this is demonstrated with a rabbit polyclonal anti-RIM1 from Synaptic Systems (left) and a mouse monoclonal against RIM1 from BD Transduction Laboratories (right). Scale bar, 25 μm. C , D ), demarcating the OPL. The section in D is from a LMOPcre + transgenic animal, which shows an intensely stained patch of Cre in the ONL that is designated as a Cre region (samples in C and D are from RIM1ff/2ff littermates, 3 weeks of age). Bottom overview cropped to the OPL is labeled “all probes” because the signals are derived from anti-RIM2 staining (rabbit polyclonal, Alexa Fluor488, shown in green), the cone marker PNA (linked to Alexa Fluor 568, shown in red), and anti-CtBP2 (Ms monoclonal, Alexa Fluor 648, shown in blue). E , F , Regions selected for presentation at higher resolution. The control condition presented in C and E and the “Cre-poor region” in F show that the RIM2 signal overlaps extensively with CtBP2/Ribeye (aqua). The “Cre-rich region” in F reveals a drop in RIM2 staining. G , H ) and Synaptotagmin1 (Syt1, ∼60 kDa) were used as presynaptic markers, whereas CtBP2 (∼45 kDa) in the context of Western analysis represents the transcription factor that is expressed throughout the retina. I , Graphical summary of the densitometry measurements from Western analysis. Only RIM2 was reduced (see text for values) and no apparent change in the intensity of the synaptic markers was witnessed at 3 weeks. Data are presented as mean ± SE.

    Article Snippet: Other antibodies used were as follows: anti-RIM2, rabbit polyclonal (Synaptic Systems, 140–303); anti-RIM1, rabbit polyclonal (Synaptic Systems, 140–003) and anti-RIM1 mouse monoclonal (BD Transduction Laboratories, 610907); anti-Fodrin/α-spectrin, clone AA6, mouse monoclonal (ICN Biomedicals); lectin PNA-Alexa Fluor 568 (Invitrogen, ): anti-Ribeye/U2656 rabbit polyclonal antibody ( ); anti-CtBP2 mouse monoclonal (BD Transduction Laboratories, 612044); anti-Cre Recombinase, rabbit polyclonal (Novagen, 69050); and anti-Cre recombinase, mouse monoclonal (Covance, MMS-106P).

    Techniques: Knock-Out, Transgenic Assay, Mouse Assay, Expressing, Mass Spectrometry, Migration, Immunostaining, Transduction, Staining, Labeling, Derivative Assay, Marker, Western Blot

    Stratified presynaptic localizations of sequence regions of Aczonin, Bassoon, Munc13, and RIM in conventional mammalian synapses. ( A ) Domain architectures of Aczonin, Bassoon, Munc13-1, RIM1, and CAST1, and sequence positions of the immunogens used in

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

    Article Title: Molecular in situ topology of Aczonin/Piccolo and associated proteins at the mammalian neurotransmitter release site

    doi: 10.1073/pnas.1101707108

    Figure Lengend Snippet: Stratified presynaptic localizations of sequence regions of Aczonin, Bassoon, Munc13, and RIM in conventional mammalian synapses. ( A ) Domain architectures of Aczonin, Bassoon, Munc13-1, RIM1, and CAST1, and sequence positions of the immunogens used in

    Article Snippet: Construct Rim1(207–366) was cloned at Synaptic Systems into a derivative of pASK-IBA37plus (IBA) and carries an N-terminal RGS-His-tag; it expresses the immunogen used at Synaptic Systems to generate a new RIM1-specific antibody (RIM1ab2, no. 140 013) and was provided by Henrik Martens (Synaptic Systems, Göttingen, Germany).

    Techniques: Sequencing

    Physiological relevance of effects of RIMs on inactivation properties of VDCCs. A , RT-PCR analysis of RIM1, RIM2, RIM3, and RIM4 RNA expression in PC12 cells treated with GAPDH siRNA ( siControl ), a combination of RIM1- and RIM2-specific siRNAs ( siRIM1  2 ), a combination of RIM3- and RIM4-specific siRNAs ( siRIM3  4 ), and a combination of RIM1-, RIM2-, RIM3-, and RIM4-specific siRNAs ( siRIM1  2  3  4 ). PCR was performed for 29 cycles. β-Actin was used as an internal control.  B , WB of essential components of release machinery and VDCC subunits in PC12 cells transfected with the indicated combination of siRNAs. Primary antibodies used are indicated on the  left. C , acceleration of inactivation by application of siRNAs specific for RIMs in VDCC currents recorded from PC12 cells. The acceleration of inactivation in RIM knockdown cells was reversed by expression of siRNA-resistant RIM cDNAs (siRIM1  2 + RIM1*  2*, and siRIM3  4 + RIM3*  4*).  Top  and  middle , normalized current traces.  Bottom  for statistical significance of the differences.

    Journal: The Journal of Biological Chemistry

    Article Title: Rab3-interacting Molecule ? Isoforms Lacking the Rab3-binding Domain Induce Long Lasting Currents but Block Neurotransmitter Vesicle Anchoring in Voltage-dependent P/Q-type Ca2+ Channels *

    doi: 10.1074/jbc.M110.101311

    Figure Lengend Snippet: Physiological relevance of effects of RIMs on inactivation properties of VDCCs. A , RT-PCR analysis of RIM1, RIM2, RIM3, and RIM4 RNA expression in PC12 cells treated with GAPDH siRNA ( siControl ), a combination of RIM1- and RIM2-specific siRNAs ( siRIM1 2 ), a combination of RIM3- and RIM4-specific siRNAs ( siRIM3 4 ), and a combination of RIM1-, RIM2-, RIM3-, and RIM4-specific siRNAs ( siRIM1 2 3 4 ). PCR was performed for 29 cycles. β-Actin was used as an internal control. B , WB of essential components of release machinery and VDCC subunits in PC12 cells transfected with the indicated combination of siRNAs. Primary antibodies used are indicated on the left. C , acceleration of inactivation by application of siRNAs specific for RIMs in VDCC currents recorded from PC12 cells. The acceleration of inactivation in RIM knockdown cells was reversed by expression of siRNA-resistant RIM cDNAs (siRIM1 2 + RIM1* 2*, and siRIM3 4 + RIM3* 4*). Top and middle , normalized current traces. Bottom for statistical significance of the differences.

    Article Snippet: After centrifugation at 6,654 × g for 15 min, the supernatant was incubated with protein A-agarose coupled to anti-RIM1/2 antibody (Synaptic Systems catalog no. 140-203) or anti-RIM3 polyclonal antibody for 6 h at 4 °C.

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Polymerase Chain Reaction, Western Blot, Transfection, Expressing

    γ-RIMs reduce the density of vesicles at the plasma membrane in PC12 cells. A , typical TIRF images of plasma membrane-docked vesicles containing NPY-Venus are shown. NPY-Venus and combinations of siRNAs and siRNA-resistant RIM cDNAs were cotransfected in PC12 cells, and live images of cells were obtained by TIRF microscopy.  Scale bar , 10 μm.  B , the vesicle density (number ( N ) μm −2 ) was determined by counting the vesicles in each image. The number of individual fluorescent spots in the area, where vesicles uniformly distributed in TIRF images, was divided by the area. Numbers of PC12 cells analyzed were 48, 51, 40, 45, 30, and 29 for transfection of siControl, siRIM1  2, siRIM3  4, siRIM1  2  3  4, siRIM1  2 + RIM1*  2*, and siRIM3  4 + RIM3*  4*, respectively. *,  p

    Journal: The Journal of Biological Chemistry

    Article Title: Rab3-interacting Molecule ? Isoforms Lacking the Rab3-binding Domain Induce Long Lasting Currents but Block Neurotransmitter Vesicle Anchoring in Voltage-dependent P/Q-type Ca2+ Channels *

    doi: 10.1074/jbc.M110.101311

    Figure Lengend Snippet: γ-RIMs reduce the density of vesicles at the plasma membrane in PC12 cells. A , typical TIRF images of plasma membrane-docked vesicles containing NPY-Venus are shown. NPY-Venus and combinations of siRNAs and siRNA-resistant RIM cDNAs were cotransfected in PC12 cells, and live images of cells were obtained by TIRF microscopy. Scale bar , 10 μm. B , the vesicle density (number ( N ) μm −2 ) was determined by counting the vesicles in each image. The number of individual fluorescent spots in the area, where vesicles uniformly distributed in TIRF images, was divided by the area. Numbers of PC12 cells analyzed were 48, 51, 40, 45, 30, and 29 for transfection of siControl, siRIM1 2, siRIM3 4, siRIM1 2 3 4, siRIM1 2 + RIM1* 2*, and siRIM3 4 + RIM3* 4*, respectively. *, p

    Article Snippet: After centrifugation at 6,654 × g for 15 min, the supernatant was incubated with protein A-agarose coupled to anti-RIM1/2 antibody (Synaptic Systems catalog no. 140-203) or anti-RIM3 polyclonal antibody for 6 h at 4 °C.

    Techniques: Microscopy, Transfection

    Effect of the binding of CAST and RIM1 on synaptic transmission. (A) Effects of the COOH-terminal regions of CAST on synaptic transmission. (a) Sequences of the CAST peptides. RID, RIM1-interacting domain; scb RID, scrambled RID. (b) Effects of the peptides (5 μM each) on the binding of HA-RIM1 to immobilized GST-CAST-4. The binding was inhibited by RID, but not by RIDΔIWA or scb RID. (c and d) Effects of the CAST peptides (1 mM each in the injection pipette) on synaptic transmission. Presynaptic neurons were stimulated every 20 s CAST peptides were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in c. Normalized and averaged EPSP amplitudes are plotted from five experiments with RID, RIDΔIWA, or scb RID peptide in d. (e) Effects of the COOH-terminal regions of CAST (5 μM each) on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST-CASTCΔIWA or GST-CASTC, followed by Western blotting using the anti-Myc and anti-HA Abs. GST-CASTC inhibited the binding but GST-CASTCΔIWA did not. (f and g) Effects of the recombinant CAST proteins (150 μM each in the injection) on synaptic transmission. Presynaptic neurons were stimulated every 20 s. Recombinant CAST proteins were introduced into presynaptic neurons at t = 0. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-CASTC or GST-CASTCΔIWA in f. (B) Effect of the PDZ domain of RIM1 on synaptic transmission. (a) Effect of the GST fusion protein containing the PDZ domain on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST alone or GST-RIM1 PDZ (5 μM each), followed by Western blotting using the anti-Myc and anti-HA Abs. GST-RIM1 PDZ inhibited the binding but GST alone did not. (b and c) Effect of GST-RIM1 PDZ on synaptic transmission. Presynaptic neurons were stimulated every 20 s GST alone or GST-RIM1 PDZ (170 μM each) were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in b. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-RIM1 PDZ or GST alone in c.

    Journal: The Journal of Cell Biology

    Article Title: Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

    doi: 10.1083/jcb.200307101

    Figure Lengend Snippet: Effect of the binding of CAST and RIM1 on synaptic transmission. (A) Effects of the COOH-terminal regions of CAST on synaptic transmission. (a) Sequences of the CAST peptides. RID, RIM1-interacting domain; scb RID, scrambled RID. (b) Effects of the peptides (5 μM each) on the binding of HA-RIM1 to immobilized GST-CAST-4. The binding was inhibited by RID, but not by RIDΔIWA or scb RID. (c and d) Effects of the CAST peptides (1 mM each in the injection pipette) on synaptic transmission. Presynaptic neurons were stimulated every 20 s CAST peptides were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in c. Normalized and averaged EPSP amplitudes are plotted from five experiments with RID, RIDΔIWA, or scb RID peptide in d. (e) Effects of the COOH-terminal regions of CAST (5 μM each) on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST-CASTCΔIWA or GST-CASTC, followed by Western blotting using the anti-Myc and anti-HA Abs. GST-CASTC inhibited the binding but GST-CASTCΔIWA did not. (f and g) Effects of the recombinant CAST proteins (150 μM each in the injection) on synaptic transmission. Presynaptic neurons were stimulated every 20 s. Recombinant CAST proteins were introduced into presynaptic neurons at t = 0. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-CASTC or GST-CASTCΔIWA in f. (B) Effect of the PDZ domain of RIM1 on synaptic transmission. (a) Effect of the GST fusion protein containing the PDZ domain on the binding of CAST and RIM1. Immunoprecipitation assays of Myc-CAST and HA-RIM1 were performed in the presence of GST alone or GST-RIM1 PDZ (5 μM each), followed by Western blotting using the anti-Myc and anti-HA Abs. GST-RIM1 PDZ inhibited the binding but GST alone did not. (b and c) Effect of GST-RIM1 PDZ on synaptic transmission. Presynaptic neurons were stimulated every 20 s GST alone or GST-RIM1 PDZ (170 μM each) were introduced into presynaptic neurons at t = 0. EPSPs from representative experiments with the injection are illustrated in b. Normalized and averaged EPSP amplitudes are plotted from five experiments with GST-RIM1 PDZ or GST alone in c.

    Article Snippet: These results, together with the above observations, indicate that CAST forms a ternary complex with RIM1 and Bassoon.

    Techniques: Binding Assay, Transmission Assay, Injection, Transferring, Immunoprecipitation, Western Blot, Recombinant

    Localization of CAST, RIM1, and Bassoon at synapses in cultured SCG neurons. Rat SCG neurons at 5–6 wk in culture were doubly stained using anti-CAST and anti-Bassoon, anti-RIM1 or antisynaptophysin Abs. Bars, 30 μm. The results are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

    doi: 10.1083/jcb.200307101

    Figure Lengend Snippet: Localization of CAST, RIM1, and Bassoon at synapses in cultured SCG neurons. Rat SCG neurons at 5–6 wk in culture were doubly stained using anti-CAST and anti-Bassoon, anti-RIM1 or antisynaptophysin Abs. Bars, 30 μm. The results are representative of three independent experiments.

    Article Snippet: These results, together with the above observations, indicate that CAST forms a ternary complex with RIM1 and Bassoon.

    Techniques: Cell Culture, Staining

    Ternary complex formation of CAST, RIM1 and Bassoon. Each expression plasmid of Myc-CAST, HA-RIM1, or EGFP-Bassoon was transfected into HEK293 cells. Each protein was extracted and mixed in the indicated combinations, followed by immunoprecipitation using the anti-GFP, anti-Myc, or anti-HA Ab. Immunoprecipitates were then analyzed by Western blotting using the indicated Abs. IP, immunoprecipitation. The results are representative of three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

    doi: 10.1083/jcb.200307101

    Figure Lengend Snippet: Ternary complex formation of CAST, RIM1 and Bassoon. Each expression plasmid of Myc-CAST, HA-RIM1, or EGFP-Bassoon was transfected into HEK293 cells. Each protein was extracted and mixed in the indicated combinations, followed by immunoprecipitation using the anti-GFP, anti-Myc, or anti-HA Ab. Immunoprecipitates were then analyzed by Western blotting using the indicated Abs. IP, immunoprecipitation. The results are representative of three independent experiments.

    Article Snippet: These results, together with the above observations, indicate that CAST forms a ternary complex with RIM1 and Bassoon.

    Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot

    Transsynaptic nanoscale alignment of AZ and PSD proteins a , Distributions of synaptic RIM1/2 and PSD-95 pair as the original localizations (left) and with NCs highlighted (right), scale 200 nm. Filled arrows indicate aligned NCs, open arrows non-aligned NCs. b , Paired correlation function (PCF) of measured RIM1/2 and PSD-95 compared to PCF with either distribution randomized. c , PCF of simulated distributions with (cyan) and without (orange) shuffling NC positions. d , Cumulative distributions of cross-correlation index (n = 143 synapses). e , RIM1/2 protein enrichment as a function of distance from translated PSD-95 NC centers (top, filled points) and PSD-95 enrichment relative to RIM1/2 NCs (bottom, open points). Simulations with same randomizations as in d – e were performed for each synapse. f , Protein density profile for enriched vs non-enriched NCs, n = 119 PSD-95 NCs, 90 RIM1/2 NCs. g , Enrichment indices for RIM1/2, Munc13, and Bassoon relative to PSD-95 NCs (filled) and for the opposite direction (open), n > 260 NCs, *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Transsynaptic nanoscale alignment of AZ and PSD proteins a , Distributions of synaptic RIM1/2 and PSD-95 pair as the original localizations (left) and with NCs highlighted (right), scale 200 nm. Filled arrows indicate aligned NCs, open arrows non-aligned NCs. b , Paired correlation function (PCF) of measured RIM1/2 and PSD-95 compared to PCF with either distribution randomized. c , PCF of simulated distributions with (cyan) and without (orange) shuffling NC positions. d , Cumulative distributions of cross-correlation index (n = 143 synapses). e , RIM1/2 protein enrichment as a function of distance from translated PSD-95 NC centers (top, filled points) and PSD-95 enrichment relative to RIM1/2 NCs (bottom, open points). Simulations with same randomizations as in d – e were performed for each synapse. f , Protein density profile for enriched vs non-enriched NCs, n = 119 PSD-95 NCs, 90 RIM1/2 NCs. g , Enrichment indices for RIM1/2, Munc13, and Bassoon relative to PSD-95 NCs (filled) and for the opposite direction (open), n > 260 NCs, *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Protein Enrichment

    Nanocluster organization of vesicle release machinery proteins in the active zone and postsynaptic AMPA receptors a , En-face (top) and side (bottom) views of local density maps of a simulated synapse with artificial NCs with 40 nm diameters, scale 100 nm. b , Autocorrelation function of simulated clusters with different sized NCs. The points represent the radius where g(r) = 1. c , Pooled data from 15 sets of simulations showing that the radius where g(r) first crosses 1 reasonably estimates the average NC diameters. d , Comparison of NC number, fraction of localization in NC, and NC volume across different developmental stages shows no significant difference, though the young DIV9 culture shows a trend toward increased NC numbers (one-way ANOVA on ranks for NC number and volume, one-way ANOVA for %localization in NC). Data were from 143 RIM NCs and 135 PSD NCs of 64 DIV9 synapses, 63 RIM NCs and 65 PSD NCs of 38 DIV14 synapses, and 44 RIM NCs and 41 PSD NCs from 28 DIV21 synapses. e , Comparison of two RIM antibodies (from left to right) in whole synaptic cluster volume, number of NCs, autocorrelation function estimating average NC diameter, and protein density relative to PSD-95 NC centers. Anti-RIM1/2 (Synaptic Systems #140–203) targets the Zn-finger domain and anti-RIM1 targets the PDZ domain of RIM1 (Synaptic Systems #140–003). These tests suggest that there is no significant difference between these two antibodies. The numbers in bars denote the group sizes. f , Local density maps of en-face (top) and side (bottom) views of an example Munc13 cluster, scale 200 nm. g , Auto-correlation functions for Munc13 distributions compared to simulated randomized distributions. h–i , Local density maps and ACF of Bassoon cluster, scale 200 nm. j , Pooled cluster volumes, normalized to PSD-95 volumes within each synapse. Each bar pair represents data from a set of RIM1/2-PSD-95, Munc13-PSD-95 or Bassoon-PSD-95 staining. The numbers in bars denote the group sizes. k , Distribution of en-face distances between NC center and synapse center. Data were normalized to the distribution of simulated clusters with the same number of NCs as the original synapse but randomized positions. l , An example synapse with RIM1/2 and Munc13 staining of the same synapse, shown in two different angles. The translucent surfaces represent the alpha shapes that define the synaptic cluster borders. m , Pooled RIM1/2 and Munc13 cluster volumes, normalized to RIM1/2 within each synapse. n , Pooled RIM1/2, Munc13 and Bassoon cluster volumes from staining of RIM1/2-Bassoon and Munc13-Bassoon, normalized to Bassoon within each synapse. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Nanocluster organization of vesicle release machinery proteins in the active zone and postsynaptic AMPA receptors a , En-face (top) and side (bottom) views of local density maps of a simulated synapse with artificial NCs with 40 nm diameters, scale 100 nm. b , Autocorrelation function of simulated clusters with different sized NCs. The points represent the radius where g(r) = 1. c , Pooled data from 15 sets of simulations showing that the radius where g(r) first crosses 1 reasonably estimates the average NC diameters. d , Comparison of NC number, fraction of localization in NC, and NC volume across different developmental stages shows no significant difference, though the young DIV9 culture shows a trend toward increased NC numbers (one-way ANOVA on ranks for NC number and volume, one-way ANOVA for %localization in NC). Data were from 143 RIM NCs and 135 PSD NCs of 64 DIV9 synapses, 63 RIM NCs and 65 PSD NCs of 38 DIV14 synapses, and 44 RIM NCs and 41 PSD NCs from 28 DIV21 synapses. e , Comparison of two RIM antibodies (from left to right) in whole synaptic cluster volume, number of NCs, autocorrelation function estimating average NC diameter, and protein density relative to PSD-95 NC centers. Anti-RIM1/2 (Synaptic Systems #140–203) targets the Zn-finger domain and anti-RIM1 targets the PDZ domain of RIM1 (Synaptic Systems #140–003). These tests suggest that there is no significant difference between these two antibodies. The numbers in bars denote the group sizes. f , Local density maps of en-face (top) and side (bottom) views of an example Munc13 cluster, scale 200 nm. g , Auto-correlation functions for Munc13 distributions compared to simulated randomized distributions. h–i , Local density maps and ACF of Bassoon cluster, scale 200 nm. j , Pooled cluster volumes, normalized to PSD-95 volumes within each synapse. Each bar pair represents data from a set of RIM1/2-PSD-95, Munc13-PSD-95 or Bassoon-PSD-95 staining. The numbers in bars denote the group sizes. k , Distribution of en-face distances between NC center and synapse center. Data were normalized to the distribution of simulated clusters with the same number of NCs as the original synapse but randomized positions. l , An example synapse with RIM1/2 and Munc13 staining of the same synapse, shown in two different angles. The translucent surfaces represent the alpha shapes that define the synaptic cluster borders. m , Pooled RIM1/2 and Munc13 cluster volumes, normalized to RIM1/2 within each synapse. n , Pooled RIM1/2, Munc13 and Bassoon cluster volumes from staining of RIM1/2-Bassoon and Munc13-Bassoon, normalized to Bassoon within each synapse. *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Staining

    Enrichment of other scaffolding proteins within nanocolumns a , Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm. b , Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Enrichment of other scaffolding proteins within nanocolumns a , Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm. b , Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm. *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Scaffolding

    Retrograde plasticity of synaptic nanoscale alignment a , Distributions of synaptic RIM1/2 and PSD-95 for control and post-LTP induction conditions with NCs highlighted. b–c , Across-condition comparison of enrichment index and percentage of NCs enriched (n = 45, 87 and 42 synapses for control, LTP, and APV, respectively). d , Distributions of RIM1/2 and PSD-95 for conditions following NMDA stimulation. Scale 100 nm. e–i , Across-conditions comparison of RIM1/2 and PSD-95. Dark red in i represents RIM1/2 NCs enriched with PSD-95 and light red the unenriched NCs. n = 61, 96, 77 and 74 synapses for control, NMDA, washout, and APV, respectively. j , Schematic summarizing the reorganization of NCs during NMDA-induced plasticity and recovery. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Retrograde plasticity of synaptic nanoscale alignment a , Distributions of synaptic RIM1/2 and PSD-95 for control and post-LTP induction conditions with NCs highlighted. b–c , Across-condition comparison of enrichment index and percentage of NCs enriched (n = 45, 87 and 42 synapses for control, LTP, and APV, respectively). d , Distributions of RIM1/2 and PSD-95 for conditions following NMDA stimulation. Scale 100 nm. e–i , Across-conditions comparison of RIM1/2 and PSD-95. Dark red in i represents RIM1/2 NCs enriched with PSD-95 and light red the unenriched NCs. n = 61, 96, 77 and 74 synapses for control, NMDA, washout, and APV, respectively. j , Schematic summarizing the reorganization of NCs during NMDA-induced plasticity and recovery. *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques:

    Protein enrichment within nanocolumns a, Enrichment index between RIM1/2 and PSD-95. The left insets are replicas of Fig. 3e , and the enrichment index is defined as the average of the first three bins in the enrichment profile (boxed), i.e. normalized localization density within 60 nm from the projection center of a given NC. Filled points show RIM1/2 relative to PSD-95 NCs, open points show PSD-95 relative to RIM1/2 NCs. Same randomizations as in Fig. 3e and depicted again in b. **p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Protein enrichment within nanocolumns a, Enrichment index between RIM1/2 and PSD-95. The left insets are replicas of Fig. 3e , and the enrichment index is defined as the average of the first three bins in the enrichment profile (boxed), i.e. normalized localization density within 60 nm from the projection center of a given NC. Filled points show RIM1/2 relative to PSD-95 NCs, open points show PSD-95 relative to RIM1/2 NCs. Same randomizations as in Fig. 3e and depicted again in b. **p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Protein Enrichment

    Plasticity within nanocolumns a , Changes in the localization density within RIM1/2 (red) and PSD-95 (blue) NCs under control, 5 min NMDA treatment, 25 min washout, and NMDA + APV treatment conditions. b–h , Reorganization of RIM1/2 and GluR2/3 under control, 5 min NMDA treatment, 25 min washout conditions examples ( b ), comparison of whole synaptic cluster sizes ( c ), NC number per synapse ( d ), localization density within NCs ( e ), enrichment indices ( f ), percentage of NCs that were enriched ( g ), and NC volumes ( h ). Note that similar to the results from the RIM1/2-PSD-95 analyses, only those RIM1/2 NCs that were enriched with GluR2/3 (dark red) were increased in volume. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Plasticity within nanocolumns a , Changes in the localization density within RIM1/2 (red) and PSD-95 (blue) NCs under control, 5 min NMDA treatment, 25 min washout, and NMDA + APV treatment conditions. b–h , Reorganization of RIM1/2 and GluR2/3 under control, 5 min NMDA treatment, 25 min washout conditions examples ( b ), comparison of whole synaptic cluster sizes ( c ), NC number per synapse ( d ), localization density within NCs ( e ), enrichment indices ( f ), percentage of NCs that were enriched ( g ), and NC volumes ( h ). Note that similar to the results from the RIM1/2-PSD-95 analyses, only those RIM1/2 NCs that were enriched with GluR2/3 (dark red) were increased in volume. *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques:

    Vesicle release proteins form subsynaptic nanoclusters a , Color-coded schematic of studied synaptic proteins. b , Synapses labeled with RIM1/2 and PSD-95 imaged using 3D-STORM (10 nm pixels) compared to wide-field composite (bottom corner, 100 nm pixels), scale 2 μm. Boxed synapse enlarged in original (top) and rotated (bottom) angles, scale 200 nm. c , En-face (top) and side (bottom) views of a RIM1/2 cluster showing all localizations and local density maps for a measured synaptic cluster compared to a simulated randomized cluster, scale 200 nm. d , Auto-correlation functions of measured RIM1/2 (n = 115), isolated non-synaptic small groups of localizations due to repetitive switching of fluorophores (n = 42), and simulated randomized (n = 115) distributions. e , RIM1/2 nanoclusters (NCs, red) within a synaptic cluster. f , Distribution of NC distances from the center of synapses normalized to randomized distribution. g , Molecule density inside NCs normalized to synaptic average. h , Average number of protein NCs per synapse. i , Cumulative distributions of NC volumes. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Vesicle release proteins form subsynaptic nanoclusters a , Color-coded schematic of studied synaptic proteins. b , Synapses labeled with RIM1/2 and PSD-95 imaged using 3D-STORM (10 nm pixels) compared to wide-field composite (bottom corner, 100 nm pixels), scale 2 μm. Boxed synapse enlarged in original (top) and rotated (bottom) angles, scale 200 nm. c , En-face (top) and side (bottom) views of a RIM1/2 cluster showing all localizations and local density maps for a measured synaptic cluster compared to a simulated randomized cluster, scale 200 nm. d , Auto-correlation functions of measured RIM1/2 (n = 115), isolated non-synaptic small groups of localizations due to repetitive switching of fluorophores (n = 42), and simulated randomized (n = 115) distributions. e , RIM1/2 nanoclusters (NCs, red) within a synaptic cluster. f , Distribution of NC distances from the center of synapses normalized to randomized distribution. g , Molecule density inside NCs normalized to synaptic average. h , Average number of protein NCs per synapse. i , Cumulative distributions of NC volumes. *p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Labeling, Isolation

    RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Article Snippet: For transsynaptic measurements, rabbit anti-Munc13, anti-RIM1/2, anti-RIM1 (1:500; Synaptic Systems No.140003) or anti-Bassoon (1:500, Cell Signaling), were used with mouse anti-PSD-95 (1:200; Neuromab), mouse anti-GluA2 (1:100, Millipore), or rabbit anti-GluR2/3 (1:100, Millipore).

    Techniques: Expressing, Immunofluorescence, Transfection, Over Expression

    Multi‐color imaging with X10 reveals synaptic ultrastructure in cell culture Three‐color imaging resolves synaptic vesicle clusters (identified by Synaptophysin), along with pre‐synaptic active zones (identified by Bassoon) and post‐synaptic densities (identified by Homer 1). The panel at the top right gives a schematic overview of the organization of a synapse, for orientation (colors as in the fluorescence images). The two panels on the bottom right provide a stereo view of the synapses. Expansion factor: 11.0×. Scale bars: 500 nm (both). Upper panels: higher magnification images show the alignment of pre‐synaptic active zones and post‐synaptic densities, as well as the distance between them, in side view. Expansion factor: 11.0×. Scale bar: 200 nm. Lower panels: a z ‐stack through an additional synapse, in face view. Expansion factor: 11.0×. Scale bar: 200 nm. Representative images of an immunostaining for pre‐synaptic RIM1/2 and post‐synaptic PSD95, two markers known to be more closely associated than Bassoon/Homer 1 24 . Arrowheads indicate nanocolumns of aligned pre‐ and post‐synaptic proteins. Expansion factor: 10.4×. Scale bars: 500 nm (upper panel), 200 nm (lower panels). Line scans through Bassoon staining (green) in pre‐synaptic active zones and through Homer 1 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Line scans through RIM1/2 staining (green) in pre‐synaptic active zones and through PSD95 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Histogram showing the distribution of Bassoon to Homer 1 distances and RIM1/2 to PSD95 distances ( n = 15 neuronal areas, with the corresponding synapses, for Bassoon and Homer 1, n = 74 neuronal areas for RIM1/2 and PSD95).

    Journal: EMBO Reports

    Article Title: X10 expansion microscopy enables 25‐nm resolution on conventional microscopes

    doi: 10.15252/embr.201845836

    Figure Lengend Snippet: Multi‐color imaging with X10 reveals synaptic ultrastructure in cell culture Three‐color imaging resolves synaptic vesicle clusters (identified by Synaptophysin), along with pre‐synaptic active zones (identified by Bassoon) and post‐synaptic densities (identified by Homer 1). The panel at the top right gives a schematic overview of the organization of a synapse, for orientation (colors as in the fluorescence images). The two panels on the bottom right provide a stereo view of the synapses. Expansion factor: 11.0×. Scale bars: 500 nm (both). Upper panels: higher magnification images show the alignment of pre‐synaptic active zones and post‐synaptic densities, as well as the distance between them, in side view. Expansion factor: 11.0×. Scale bar: 200 nm. Lower panels: a z ‐stack through an additional synapse, in face view. Expansion factor: 11.0×. Scale bar: 200 nm. Representative images of an immunostaining for pre‐synaptic RIM1/2 and post‐synaptic PSD95, two markers known to be more closely associated than Bassoon/Homer 1 24 . Arrowheads indicate nanocolumns of aligned pre‐ and post‐synaptic proteins. Expansion factor: 10.4×. Scale bars: 500 nm (upper panel), 200 nm (lower panels). Line scans through Bassoon staining (green) in pre‐synaptic active zones and through Homer 1 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Line scans through RIM1/2 staining (green) in pre‐synaptic active zones and through PSD95 staining (magenta) in the corresponding post‐synaptic densities reveal the distance between the two. The image inset shows three example line scans and identifies them by number. Histogram showing the distribution of Bassoon to Homer 1 distances and RIM1/2 to PSD95 distances ( n = 15 neuronal areas, with the corresponding synapses, for Bassoon and Homer 1, n = 74 neuronal areas for RIM1/2 and PSD95).

    Article Snippet: The following primary antibodies were used: rat monoclonal anti‐tubulin (MA1‐80017; Thermo Fisher Scientific, Waltham, MA, USA), anti‐tubulin single‐chain variable fragments (scFv) directly conjugated to Alexa Fluor 488 for X10 or Atto647N for STED imaging (self‐produced, see above), guinea pig polyclonal anti‐Synaptophysin (101 004; Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti‐Homer1 (160 003; Synaptic Systems), mouse monoclonal anti‐Bassoon (SAP7F407; Enzo, Farmingdale, NY, USA), rabbit polyclonal anti‐VDAC (sc‐98708; Santa Cruz, Heidelberg, Germany), mouse monoclonal anti‐PSD95 (MA1‐046; Thermo Fisher Scientific, Waltham, MA, USA), and rabbit anti‐RIM1 (140 003; Synaptic Systems).

    Techniques: Imaging, Cell Culture, Fluorescence, Immunostaining, Staining

    Transsynaptic nanoscale alignment of AZ and PSD proteins a , Distributions of synaptic RIM1/2 and PSD-95 pair as the original localizations (left) and with NCs highlighted (right), scale 200 nm. Filled arrows indicate aligned NCs, open arrows non-aligned NCs. b , Paired correlation function (PCF) of measured RIM1/2 and PSD-95 compared to PCF with either distribution randomized. c , PCF of simulated distributions with (cyan) and without (orange) shuffling NC positions. d , Cumulative distributions of cross-correlation index (n = 143 synapses). e , RIM1/2 protein enrichment as a function of distance from translated PSD-95 NC centers (top, filled points) and PSD-95 enrichment relative to RIM1/2 NCs (bottom, open points). Simulations with same randomizations as in d – e were performed for each synapse. f , Protein density profile for enriched vs non-enriched NCs, n = 119 PSD-95 NCs, 90 RIM1/2 NCs. g , Enrichment indices for RIM1/2, Munc13, and Bassoon relative to PSD-95 NCs (filled) and for the opposite direction (open), n > 260 NCs, *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Transsynaptic nanoscale alignment of AZ and PSD proteins a , Distributions of synaptic RIM1/2 and PSD-95 pair as the original localizations (left) and with NCs highlighted (right), scale 200 nm. Filled arrows indicate aligned NCs, open arrows non-aligned NCs. b , Paired correlation function (PCF) of measured RIM1/2 and PSD-95 compared to PCF with either distribution randomized. c , PCF of simulated distributions with (cyan) and without (orange) shuffling NC positions. d , Cumulative distributions of cross-correlation index (n = 143 synapses). e , RIM1/2 protein enrichment as a function of distance from translated PSD-95 NC centers (top, filled points) and PSD-95 enrichment relative to RIM1/2 NCs (bottom, open points). Simulations with same randomizations as in d – e were performed for each synapse. f , Protein density profile for enriched vs non-enriched NCs, n = 119 PSD-95 NCs, 90 RIM1/2 NCs. g , Enrichment indices for RIM1/2, Munc13, and Bassoon relative to PSD-95 NCs (filled) and for the opposite direction (open), n > 260 NCs, *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Protein Enrichment

    Nanocluster organization of vesicle release machinery proteins in the active zone and postsynaptic AMPA receptors a , En-face (top) and side (bottom) views of local density maps of a simulated synapse with artificial NCs with 40 nm diameters, scale 100 nm. b , Autocorrelation function of simulated clusters with different sized NCs. The points represent the radius where g(r) = 1. c , Pooled data from 15 sets of simulations showing that the radius where g(r) first crosses 1 reasonably estimates the average NC diameters. d , Comparison of NC number, fraction of localization in NC, and NC volume across different developmental stages shows no significant difference, though the young DIV9 culture shows a trend toward increased NC numbers (one-way ANOVA on ranks for NC number and volume, one-way ANOVA for %localization in NC). Data were from 143 RIM NCs and 135 PSD NCs of 64 DIV9 synapses, 63 RIM NCs and 65 PSD NCs of 38 DIV14 synapses, and 44 RIM NCs and 41 PSD NCs from 28 DIV21 synapses. e , Comparison of two RIM antibodies (from left to right) in whole synaptic cluster volume, number of NCs, autocorrelation function estimating average NC diameter, and protein density relative to PSD-95 NC centers. Anti-RIM1/2 (Synaptic Systems #140–203) targets the Zn-finger domain and anti-RIM1 targets the PDZ domain of RIM1 (Synaptic Systems #140–003). These tests suggest that there is no significant difference between these two antibodies. The numbers in bars denote the group sizes. f , Local density maps of en-face (top) and side (bottom) views of an example Munc13 cluster, scale 200 nm. g , Auto-correlation functions for Munc13 distributions compared to simulated randomized distributions. h–i , Local density maps and ACF of Bassoon cluster, scale 200 nm. j , Pooled cluster volumes, normalized to PSD-95 volumes within each synapse. Each bar pair represents data from a set of RIM1/2-PSD-95, Munc13-PSD-95 or Bassoon-PSD-95 staining. The numbers in bars denote the group sizes. k , Distribution of en-face distances between NC center and synapse center. Data were normalized to the distribution of simulated clusters with the same number of NCs as the original synapse but randomized positions. l , An example synapse with RIM1/2 and Munc13 staining of the same synapse, shown in two different angles. The translucent surfaces represent the alpha shapes that define the synaptic cluster borders. m , Pooled RIM1/2 and Munc13 cluster volumes, normalized to RIM1/2 within each synapse. n , Pooled RIM1/2, Munc13 and Bassoon cluster volumes from staining of RIM1/2-Bassoon and Munc13-Bassoon, normalized to Bassoon within each synapse. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Nanocluster organization of vesicle release machinery proteins in the active zone and postsynaptic AMPA receptors a , En-face (top) and side (bottom) views of local density maps of a simulated synapse with artificial NCs with 40 nm diameters, scale 100 nm. b , Autocorrelation function of simulated clusters with different sized NCs. The points represent the radius where g(r) = 1. c , Pooled data from 15 sets of simulations showing that the radius where g(r) first crosses 1 reasonably estimates the average NC diameters. d , Comparison of NC number, fraction of localization in NC, and NC volume across different developmental stages shows no significant difference, though the young DIV9 culture shows a trend toward increased NC numbers (one-way ANOVA on ranks for NC number and volume, one-way ANOVA for %localization in NC). Data were from 143 RIM NCs and 135 PSD NCs of 64 DIV9 synapses, 63 RIM NCs and 65 PSD NCs of 38 DIV14 synapses, and 44 RIM NCs and 41 PSD NCs from 28 DIV21 synapses. e , Comparison of two RIM antibodies (from left to right) in whole synaptic cluster volume, number of NCs, autocorrelation function estimating average NC diameter, and protein density relative to PSD-95 NC centers. Anti-RIM1/2 (Synaptic Systems #140–203) targets the Zn-finger domain and anti-RIM1 targets the PDZ domain of RIM1 (Synaptic Systems #140–003). These tests suggest that there is no significant difference between these two antibodies. The numbers in bars denote the group sizes. f , Local density maps of en-face (top) and side (bottom) views of an example Munc13 cluster, scale 200 nm. g , Auto-correlation functions for Munc13 distributions compared to simulated randomized distributions. h–i , Local density maps and ACF of Bassoon cluster, scale 200 nm. j , Pooled cluster volumes, normalized to PSD-95 volumes within each synapse. Each bar pair represents data from a set of RIM1/2-PSD-95, Munc13-PSD-95 or Bassoon-PSD-95 staining. The numbers in bars denote the group sizes. k , Distribution of en-face distances between NC center and synapse center. Data were normalized to the distribution of simulated clusters with the same number of NCs as the original synapse but randomized positions. l , An example synapse with RIM1/2 and Munc13 staining of the same synapse, shown in two different angles. The translucent surfaces represent the alpha shapes that define the synaptic cluster borders. m , Pooled RIM1/2 and Munc13 cluster volumes, normalized to RIM1/2 within each synapse. n , Pooled RIM1/2, Munc13 and Bassoon cluster volumes from staining of RIM1/2-Bassoon and Munc13-Bassoon, normalized to Bassoon within each synapse. *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Staining

    Enrichment of other scaffolding proteins within nanocolumns a , Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm. b , Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Enrichment of other scaffolding proteins within nanocolumns a , Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm. b , Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm. *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Scaffolding

    Retrograde plasticity of synaptic nanoscale alignment a , Distributions of synaptic RIM1/2 and PSD-95 for control and post-LTP induction conditions with NCs highlighted. b–c , Across-condition comparison of enrichment index and percentage of NCs enriched (n = 45, 87 and 42 synapses for control, LTP, and APV, respectively). d , Distributions of RIM1/2 and PSD-95 for conditions following NMDA stimulation. Scale 100 nm. e–i , Across-conditions comparison of RIM1/2 and PSD-95. Dark red in i represents RIM1/2 NCs enriched with PSD-95 and light red the unenriched NCs. n = 61, 96, 77 and 74 synapses for control, NMDA, washout, and APV, respectively. j , Schematic summarizing the reorganization of NCs during NMDA-induced plasticity and recovery. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Retrograde plasticity of synaptic nanoscale alignment a , Distributions of synaptic RIM1/2 and PSD-95 for control and post-LTP induction conditions with NCs highlighted. b–c , Across-condition comparison of enrichment index and percentage of NCs enriched (n = 45, 87 and 42 synapses for control, LTP, and APV, respectively). d , Distributions of RIM1/2 and PSD-95 for conditions following NMDA stimulation. Scale 100 nm. e–i , Across-conditions comparison of RIM1/2 and PSD-95. Dark red in i represents RIM1/2 NCs enriched with PSD-95 and light red the unenriched NCs. n = 61, 96, 77 and 74 synapses for control, NMDA, washout, and APV, respectively. j , Schematic summarizing the reorganization of NCs during NMDA-induced plasticity and recovery. *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques:

    Protein enrichment within nanocolumns a, Enrichment index between RIM1/2 and PSD-95. The left insets are replicas of Fig. 3e , and the enrichment index is defined as the average of the first three bins in the enrichment profile (boxed), i.e. normalized localization density within 60 nm from the projection center of a given NC. Filled points show RIM1/2 relative to PSD-95 NCs, open points show PSD-95 relative to RIM1/2 NCs. Same randomizations as in Fig. 3e and depicted again in b. **p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Protein enrichment within nanocolumns a, Enrichment index between RIM1/2 and PSD-95. The left insets are replicas of Fig. 3e , and the enrichment index is defined as the average of the first three bins in the enrichment profile (boxed), i.e. normalized localization density within 60 nm from the projection center of a given NC. Filled points show RIM1/2 relative to PSD-95 NCs, open points show PSD-95 relative to RIM1/2 NCs. Same randomizations as in Fig. 3e and depicted again in b. **p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Protein Enrichment

    Plasticity within nanocolumns a , Changes in the localization density within RIM1/2 (red) and PSD-95 (blue) NCs under control, 5 min NMDA treatment, 25 min washout, and NMDA + APV treatment conditions. b–h , Reorganization of RIM1/2 and GluR2/3 under control, 5 min NMDA treatment, 25 min washout conditions examples ( b ), comparison of whole synaptic cluster sizes ( c ), NC number per synapse ( d ), localization density within NCs ( e ), enrichment indices ( f ), percentage of NCs that were enriched ( g ), and NC volumes ( h ). Note that similar to the results from the RIM1/2-PSD-95 analyses, only those RIM1/2 NCs that were enriched with GluR2/3 (dark red) were increased in volume. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Plasticity within nanocolumns a , Changes in the localization density within RIM1/2 (red) and PSD-95 (blue) NCs under control, 5 min NMDA treatment, 25 min washout, and NMDA + APV treatment conditions. b–h , Reorganization of RIM1/2 and GluR2/3 under control, 5 min NMDA treatment, 25 min washout conditions examples ( b ), comparison of whole synaptic cluster sizes ( c ), NC number per synapse ( d ), localization density within NCs ( e ), enrichment indices ( f ), percentage of NCs that were enriched ( g ), and NC volumes ( h ). Note that similar to the results from the RIM1/2-PSD-95 analyses, only those RIM1/2 NCs that were enriched with GluR2/3 (dark red) were increased in volume. *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques:

    Vesicle release proteins form subsynaptic nanoclusters a , Color-coded schematic of studied synaptic proteins. b , Synapses labeled with RIM1/2 and PSD-95 imaged using 3D-STORM (10 nm pixels) compared to wide-field composite (bottom corner, 100 nm pixels), scale 2 μm. Boxed synapse enlarged in original (top) and rotated (bottom) angles, scale 200 nm. c , En-face (top) and side (bottom) views of a RIM1/2 cluster showing all localizations and local density maps for a measured synaptic cluster compared to a simulated randomized cluster, scale 200 nm. d , Auto-correlation functions of measured RIM1/2 (n = 115), isolated non-synaptic small groups of localizations due to repetitive switching of fluorophores (n = 42), and simulated randomized (n = 115) distributions. e , RIM1/2 nanoclusters (NCs, red) within a synaptic cluster. f , Distribution of NC distances from the center of synapses normalized to randomized distribution. g , Molecule density inside NCs normalized to synaptic average. h , Average number of protein NCs per synapse. i , Cumulative distributions of NC volumes. *p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: Vesicle release proteins form subsynaptic nanoclusters a , Color-coded schematic of studied synaptic proteins. b , Synapses labeled with RIM1/2 and PSD-95 imaged using 3D-STORM (10 nm pixels) compared to wide-field composite (bottom corner, 100 nm pixels), scale 2 μm. Boxed synapse enlarged in original (top) and rotated (bottom) angles, scale 200 nm. c , En-face (top) and side (bottom) views of a RIM1/2 cluster showing all localizations and local density maps for a measured synaptic cluster compared to a simulated randomized cluster, scale 200 nm. d , Auto-correlation functions of measured RIM1/2 (n = 115), isolated non-synaptic small groups of localizations due to repetitive switching of fluorophores (n = 42), and simulated randomized (n = 115) distributions. e , RIM1/2 nanoclusters (NCs, red) within a synaptic cluster. f , Distribution of NC distances from the center of synapses normalized to randomized distribution. g , Molecule density inside NCs normalized to synaptic average. h , Average number of protein NCs per synapse. i , Cumulative distributions of NC volumes. *p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Labeling, Isolation

    RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Journal: Nature

    Article Title: A transsynaptic nanocolumn aligns neurotransmitter release to receptors

    doi: 10.1038/nature19058

    Figure Lengend Snippet: RIM1-mEos3.1 PALM identifies NCs a. Neurons coexpressing RIM1-mVenus (a generous gift from Pascal Kaesar) and Syn1a-CFP colocalize to the same boutons. Right panels show enlargement of areas within the white boxes, scale 5 μm (left) and 1 μm (right). b. Neurons expressing RIM1-mVenus immunostained for RIM1/2 and Bassoon. Arrowheads point to some colocalized AZs, scale 2 μm. c. Immunofluorescence intensity of transfected cells normalized to nearby untransfected cells show 3.74 ± 0.11-fold overexpression of RIM and 1.24 ± 0.03-fold increase in Bassoon (n = 262 synapses/7 cells). d. Photon count distribution of RIM1-mEos3.1 (3997 localizations). e. Same boutons shown in Fig. 2 visualized using 5 × Nearest Neighbor Density (NND) as a measure of local density. f–h. Cumulative distributions of PALMed RIM1 NCs diameter, area, and number, respectfully, identified using adapted Tesseler analysis and 5 × NND analysis (n = 65/13). i. RIM1 localization density as a function of radial distance from pHuse localizations. (See Supplementary Tables for statistics.) j. Mean distance from pHuse localizations as a function of local density measured by 5 × NND (Raw data R = 0.23***, n = 26/13). k. Proportion of pHuse localizations within 40 nm of a RIM1 localization as a function of RIM1 local density measured by 5 × NND (R = 0.35***). n given in synapses/experiments unless otherwise specified, ***p

    Article Snippet: For comparisons of Munc13 or RIM1/2 with Bassoon made using 3D-STORM, mouse anti-Bassoon (1:500, Enzo) was used with either rabbit anti-RIM1/2 (1:500; Synaptic Systems No. 140203) or rabbit anti-Munc13 (1:500; Synaptic Systems No. 126103).

    Techniques: Expressing, Immunofluorescence, Transfection, Over Expression

    Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, Rim1, and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.

    Journal: The Journal of Neuroscience

    Article Title: A Protein Interaction Node at the Neurotransmitter Release Site: Domains of Aczonin/Piccolo, Bassoon, CAST, and Rim Converge on the N-Terminal Domain of Munc13-1

    doi: 10.1523/JNEUROSCI.1255-09.2009

    Figure Lengend Snippet: Aczonin binds native Bassoon, CAST, Munc13, and Rim. A , B , Aczonin, Bassoon, CAST, Munc13, and Rim mutually coimmunoprecipitate from brain lysate. Brain lysate was subjected to immunoprecipitation with antibodies against Aczonin, Bassoon, Munc13, Rim1, and controls [preimmune sera and antibodies against cadherin, Nbea and PKA–RIIα], as indicated at the top, and Western blots of precipitates were probed with antibodies as indicated on the sides. See Materials and Methods for antibody details. In A , the weak background signals in the RIIα panel (anti-Acz and anti-Nbea precipitate lanes) are the Ig heavy-chain bands; in the top of B , Aczonin signals in the anti-Rim and anti-Munc13 precipitate lanes are weak and do not reproduce well. C , D , The recombinant second coiled-coil region of Aczonin pulls down native Bassoon, and the third coiled-coil region of Aczonin pulls down native CAST, Munc13, and Rim. Aczonin sequence regions were expressed as GST or His-tagged fusion proteins (overview in C , with construct designations and amino acid intervals) and used in pull-down experiments with brain lysate. Pull-down pellets were analyzed by Western blotting ( D ) with antibodies against the proteins indicated on the panels. Pull-down constructs to the left of the vertical line in D were His-tagged fusion proteins immobilized by covalent coupling, and constructs to the right were GST fusion proteins immobilized by glutathione affinity binding. The smear in the Aczp18p19 lane is attributable to residual fusion protein left behind in the gel, because the same Aczonin sequence was used for pull down and for generating the antibody with which the blot was probed; the lane was blank when the blot was reprobed with an antibody against a different Aczonin sequence (data not shown). GST constructs with the Munc13-binding zinc-finger domain of Rim1 (Rim43, Rim5/8) were used as positive controls. Constructs also used for immunization are boxed in C . The schematic of the Aczonin molecule represents sequences with low interspecies conservation as narrow bars and sequences with high interspecies conservation as wide bars; sequences partially conserved between Aczonin and Bassoon are shaded. Zing-finger (Zn), coiled-coil (CC1–CC3), polyproline (PP), PDZ, C2A and C2B domains, and the short splice variant ending with a SKRRK amino acid sequence are also indicated.

    Article Snippet: A Rim1 monoclonal antibody (clone 26; immunogen, amino acids 602-723 of rat Rim1) was from BD Biosciences (“Rim1-BD”), and Munc13 monoclonal antibodies were purchased from BD Biosciences [clone 32; immunogen, amino acids 621-834 of rat Munc13-1 (“Munc13-BD”); described by the vendor as panMunc13 reactive] and Synaptic Systems [immunogen, amino acids 1399-1736 of rat Munc13-1 (“Munc13-SS”); described by the vendor as Munc13-1 specific].

    Techniques: Immunoprecipitation, Western Blot, Recombinant, Sequencing, Construct, Binding Assay, Variant Assay

    Conditional knock-out of RIM2 in rod photoreceptors is under way as early as 3 weeks of age. A , Forms of RIM1 and RIM2 found in the brain and retina of wild-type and transgenic RIM1ff/2ff mice. Expression of RIM 1α ≫ 1β in the retina, which is similar to the proportions in whole-brain lysates (probed with Ms monoclonal anti-RIM1). The floxed RIM2ff ) and therefore all of the RIM2 in the transgenic animal is RIM2-cfp, which is revealed as an upward shift in its migration compared with brain lysates from wild-type animals (the cfp tag is nonfluorescent in the retina). Relative RIM2 expression levels in the brain are RIM 2α > 2β ∼ 2α-splice variants (α-sp.) and, in the retina, 2α and 2α-sp are equally expressed, but 2β is not detected. The regional distribution of RIM1/2 was examined by immunostaining cryosections of retina ∼10 μm thick ( B – F ). B , Expression of RIM1 is largely confined to the IPL and this is demonstrated with a rabbit polyclonal anti-RIM1 from Synaptic Systems (left) and a mouse monoclonal against RIM1 from BD Transduction Laboratories (right). Scale bar, 25 μm. C , D ), demarcating the OPL. The section in D is from a LMOPcre + transgenic animal, which shows an intensely stained patch of Cre in the ONL that is designated as a Cre region (samples in C and D are from RIM1ff/2ff littermates, 3 weeks of age). Bottom overview cropped to the OPL is labeled “all probes” because the signals are derived from anti-RIM2 staining (rabbit polyclonal, Alexa Fluor488, shown in green), the cone marker PNA (linked to Alexa Fluor 568, shown in red), and anti-CtBP2 (Ms monoclonal, Alexa Fluor 648, shown in blue). E , F , Regions selected for presentation at higher resolution. The control condition presented in C and E and the “Cre-poor region” in F show that the RIM2 signal overlaps extensively with CtBP2/Ribeye (aqua). The “Cre-rich region” in F reveals a drop in RIM2 staining. G , H ) and Synaptotagmin1 (Syt1, ∼60 kDa) were used as presynaptic markers, whereas CtBP2 (∼45 kDa) in the context of Western analysis represents the transcription factor that is expressed throughout the retina. I , Graphical summary of the densitometry measurements from Western analysis. Only RIM2 was reduced (see text for values) and no apparent change in the intensity of the synaptic markers was witnessed at 3 weeks. Data are presented as mean ± SE.

    Journal: The Journal of Neuroscience

    Article Title: RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors

    doi: 10.1523/JNEUROSCI.0658-15.2015

    Figure Lengend Snippet: Conditional knock-out of RIM2 in rod photoreceptors is under way as early as 3 weeks of age. A , Forms of RIM1 and RIM2 found in the brain and retina of wild-type and transgenic RIM1ff/2ff mice. Expression of RIM 1α ≫ 1β in the retina, which is similar to the proportions in whole-brain lysates (probed with Ms monoclonal anti-RIM1). The floxed RIM2ff ) and therefore all of the RIM2 in the transgenic animal is RIM2-cfp, which is revealed as an upward shift in its migration compared with brain lysates from wild-type animals (the cfp tag is nonfluorescent in the retina). Relative RIM2 expression levels in the brain are RIM 2α > 2β ∼ 2α-splice variants (α-sp.) and, in the retina, 2α and 2α-sp are equally expressed, but 2β is not detected. The regional distribution of RIM1/2 was examined by immunostaining cryosections of retina ∼10 μm thick ( B – F ). B , Expression of RIM1 is largely confined to the IPL and this is demonstrated with a rabbit polyclonal anti-RIM1 from Synaptic Systems (left) and a mouse monoclonal against RIM1 from BD Transduction Laboratories (right). Scale bar, 25 μm. C , D ), demarcating the OPL. The section in D is from a LMOPcre + transgenic animal, which shows an intensely stained patch of Cre in the ONL that is designated as a Cre region (samples in C and D are from RIM1ff/2ff littermates, 3 weeks of age). Bottom overview cropped to the OPL is labeled “all probes” because the signals are derived from anti-RIM2 staining (rabbit polyclonal, Alexa Fluor488, shown in green), the cone marker PNA (linked to Alexa Fluor 568, shown in red), and anti-CtBP2 (Ms monoclonal, Alexa Fluor 648, shown in blue). E , F , Regions selected for presentation at higher resolution. The control condition presented in C and E and the “Cre-poor region” in F show that the RIM2 signal overlaps extensively with CtBP2/Ribeye (aqua). The “Cre-rich region” in F reveals a drop in RIM2 staining. G , H ) and Synaptotagmin1 (Syt1, ∼60 kDa) were used as presynaptic markers, whereas CtBP2 (∼45 kDa) in the context of Western analysis represents the transcription factor that is expressed throughout the retina. I , Graphical summary of the densitometry measurements from Western analysis. Only RIM2 was reduced (see text for values) and no apparent change in the intensity of the synaptic markers was witnessed at 3 weeks. Data are presented as mean ± SE.

    Article Snippet: Other antibodies used were as follows: anti-RIM2, rabbit polyclonal (Synaptic Systems, 140–303); anti-RIM1, rabbit polyclonal (Synaptic Systems, 140–003) and anti-RIM1 mouse monoclonal (BD Transduction Laboratories, 610907); anti-Fodrin/α-spectrin, clone AA6, mouse monoclonal (ICN Biomedicals); lectin PNA-Alexa Fluor 568 (Invitrogen, ): anti-Ribeye/U2656 rabbit polyclonal antibody ( ); anti-CtBP2 mouse monoclonal (BD Transduction Laboratories, 612044); anti-Cre Recombinase, rabbit polyclonal (Novagen, 69050); and anti-Cre recombinase, mouse monoclonal (Covance, MMS-106P).

    Techniques: Knock-Out, Transgenic Assay, Mouse Assay, Expressing, Mass Spectrometry, Migration, Immunostaining, Transduction, Staining, Labeling, Derivative Assay, Marker, Western Blot