er-gcamp6f  (Alstem Inc)

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: FT293 cells were transfected with GCaMP6f 24 hr before imaging. Cells were then incubated with 3 µM DRAQ7 and with or without 50 ng/mL doxycycline to induce APOL1 expression. Cells were imaged via widefield from 4.5 to 30 hr post induction, and dual color images were taken every 10 min. Scale bars = 50 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: HEK293 cells were co-transfected with RUSH-APOL1 and GCaMP6f for 24 hr. Prior to imaging, 3 µM DRAQ7 was added and cells were treated with or without 80 µM biotin to release APOL1 from the ER. Cells were imaged via widefield from 1 to 18 hr post-biotin treatment, and dual color images were taken every 5 min. Scale bars = 20 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: CHO cells were co-transfected with RUSH-APOL1 and GCaMP6f for 24 hr. Prior to imaging, cells were treated with or without 80 µM biotin to release APOL1 from the ER. Cells were imaged via widefield from 1 to 12 hr post biotin treatment, and images were taken every 5 min. Scale bars = 20 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: CHO cells were co-transfected with RUSH-APOL1, GCaMP6f, and ER-LAR-GECO for 24 hr prior to imaging. On the day of the experiment cells were treated with 80 µM biotin and imaged for 0.5–12 hr post treatment. Cells that displayed the established phenotype of Ca 2+ influx followed by cell swelling were selected. Dual color images were taken every 5 min. Scale bars = 20 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: CHO cells were co-transfected with RUSH-G2, GCaMP6f, and FliCR for 24 hr prior to imaging. On the day of the experiment cells were treated with 80 µM biotin and imaged every 5 min from 0.5 to 12 hr post-biotin (the cells represented here, from Figure 5 , underwent lysis by 5 hr). Cells that displayed the established phenotype of Ca 2+ influx followed by cell swelling were selected. Scale bar = 20 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: CHO cells were co-transfected with RUSH-G2 and GCaMP6f for 24 hr prior to imaging. On the day of the experiment cells were treated with 80 µM biotin and incubated in media containing 150 mM Na + (130 mM NaCl), 85 mM Na + and 65 mM choline + , or 85 mM Na + and 65 mM K + . The G2-mediated Ca 2+ influx was unaffected by reduced Na + . 3 different fields are shown for each condition. Cells were imaged every 5 min from 0.5 to 12 hr post-biotin. Scale bar = 100 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques:

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: ( a ) Fluorescence traces of representative GCaMP6f-positive cells demonstrating that G1 and G2 cause a Ca 2+ influx prior to cell swelling. GCaMP6f-transfected FT293 cells were incubated with DRAQ7 followed by 50 ng/mL doxycycline to induce APOL1 expression and then imaged via widefield every 10 min for 4.5–30 hr post induction. Traces represent levels of cytoplasmic Ca 2+ over time as measured by GCaMP6f fluorescence (no DRAQ7 was observed in depicted cells). Cells are from . Scale bars = 20 μm. ( b ) High-throughput analysis revealed a significant increase of cytoplasmic Ca 2+ levels driven by G1 and G2 compared to G0. Each point is the ∆F/F 0 for an individually tracked cell and bars represent the cell population mean of GCaMP6f fluorescence. Cells were analyzed from 4 fields of view per condition, n = 1748. A one-way ANOVA multiple comparisons test was performed to compare the RRVs with G0 at the indicated timepoints. Figure 3—source data 1. FT283 cells GCaMP6f microscopy, 30 hours after induction one way ANOVA.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Fluorescence, Transfection, Incubation, Expressing, High Throughput Screening Assay, Microscopy

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: ( a ) Schematic of the RUSH system. Streptavidin was expressed with a signal peptide and KDEL allowing for localization and retention in the ER lumen along with streptavidin-binding protein (SBP) tagged APOL1. SBP binds to streptavidin causing APOL1 to be retained in the ER until synchronous release is initiated by the addition of biotin. ( b ) Time course showing that RRV cytotoxicity requires trafficking from the ER. 24 hr after transfection, HEK293 cells were treated with or without (0 hr) 80 µM biotin at the indicated times. 48 hr post-transfection, cytotoxicity was measured via release of lactate dehydrogenase. To compare cytotoxicity between biotin treated and untreated (0 hr) for respective genotypes, a two-way ANOVA with multiple comparisons was performed (n = 6). ( c ) Fluorescence traces of GCaMP6f-positive HEK293 cells showing that the G1 and G2-mediated Ca 2+ influx occurs after trafficking from the ER. GCaMP6f-transfected cells were incubated with DRAQ7 followed by 80 µM biotin to release APOL1 and were then imaged via widefield every 5 min for 1–18 hr post treatment. Cells are from . Scale bars = 20 μm. ( d ) High-throughput imaging and analysis was performed as in , demonstrating that the G1 and G2-mediated Ca 2+ influx requires trafficking from the ER. Each point is the ∆F/F 0 for an individually tracked cell and bars represent the cell population mean of GCaMP6f fluorescence. Cells were analyzed from 3 fields of view per condition, n = 1657. A one-way ANOVA multiple comparisons test was performed to compare the RRVs with G0 at the indicated timepoints.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Binding Assay, Transfection, Fluorescence, Incubation, High Throughput Screening Assay, Imaging

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: ( a ) Western blot of whole cell lysates displaying protein expression of RUSH-APOL1 in HEK293 cells 24 hr after transfection. Cells were not treated with biotin. ( b ) Fluorescent traces of all GCaMP6f-positive HEK293 cells in and ( c ) High throughput microscopy and analysis was performed as in and , validating in CHO cells the requirement of G1 and G2 trafficking from the ER to mediate a Ca 2+ influx and cell swelling. RUSH-APOL1 transfected CHO cells were treated with or without 80 µM biotin and imaged via widefield every 5 min for 1–12 hr post treatment. Each point is the ∆F/F 0 for an individually tracked cell and bars represent the cell population mean of GCaMP6f fluorescence. Representative cells from this analysis can be viewed in . Cells were analyzed from 3 different fields of view per condition, n = 882. A one-way ANOVA multiple comparisons test was performed to compare G1 and G2 with G0 at the indicated timepoints. ( d ) Fluorescent traces of all GCaMP6f-positive CHO cells from . ( e ) All cells from 4 hr after +/- biotin treatment in were directly compared via one-way ANOVA.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Western Blot, Expressing, Transfection, High Throughput Screening Assay, Microscopy, Fluorescence

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: ( a ) Validation for the simultaneous use of Ca 2+ sensors GCaMP6f and ER-LAR-GECO from a representative cell. Co-transfected cells were treated with 10 µM thapsigargin to prevent Ca 2+ reuptake in the ER, which increases cytoplasmic Ca 2+ levels (GCaMP6f) while concurrently depleting ER Ca 2+ (ER-LAR-GECO). Cells were imaged via widefield. ( b–e ) Fluorescence traces revealing that there is no ER Ca 2+ release during G1 and G2 mediated cytotoxicity. CHO cells were co-transfected with RUSH-APOL1, GCaMP6f, and ER-LAR-GECO, then treated with 80 µM biotin and imaged via widefield every 5 min for 0.5–12 hr post treatment. Cells that displayed the established phenotype of Ca 2+ influx followed by cell swelling were selected for analysis. Representative cells are from . A minimum of 5 cells were analyzed per genotype. Scale bars = 10 µm.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Biomarker Discovery, Transfection, Fluorescence

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: ( a ) Schematic of the planar lipid bilayer setup showing the sequence of cis buffer perfusions. ( b ) The APOL1 channel is readily permeable to Na + , but not choline + . In symmetrical KCl solutions E rev was determined as + 1 mV. Then, after cis perfusion with equimolar NaCl buffer (pH 7.2, horizontal bar) there was only a slight change in E rev (E rev = −2 mV; 4 mV scale). In contrast, E rev increased to + 60 mV after exchanging the cis solution for chamber buffer containing equimolar choline chloride. Substituting into the Goldman-Hodgkin-Katz equation (assuming zero permeability to chloride) gives K:Na and K:choline permeability ratios of 1.0:1.1 and 1.0:0.1 respectively. There are two breaks in the record (indicated by //), during which the perfuser was recharged with the appropriate solution. ( c ) The cytotoxicity of the RRVs in RUSH transfected HEK293 cells is significantly reduced by lowering extracellular Na + from 150 mM to 85 mM. The rescue from cytotoxicity was indistinguishable between replacement with either K + or choline + (n = 9). ( d ) RRV cytotoxicity was reduced by lowering extracellular Ca 2+ from 1.8 mM to 0.45 or 0.1125 mM (n = 12). ( e ) Reduction of both extracellular Ca 2+ and Na + (replaced by choline + ) has an additive effect in lowering RRV cytotoxicity, as seen by further rescue from cell death with 0.45 mM Ca 2+ combined with 85 mM Na + (n = 13). ( c–e ) Cell death was assayed 12 hr post-biotin treatment with the Promega MultiTox fluorescent assay. Two-way ANOVAs with multiple comparisons were performed. ( f ) RRV mediated cytotoxicity is driven by the concurrent influx of both Ca 2+ and Na + . CHO cells were co-transfected with either RUSH-G0 or G2, GCaMP6f, and the membrane voltage sensor FliCR. G2 cells exhibiting the established phenotype of Ca 2+ influx followed by cell swelling were analyzed for changes in membrane voltage (used as a surrogate for the influx of Na + ) (n = 21). G0 cells treated with biotin were analyzed for comparison (n = 7). Blue boxes and arrows indicate when the sustained increase in Ca 2+ initiates. The representative cells from this figure can be viewed in .

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Sequencing, Permeability, Transfection, Fluorescence, Membrane, Comparison

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet: Release of RUSH-G0 from the ER does not elicit a sustained increase in cytoplasmic Ca 2+ or membrane depolarization. ( a ) Fluorescent trace of a representative CHO cell transfected with the plasma membrane voltage sensor FliCR. Cells were depolarized by adding 50 mM KCl to the media while imaging. ( b ) RUSH-G0 does not lead to a sustained increase in GCaMP6f or FliCR fluorescence after biotin treatment (n = 7). Some cells elicit temporary increases in cytoplasmic Ca 2+ and membrane voltage, which may be due to signaling events or passage through the cell cycle.

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Membrane, Transfection, Clinical Proteomics, Imaging, Fluorescence

Journal: eLife

Article Title: Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

doi: 10.7554/eLife.51185

Figure Lengend Snippet:

Article Snippet: Cells were co-transfected with APOL1 RUSH vectors and one or a combination of the following Ca 2+ sensors: GCaMP6f, CMV-ER-LAR-GECO1 (A gift from Robert Campbell, Addgene 61244) , or plasma membrane voltage sensor CMV-FliCR (A gift from Robert Campbell, Addgene 74142) ( ).

Techniques: Recombinant, Construct, Mutagenesis, Sequencing, Transfection, Clone Assay, Plasmid Preparation, Stable Transfection, Microscopy, Lactate Dehydrogenase Assay, Cytotoxicity Assay, Multiplex Assay, Software