otoferlin amino acids 1  (Addgene inc)

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Schematic of sensory hair cell and neuronal presynapses. (A) Synaptic ribbons within the sensory hair cells of the cochlea position synaptic vesicles proximal to the presynaptic membrane. Otoferlin resides on synaptic vesicles, whereas Cav1.3 localizes to the presynapse. (B) The synaptic vesicles of neurons typically harbor synaptotagmin I/II and an N- or P-type calcium channel (Cav2.1 or Cav2.2). (C) Diagram of otoferlin depicting six C2 domains, labeled C2A–C2F, and the transmembrane domain (TMD).

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Membrane, Labeling

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin interacts with Loop1.3. (A, Upper) Representative SDS/PAGE showing results of immunoprecipitation of otoferlin from lysate of HEK293 cells cotransfected with YFP-otoferlin and mCherry-Loop1.3. Both otoferlin and Loop1.3 precipitate. (A, Lower) Representative SDS/PAGE showing results of immunoprecipitation of YFP from lysate of HEK293 cotransfected with YFP and mCherry-Loop1.3. YFP but not Loop1.3 precipitates. IP, immunoprecipitated pellet; total lysate, lysate before immunoprecipitation. (B) Quantitation of coimmunoprecipitation showing the fraction of each protein in the IP and supernatant (n = 3 biological replicates; error = SE). (C) Cartoon depicting smCoBRA. Titration of a fluorescently labeled ligand onto immobilized YFP-otoferlin results in colocalized fluorescent puncta. The resulting saturation curve is fitted to obtain a dissociation constant Kd. (D) Dose–response for YFP-otoferlin titrated with Loop1.3 at increasing concentrations (0.1–50 µM) of free calcium. Each data point represents the mean of three biological replicates (n = 3). Experimental data are fit with a Langmuir isotherm (solid lines). Inset depicts 0–10 µM for clarity. (E) Determined Mander’s colocalization coefficients M1 (black) and M2 (red) for a dose–response of Loop1.3 titrated onto YFP-otoferlin (0.1 µM calcium). Inset depicts 0–10 µM. (F) Dose–response for immobilized synaptotagmin I titrated with Loop1.3 in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between Loop1.3 and YFP-otoferlin is included for comparison. (G) Dose–response for immobilized synaptotagmin I titrated with the loop II–III region of Cav2.2 in in the presence of 0.1–50 μM calcium (n = 3). Inset depicts 0–10 µM for clarity. MCC, Mander's correlation coefficients.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: SDS Page, Immunoprecipitation, Quantitation Assay, Titration, Labeling, Comparison

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Loop1.3 interacts with regions of the N and C termini of otoferlin. Bead-immobilized GST-Loop1.3 cosedimented with recombinant forms of otoferlin composed of the first three C2 domains (C2ABC; Upper) or the last three domains (C2DEF; Lower). Bead-immobilized GST served as a control. Results represent the mean of three sample preparations (n = 3). Error = SEM.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Recombinant, Control

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: The pathogenic mutation L1010 reduces otoferlin–Loop1.3 interaction. (A) Titration curve of Loop1.3 with immobilized YFP-otoferlinL1010P in 50 μM free calcium. (B) Representative photobleaching time course for colocalized YFP-otoferlinL1010P-mCherry-Loop1.3 puncta. Green arrowheads represent individual bleaching steps. (C) Photobleaching distributions for YFP-otoferlinL1010P-mCherry-Loop1.3 puncta in EDTA and calcium (n =2,300 puncta). (D) Dose–response for immobilized YFP-C2DL1010P titrated with t-SNAREs in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between t-SNAREs and YFP-otoferlin is included for comparison. (E) Dose–response for immobilized YFP-C2DL1010P titrated with Loop1.3 in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between Loop1.3 and YFP-otoferlin is included for comparison. (F) CD spectrum for WT C2D and C2DL1010P.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Mutagenesis, Titration, Comparison

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin binds multiple Loop1.3 molecules. (A) Representative single-molecule photobleaching traces for Loop1.3-mCherry bound to YFP-otoferlin in the presence of 100 µM EDTA (Upper) and 200 µM calcium (Lower). Green arrowheads denote photobleaching events. (B) Single-molecule photobleaching distributions for EDTA and calcium conditions (n = 2,300).

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques:

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Multiple C2 domains mediate otoferlin–Loop1.3 interaction. (A–D) Binding curves for immobilized YFP-otoferlin C2A (A), C2B (B), C2D (C), and C2F (D) domains titrated against Loop1.3 at 0.1–50 µM free calcium. Data are fit with a Langmuir isotherm (solid lines). Insets depict 0–10 µM for clarity. Each experimental data point represents the mean value of n = 3. (E and F) Binding curves for immobilized YFP-otoferlin C2C and C2E domains titrated against Loop1.3. Colocalization between Loop1.3 and YFP-otoferlin (otoferlin amino acids 1–1,885) is included for comparison.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Binding Assay, Comparison

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin binds t-SNAREs. t-SNARE binding curve in the presence of increasing free calcium concentrations (0.1–50 µM). (A) Experimental data are fit with a Langmuir isotherm (solid lines).Each experimental data point represents the mean value of n = 3. Inset depicts 0–10 μM for clarity. (B) Mander’s coefficients M1 (black) and M2 (red) for YFP-otoferlin–t-SNARE colocalization. Inset depicts 0–10 μM for clarity. (C) Titration of t-SNARE with immobilized YFP in the presence of 0.1 or 30 µM free calcium. Each experimental data point represents the mean value of n = 3. (D) Representative single-molecule photobleaching traces for mCherry–t-SNARE bound to YFP-otoferlin in the presence of 100 µM ethylenediaminetetraacetic acid (EDTA) (Upper) and 200 µM calcium (Lower). Green arrowheads denote photobleaching events. a.u., arbitrary units. (E) Single-molecule photobleaching distributions for EDTA and calcium conditions (n = 2,300).

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Binding Assay, Titration

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Determined binding constants for the C2 domains of otoferlin with Loop1.3 or t-SNARE

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Binding Assay

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Titration of Loop1.3 or t-SNARE onto heteromeric complexes. (A) Titration of Loop1.3 with immobilized t-SNARE–YFP-otoferlin complexes at indicated calcium concentration (0.1–50 µM). (B) Titration of t-SNARE with immobilized Loop1.3-YFP-otoferlin complexes at indicated calcium concentration (0.1–50 µM). (C) Dissociation constants for YFP-otoferlin binding to either t-SNARE or Loop1.3 individually plotted as a function of calcium. (D) Dissociation constants for YFP-otoferlin heteromeric complex binding to t-SNARE or Loop1.3 plotted as a function of calcium. The shaded gray and red areas represent the calcium concentration ranges of hair cell synapses during inactive and active states, respectively.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: Titration, Concentration Assay, Binding Assay

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

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Mander’s colocalization coefficients associated with Fig. 7. M1 (black) represents the fraction of analyte prebound complex (otoferlin with t-SNARE dimer) colocalized with the titrant (Loop1.3). Colocalization coefficient M2 (red) represents the fraction of Loop1.3 titrant colocalized with the analyte prebound complex. Results depicted are in the presence of 1 μM calcium. Normalized MCC refers to the normalized Mander's correlation coefficient. Inset depicts 0–10 μM for clarity.

Article Snippet: Otoferlin amino acids 1–1,885 were amplified via PCR from a PCDNA3 vector and subcloned into the HindIII and BamHI sites of the CKAR vector acquired from Addgene (plasmid 14860).

Techniques: