Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: The K 2P channel TASK-1 interacts with the SNARE protein syntaxin-8. (A) The topology of TASK-1 and stx8. (B) Membrane yeast two-hybrid screen with TASK-1 or TASK-3 as bait and stx8 or mutants thereof as prey. The Q179A mutant of stx8 cannot assemble with other SNARE proteins; in the Δ100–140 mutant the linker between the Hc domain and the SNARE domain was excised. (C) RT-PCR analysis of TASK-1 and endosomal SNARE proteins in human brain and in A549 cells. Asterisks represent nonspecific PCR products. (D) Coimmunoprecipitation of stx8 and TASK-1 endogenously expressed in A549 cells. The complex containing TASK-1 was precipitated from cell lysate with a TASK-1–specific antibody from Alomone (APC-024), and a Western blot of the precipitate was probed with TASK-1, stx8, and stx7 antibodies (left); the cell lysate (input) was used as positive control. Coimmunoprecipitation with an unrelated immunoglobulin G antibody (Santa Cruz Biotechnology) was used as a negative control (right).

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Two Hybrid Screening, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Negative Control

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Coexpression of TASK-1 with stx8 or stx7 in CHO cells and Xenopus oocytes. (A) Current–voltage relation of rTASK-1 expressed in CHO cells. The currents were measured using voltage ramps from −120 to +40 mV at pH 7.4 (black curve) and 6.0 (red curve). (B) TASK-1 current–voltage relation measured in the same batches of CHO cells 48 h after transfection of TASK-1 alone (black curve) and after cotransfection of TASK-1 with stx8 (green curve); mean values ± SEM of n = 28 cells. (C) Mean outward currents ± SEM measured in CHO cells at 0 mV after transfection with rat TASK‑1 alone (black) and after cotransfection of TASK-1 with stx8 (green) or stx7 (orange). (D) Typical current–voltage relation measured 48 h after injection of human TASK-1 cRNA (black curve) and after coinjection of TASK-1 and stx8 cRNA. For experiments with human TASK-1 we used the NQ TASK-1 mutant, which displays a higher current amplitude ( Zuzarte et al. , 2009 ; Materials and Methods ). (E) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV after injection of hTASK‑1 or hTASK-3 cRNA alone (black) or together with 1.5, 3, or 6 ng stx8 cRNA per oocyte as indicated. (F) Mean outward currents ± SEM measured in Xenopus oocytes at 0 mV measured after injection of hTASK‑1 cRNA alone (black) or together with 6 ng cRNA encoding stx8, VAMP8, vti1b, or stx7. (G) Mean surface expression of HA-tagged hTASK-1 channels (measured in relative light units [RLUs]) in Xenopus oocytes after injection of TASK-1 cRNA alone or together with 6 ng of stx8 or stx8 Q179A . (H) Mean hTASK-1 current measured in Xenopus oocytes after injection of hTASK-1 cRNA alone or together with stx8 or stx8 Q179A . In all bar graphs the number of oocytes or CHO cells from which the data were obtained is indicated in brackets. Note that in the series of experiments shown in E, F, and H, coinjection of 6 ng of stx8 cRNA caused a reduction of TASK-1 current to values between 13 and 23% of control, illustrating that there was a certain degree of variability among different batches of oocytes. For this reason, TASK-1 (and other) currents with and without coinjection of a second cRNA were always compared in the same batch of oocytes (measured on the same day); normalized current amplitudes of at least three different batches are combined in the bar graphs.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Transfection, Cotransfection, Injection, Mutagenesis, Expressing

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Dissection of the interacting regions of stx8 and TASK-1. (A) Topology of stx8, stx7, and the stx8/stx7 chimeras. (B) TASK-1 currents measured in Xenopus oocytes expressing TASK-1 and stx8 or stx8/stx7 chimeras. (C) Normalized hTASK-1 currents measured in Xenopus oocytes expressing hTASK-1 and stx8 or deletion mutants of stx8. (D) Normalized currents measured in Xenopus oocytes expressing TASK-3/TASK-1 or TASK-1/TASK-3 chimeras alone or together with stx8 or stx8Q179A. (E) Schematic drawing of TASK-3/TASK-1 and TASK-1/TASK-3 chimeras.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Dissection, Expressing

Journal: Molecular Biology of the Cell

Article Title: Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

doi: 10.1091/mbc.E13-10-0592

Figure Lengend Snippet: Analysis of endocytosis of TASK-1 using an antibody uptake assay. (A) COS-7 cells expressing HA epitope–tagged TASK-1 (or TASK-1 mutants) and stx8 (or stx8 mutants) were incubated with an anti-HA antibody at 4°C to label the channels at the cell surface and then warmed to 37°C to initiate internalization. After incubation at 37°C for 30 min, the anti-HA–labeled channels that remained on the surface were detected with a secondary antibody labeled with Alexa Fluor 594 (red). Then the cells were permeabilized, and the internalized channels were detected with a different secondary antibody, labeled with Alexa Fluor 488 (green). The measurements were carried out 48 h after transfection of TASK-1, TASK-1 mutants, stx8, stx8 mutants, or stx7. Note that rTASK‑1 Y317A corresponds to hTASK-1 Y300A . (B) Statistical evaluation of the antibody uptake assay under different conditions. The ratio between the fluorescence of internalized channels (green) and channels at the cell surface (red) was calculated at 0 and 30 min after heating to 37°C; number of cells is indicated in brackets. Scale bars, 50 μm.

Article Snippet: Proteins were transferred to nitrocellulose membrane and probed with either mouse anti-myc antibody (1:1000; Santa Cruz Biotechnology), rabbit anti-GFP antibody (1:1000; Abcam, Cambridge, United Kingdom), anti–TASK-1 (1:1000; Alomone), anti-stx8 (1:1000; BD Biosciences, Heidelberg, Germany), or anti-stx7 (1:1000; Acris Antibodies, Herford, Germany).

Techniques: Expressing, Incubation, Labeling, Transfection, Fluorescence

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Localization of SNAREs on early endosomes by using immunocytochemistry. (a) Fluorescence micrographs showing endosomes labeled with dextran-Alexa 488 (green channel) immunostained for syntaxin 13 (Left) and use 1 (Right) (red channel). To determine colocalization, linescans were performed through the intensity centers of green endosomes (examples indicated by white lines). (b) Representative linescan analysis, obtained from the images shown in a, showing intensity profiles of green (endosomes) and red (antibody-staining) signals. (c) Colocalization between early endosomes and SNARE proteins as determined by linescan analysis and correlation (see Materials and Methods). A correlation coefficient of 1 represents complete colocalization, whereas a correlation coefficient of ≈0 represent independent distribution. Coint, simultaneous labeling with dextran-Alexa 488 and dextran-Alexa 594 (positive control); 2nd AB, omission of the primary antibody (negative control); syphy, synaptophysin; syx, syntaxin; eb, endobrevin/VAMP8; β-tub, β-tubulin; tfr, transferrin receptor. Values are means of two independent experiments with 60 analyzed endosomes each. Error bars indicate the range of values.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Immunocytochemistry, Fluorescence, Labeling, Staining, Positive Control, Negative Control

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Effects of adding recombinant SNAREs on fusion of early endosomes. (a) Effects of Q-SNAREs considered to be involved in the fusion of early endosomes, including syntaxin 16 (syx 16), syntaxin 13 (syx 13), vti1a, and syntaxin 6 (syx 6). Values are means ± SEM of 7–10 independent experiments. (b) Effects of late endosomal and neuronal Q-SNAREs, including syntaxin 7 (syx 7), vti1b, syntaxin 8 (syx 8), syntaxin 1 (syx 1), and SNAP-25. Values are means ± SEM of 7–13 independent experiments. (c) Effects of R-SNAREs, including VAMP4, synaptobrevin/VAMP2 (syb), endobrevin/VAMP8 (eb), cellubrevin/VAMP3, Ti-VAMP/VAMP7, VAMP5, ykt6, and sec22. Values are means ± SEM of three to seven independent experiments. Individual Q-SNAREs were used at 12 μM, with R-SNAREs tested at 25 μM.

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Recombinant

Journal:

Article Title: Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

doi: 10.1073/pnas.0511138103

Figure Lengend Snippet: Proteoliposomes containing the Q-SNAREs syntaxin 13, syntaxin 6, and vti1a show no specificity for R-SNAREs in fusion. (a) Fusion with liposomes containing VAMP4, endobrevin, or synaptobrevin. Fusion was monitored by fluorescence dequenching due to dilution of labeled phospholipids with unlabeled phospholipids during fusion (see Materials and Methods) and normalized to maximal fluorescence measured after adding detergent at the end of the reaction. As control, Q-SNARE liposomes were preincubated for 1 h at room temperature with purified endobrevin or synaptobrevin lacking the transmembrane domain (soluble fragment, final concentration of 30 μM) before starting the fusion reaction. (b–d) Dose-dependent inhibition by soluble R-SNAREs of fusion between liposomes containing the Q-SNAREs syntaxin13, vti1a, and syntaxin 6 and liposomes containing the R-SNARE VAMP4. Measurements were performed at 37°C with an overall protein concentration of 3 μM in the liposomes. The soluble R-SNAREs were added simultaneously with the acceptor liposomes at the start of the reaction (concentrations represent final assay concentrations).

Article Snippet: The following antibodies were described previously: rabbit sera specific for syntaxin 7 and syntaxin 8 ( 37 ), endobrevin ( 18 ), and vti1b ( 43 ), mouse monoclonal antibodies specific for synaptophysin Cl 7.2 ( 44 ), synaptobrevin Cl 69.1 ( 45 ), and SNAP-25 Cl 71.1 ( 46 ) (all available from Synaptic Systems, Göttingen, Germany), and syntaxin 1 HPC-1 ( 47 ).

Techniques: Fluorescence, Labeling, Purification, Concentration Assay, Inhibition, Protein Concentration