(A) ICC experimental timeline: neurons were plated on day in vitro (DIV) 0, virally transduced on DIV 2, and immunostained at DIV 15, DIV 22, or DIV 27. (B) Representative images of primary …
(A) Synaptic transmission recordings experimental timeline: neurons were plated on DIV 0, virally transduced on DIV 2, electrophysiologically recorded on DIV 19–21. (B) Representative traces of …
(A) Calcium imaging experimental timeline: neurons were plated on DIV 0, co-transfected on DIV 14 with GCaMP6f calcium indicator and either BIN1-mKate2, BIN1-ΔBAR-mKate2, or mKate2 control …
(A) Localization of endogenous BIN1, LVGCC-β1, and endogenous BIN1-LVGCC-β1 interaction detected by PLA. Scale bar = 10 µm. (B) Experimental timeline: neurons were plated on DIV 0, transduced with …
(A) Model of Tau-dependent BIN1–LVGCC interaction. BIN1’s BAR domain localizes BIN1 to the plasma membrane, and PxxP motifs in Tau’s central proline-rich domain interact with the SH3 domains of BIN1 …
RMP, mV | Rin, MΩ | N, Cells | |
---|---|---|---|
Untransduced | –60.43 ± 5.36 | 843.04 ± 55.11 | 4 |
AAV-mKate2 | –59.90 ± 3.70 | 834.83 ± 41.09 | 6 |
AAV-BIN1-mKate2 | –62.40 ± 2.24 | 790.18 ± 15.74 | 7 |
One-way ANOVA, p | 0.85 | 0.36 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo sapiens) | BIN1 | NCBI | Gene ID 274 | |
Antibody | Anti-NeuN Rabbit polyclonal | abcam | Cat# ab104225; RRID:AB_10711153 | ICC (1:500), Lot #GR3321966-1 |
Antibody | Anti-GAD67 Mouse monoclonal | Millipore Sigma | Cat# MAB5406; RRID:AB_2278725 | ICC (1:500), Lot #3015328 |
Antibody | Anti-BIN1 Rabbit polyclonal | Santa Cruz | Cat# sc-30099; RRID:AB_2243399 | ICC/PLA/IP (1:500), Lot #K1605; H-100 |
Antibody | Anti-LVGCC-β1 Mouse monoclonal | abcam | Cat# ab85020; RRID:AB_1861569 | ICC/PLA/IP (1:1000), Lot #413-8RR-52 |
Antibody | Anti-Tau Rabbit polyclonal | DAKO | Cat# A0024; RRID:AB_10013724 | ICC/IP (1:1000), Lot #20031827 |
Antibody | Anti-BIN1 Mouse monoclonal | Santa Cruz | Cat# sc-13575; RRID:AB_626753 | ICC/IP (1:1000), Lot #L3014; 99D |
Cell line (Rattus norvegicus) | Primary neuron | Charles River | Fresh from E19 albino Sprague Dawley rats | |
Genetic reagent | AAV-BIN1-mKate2 | UPenn Vector Core | AAV2 | |
Genetic reagent | AAV-mKate2 | UPenn Vector Core | AAV2 | |
Sequence-based reagent | Tau ASO | PMID:23904623 IDT | 5-ATCACTGATTTTGAAGTCCC-3 | |
Sequence-based reagent | Scrambled ASO | PMID:23904623 IDT | 5-CCTTCCCTGAAGGTTCCTCC-3 | |
Commercial assay, kit | Duolink PLA kit | Millipore Sigma | Cat#s DUO92014; DUO92002; DUO92004 | |
Transfected construct | GCaMP6f | Addgene | RRID:Addgene_40755 | |
Transfected construct | mKate2 | PMID:25156556 Evrogen | Cat# FP184 | Actin was removed from the construct obtained |
Transfected construct (Homo sapiens) | BIN1 | Horizon Discovery ORFeome Collaboration Clones | OHS5894-202501160 | Isoform 1 |
Cell line (Cricetulus griseus) | CHO-K1 | Millipore Sigma | Cat# 85051005-1VL | Chinese Hamster Ovary cell line |
Transfected construct | mKate2-Tau-mKate2 | PMID:25156556 | ||
Transfected construct | Fyn-SH3-CBG | PMID:25156556 | BIN1-SH3 or LVGCC-β1-SH3 was cloned in replacing Fyn-SH3 | |
Transfected construct (Homo sapiens) | BIN1-SH3 | IDT | AAC28646.1 | Codon optimized |
Transfected construct (Homo sapiens) | LVGCC-β1-SH3 | IDT | AAA35632.1 | Codon optimized |
mKate2 Frequency, mean ±SEM | BIN1-mKate2 Frequency, mean ±SEM | mKate2 Amplitude, mean ±SEM | BIN1-mKate2 Amplitude, mean ±SEM | |
---|---|---|---|---|
sEPSCs | 1.65 ± 26.59 Hz | 3.53 ± 65.58 Hz | 31.15 ± 0.40 pA | 29.02 ± 0.53 pA |
sIPSCs | 6.82 ± 37.61 Hz | 7.57 ± 15.25 Hz | 83.07 ± 1.49 pA | 67.73 ± 0.55 pA |
E/I ratio | 0.24 ± 0.71 | 0.47 ± 4.30 | 0.37 ± 0.27 | 0.43 ± 0.97 |