Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Expression of SFK members after mitochondrial fractionation of HEK293T cells. Argonaute 2 (Argo2) was used as a marker for the cytosolic fraction (Cyto). Voltage- dependent anion channel (VDAC), cytochrome c oxidase subunit 4 (COX4), and optic atrophy-1 (OPA1) were used as markers for the mitochondrial fraction (Mito). WCL, whole cell lysates. Representative images from 3 independent experiments. B. CSK-KD increases SFK activity in mitochondria. Representative immunoblotting images from HEK293T cells stably overexpressing CSK-shRNA (CSK-KD cells). Control cells (CTR) were prepared by overexpression of PLKO.1 empty vector. Immunoreactive bands were visualized by chemiluminescent immunoassay. Argo2 was used as a marker for the Cyto. VDAC, and COX4 were used as markers for the Mito. Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used for loading control for the WCL. IP, immunoprecipitation. IB, immunoblotting. C. Increased P-Tyr of Mfn2 in HEK293T cells by CSK knockdown. CTR and CSK-KD cells were transiently transfected with either Mfn2-HA or empty vector. Immunoprecipitation (IP) was performed with HA antibody, and P-Tyr of Mfn2 was detected by a general P-Tyr antibody. D. Summary data of C ( n =4). Band intensity of immunoprecipitated P-Tyr was normalized to Mfn2. * p <0.05. E. Overexpression of c-Src increases P-Tyr of Mfn2. HEK293T cells stably overexpressing Mfn2-HA was transiently transfected with either empty vector (pcDNA3.1(+) as a control), c-Src-WT, or c-Src-DN. IP was performed with HA antibody, and P-Tyr of Mfn2 was detected by a general P-Tyr antibody. Band intensity of immunoprecipitated P-Tyr was normalized to Mfn2. F. Summary data of E ( n =4). * p <0.05. G. Overexpression of c- Src, but not other SFK members increases P-Tyr of Mfn2. HEK293T cells stably overexpressing Mfn2-HA were transiently transfected with either empty vector (pWZL- Neo-Myr-Flag-DEST as a control), c-Src, CSK, Fyn, Lyn, or Fgr. IP was performed with HA antibody, and P-Tyr of Mfn2 was detected by a general P-Tyr antibody. H. Summary data of G ( n =9). Band intensity of the immunoprecipitated P-Tyr band was normalized by that in HA. * p <0.05. I. Submitochondrial localizations of marker proteins for mitochondria digestion assay. Red dots indicate the antibody binding sites. IMM, inner mitochondrial membrane; IMS, intermembrane space of mitochondria; Omi, human HTRA2; CyD cyclophilin D. J. Representative immunoblotting pattern of mitochondria digestion for umtiochodrial localization of c-Src. PK, Proteinase K; Dig, digitonin.

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Expressing, Fractionation, Marker, Activity Assay, Western Blot, Stable Transfection, shRNA, Over Expression, Plasmid Preparation, Immunoprecipitation, Transfection, Binding Assay

Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Schematic diagram of a FRET donor and acceptor for assessing ER-mitochondria interaction. TMD, trans membrane domain. B. Expression of FRET sensors in HEK293T cells. Representative images of FRET detection in HEK293T cells transiently transfected with mt-CFP, ER-YFP, or both constructs. Scale bar, 20 µm. C. (Top): Representative near-infrared fluorescence immunoblotting of whole cell lysates obtained from HEK293T stably overexpressing Mfn2-shRNA (Mfn2-KD) or PLKO.1 (control; CTR). IP, immunoprecipitation. IB, immunoblotting. (Bottom): Summary data ( n =3). Mfn2 band intensity was normalized by that in tubulin. * p <0.05. D. Mfn2 knockdown decreases ER-mitochondria interaction. Representative images of FRET detection in Mfn2-KD or CTR cells transfected with mt-CFP and ER-YFP. Scale bar, 20 µm. E. Summary of normalized FRET/CFP ratio from panel D ( n =104 and 100, for CTR and Mfn2-KD, respectively). * p <0.05. F. CSK knockdown decreases ER-mitochondria interaction. Representative images of FRET detection in CSK-KD or CTR cells transfected with mt-CFP and ER-YFP. Scale bar, 20 µm. G. Summary of normalized FRET/CFP ratio from panel E ( n =132 and 135, for CTR and Mfn2-KD, respectively). * p <0.05. H . Effect of a SFK inhibitor PP2 on FRET/CFP ratio. CSK-KD or CTR HEK293T cells were co-transfected with FRET biosensors and treated with PP2 (30 µM) or DMSO (vehicle) for 30 min before FRET measurements ( n =58, 50, 54, and 26 for CTR+DMSO, CTR+PP2, CSK-KD+DMSO, and CSK-KD+PP2, respectively). * p <0.05. I. Effect of a c-Src on FRET/CFP ratio. c-Src-WT, c-Src-DN, or pWZL empty vector (as a control) were co-transfected with FRET biosensors into HEK293T cells. ( n =110, 117, and 118 for CTR, c-Src-WT and c-Src-DN respectively). * p <0.05. J. Effect of a c-Src on FRET/CFP ratio in Mfn2-KD cells. c-Src-WT, or pWZL (as a control) were co-transfected with FRET biosensors into HEK293T cells. ( n =110, 117, and 118 for CTR, c-Src-WT and c-Src-DN respectively). * p <0.05.

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Expressing, Transfection, Construct, Fluorescence, Western Blot, Stable Transfection, shRNA, Immunoprecipitation, Plasmid Preparation

Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Representative images of transmission electron microscopy (TEM) in HEK293T cells. White lines, green lines, yellow area, and blue area represent OMM, MAM interface, ER area, and mitophagosome area, respectively. MAM distances from three points (Red lines) were averaged to estimate the distance between ER and OMM. Scale bars, 500 nm. B. Representative TEM images in CSK-KD and CTR HEK293T cells. Scale bar, 500 nm. Arrows point to the ER and mitochondria interaction sites. C. Summary data from B. Number in parentheses indicates the total number of images analyzed in each group * p <0.05.

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Summary data of mitochondrial size, form factor (FF), and AR in CTR and CSK-KD HEK293T cells ( n =4249 and 4394, respectively). Cells were transfected with matrix- targeted DsRed (mt-RFP), and analyzed with live cell imaging using confocal microscopy. N.S., not significant. B. Comparison of AR and FF of individual mitochondria in CSK-KD cells vs. CTR cells. C. (Top) : Representative immunoblotting of LC3-I/LC3-II obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with Torin1 are shown as a positive control that changes LC3-I/LC3-II ratio. (Bottom): Summary data for LC3-1/LC3-II ratio ( n =4). D. Mitophagosome number counted from TEM images of CTR and CSK-KD HEK293T cells ( n =60, and 83, respectively) (see also ). E. SFK activation does not promote ER stress. (Top): Immunoblotting of ER stress markers, Grp94, Grp78, and CHOP obtained from CTR and CSK-KD HEK293T cells. Lysates from HEK293T cells treated with thapsigargin (TG) were shown as a positive control that increases ER stress. (Bottom): Summary data ( n =4). Grp94 and Grp78 band intensities were normalized to tubulin. * p <0.05.

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Transfection, Live Cell Imaging, Confocal Microscopy, Western Blot, Positive Control, Activation Assay

Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Expression of mitochondria-targeted Ca 2+ -sensitive biosensor, mt-RCaMP1h, in HEK293T cells co-transfected with mitochondrial matrix-targeted GFP (mt-GFP). B. Representative traces of changes in Ca 2+ concentration in the mitochondrial matrix ([Ca 2+ ] m ) uptake trace in CTR or CSK-KD HEK293T cells in response to G αq/11 protein- coupled P2Y receptor stimulation by 1 mM ATP. [Ca 2+ ] m was assessed by mt- RCamp1h. C. Summary data of B ( n =36 and n=56 for CTR and CSK-KD cells, respectively). * p<0.05. D. Averaged traces of the changes in the fluorescence intensity of tetramethylrhodamine, ethyl ester (TMRE) in response to the treatment with CCCP in CTR and CSK-KD HEK293T cells. Initial TMRE intensity before CCCP treatment was normalized with the value after CCCP treatment. E. Summary data of A ( n =50 and n=65 for CTR and CSK-KD cells, respectively). * p <0.05

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Expressing, Transfection, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Tyrosine phosphorylation of mitofusin 2 regulates endoplasmic reticulum-mitochondria tethering

doi: 10.1101/2022.02.21.481295

Figure Lengend Snippet: A. Representative time-lapse images (top) and traces (bottom) of HEK293T cells transfected with mitochondrial targeted ro-GFP2-Orp1 (mt-roGFP2-Orp1) upon stimulation with an oxidative stress inducer, tert-butyl hydroperoxide (T-BH, 10 mM) and a reducing agent, dithiothreitol (DTT, 100 mM). B. Summary data of A ( n =32, and 22 for CTR and CSK-KD cells, respectively). * p <0.05. C. Oxygen consumption rate (OCR) measurements obtained over time (min) using an extracellular flux analyzer in CTR and CSK-KD cells. Data from 3 independent experiments. D. Summary data of C ( n =3 for each group). * p <0.05.

Article Snippet: HEK293T cells (kindly provided by Dr. Keigi Fujiwara (University Texas MD Anderson, Houston TX) and IP 3 R1-null HEK293 cells (Kerafast, Inc., Boston, MA) [ ] were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone GE Healthcare, Little Chalfont, UK) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate and 1% L-glutamine, 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U/mL penicillin, 100 µg/mL streptomycin (Genesee Scientific, El Cajon, CA) at 37°C with 5% CO 2 in a humidified incubator.

Techniques: Transfection

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Endogenous IP 3 R1s form puncta. a In-gel fluorescence of lysates from EGFP-IP 3 R1 HeLa cells (GR) and control (WT) cells demonstrates that the only fluorescence is associated with EGFP-IP 3 R1 (green arrow). Results typical of four gels. Positions of selected M r markers (kDa) are shown ( a , c , d ). b TIRFM images of EGFP-IP 3 R1 HeLa cells showing a marker for the ER lumen (mCherry-ER). The merged image and an enlargement of the boxed area show co-localization of EGFP-IP 3 R1 with mCherry-ER (Pearson’s coefficient with Costes’ automatic threshold = 0.93 ± 0.02; Costes P value = 1.00, n = 4 cells). Scale bar = 5 µm (2 µm for enlargement). c Western blots (WBs) for IP 3 R1-3 show expression of tagged (green arrow, ~290 kDa) and untagged (black arrow, ~260 kDa) IP 3 R1 in GR and WT cells, respectively. Expression of IP 3 R subtypes in GR cells is shown relative to control (WT) cells (%, mean ± SD, n = 3 for IP 3 R2 and IP 3 R3, n = 4 for IP 3 R1). Comparisons of band intensities using paired Student’s t -tests indicated no significant differences between WT and EGFP-IP 3 R1 cells. d WB (IP 3 R1-3 antibodies) from lysates of EGFP-IP 3 R1 HeLa cells after immunoprecipitation with GFP-Trap. Eluate lanes were loaded with sample equivalent to 1.5 times the amounts loaded in the lysate lanes. Numbers show % of each subtype detected in the pull-down ( n = 2). e Photobleaching of a punctum showing the final bleaching step (bracket) and the initial fluorescence (dashed line) used to calculate the total number of fluorophores ( n ). FU, fluorescence units. f Single-step photobleaching results (284 puncta from five cells, Supplementary Fig. ) were used to calculate the number of tetrameric IP 3 Rs per punctum (8.4 ± 7)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence, Marker, Western Blot, Expressing, Immunoprecipitation

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs form mobile and immobile puncta. a Time-lapse TIRFM images (0.6-s intervals) of EGFP-IP 3 R1 in cells expressing mCherry-ER. Track of a single particle, with the first and last positions shown by white and yellow arrows, respectively. Scale bar = 5 µm. b Representative epifluorescence image of an EGFP-IP 3 R1 HeLa cell with perinuclear (blue) and peripheral (magenta) regions highlighted for FRAP analysis (circular bleached area, radius = 1.84 μm). The boxed area is enlarged to show pre- and post-bleach (after 120 s) images of the peripheral region. Scale bars = 5 µm. c Normalized fluorescence intensities recorded from peripheral or perinuclear regions in a typical FRAP experiment with live and fixed EGFP-IP 3 R1 HeLa cells. d , e Summary results show mobile fractions ( M f , mean ± SEM) ( d ) and diffusion coefficients ( D , mean and all values) ( e ) for perinuclear (25 cells) and peripheral regions (26 cells). **** P < 0.0001, * P < 0.05, two-tailed Student’s t -test. f Distribution of fluorescence intensities for individual mobile and immobile puncta. Inset shows distribution for the brightest immobile puncta. g Relative numbers of mobile and immobile puncta, and distribution of fluorescence between them (%). Results ( f , g ) are from time-lapse TIRFM images of 10 cells (mean ± SD). h , i Analysis of bleaching steps of brightest and dimmest puncta in fixed cells was used to determine the step amplitude ( h ) and number of steps ( i ) for each punctum (Supplementary Fig. ). Mean ± SD, with 23–25 puncta analysed in each of two cells. *** P < 0.001, **** P < 0.0001, Student’s t -test

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Expressing, Single Particle, Fluorescence, Diffusion-based Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs within mobile puncta do not exchange with immobile puncta. a TIRFM images of FRAP experiment show images before, immediately after and 60 min after bleaching. Bleached area shown by white rectangle. Times shown as h:min:s. b , c Enlarged images of yellow boxed area in a . Immobile puncta were identified by overlaying two frames (30 s apart) using pseudocolours for each (green and magenta), such that immobile puncta appear white in the overlay (iii, iv) (Supplementary Fig. ). Images were captured before photobleaching ( b ) and after recovery for 15 min ( c ). Enlarged areas (red boxes in iii) are shown in (iv). Scale bars ( a – c ) = 10 µm. d Enlargements (blue boxes in b iv and c iv) show images before and 15 min after bleaching. Scale bar = 2 µm. Abundant green and magenta puncta in the post-bleach images ( c , and lower in d ) indicate rapid exchange of mobile EGFP-IP 3 R, while the scarcity of white puncta suggests very slow exchange of immobile EGFP-IP 3 R1. e Summary results show fluorescence recovery ( F / F 0 , %, mean ± SEM) from three cells (26 ± 15 immobile puncta per cell were identified in the initial images). f Example trace from a region centred on an immobile punctum (circled in first image) shows stepwise increase and decrease in fluorescence intensity as a mobile punctum (arrows) moves through the immobile punctum (Supplementary Movie ). Scale bar = 1 μm

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: EGFP-IP 3 Rs are diffusely distributed within puncta. a Examples of TIRFM and subsequent STORM images of EGFP-IP 3 R1 puncta. Scale bar = 0.5 µm. b , c Before STORM, mobile and immobile puncta were identified using TIRFM and by overlaying pseudocoloured images collected at 10-s intervals (Supplementary Fig. ). Examples of TIRFM and STORM images show immobile ( b ) and mobile puncta ( c ). Overlays show that EGFP-IP 3 R1s are diffusively distributed within mobile and immobile puncta. Scale bars = 100 nm. The yellow squares show approximate dimensions of an IP 3 R (20 nm × 20 nm). d Analysis of STORM images shows the area enclosing all EGFP-IP 3 R1s within each punctum and the density of localization events within the punctum for mobile and immobile puncta (mean ± SD for 4–6 puncta). * P < 0.05, Student’s t -test, relative to immobile puncta. Results show that mobile puncta are smaller and contain fewer IP 3 Rs than immobile puncta. e Many IP 3 Rs within puncta are too diffusively distributed to allow direct interactions between them. Although we used a non-oligomerizing EGFP to tag IP 3 R1 and minimize potential artefacts, the diffuse spacing of some EGFP-IP 3 R1s confirms that puncta are not formed by interactions between EGFP

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques:

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: IP 3 Rs move by diffusion and by directed motion along microtubules. a TIRFM image used for single-particle tracking shows characteristic reticular ER. Scale bar = 5 µm. b Trajectories of all puncta from the region defined by white lines in a . Colours indicate different trajectories. c Enlargement showing trajectories within the boxed area in a . d Average mean squared displacement (MSD) for all puncta within each cell plotted against time. Results are from five cells, with 225–926 puncta analysed in each. e Pooled results from five cells (2,698 tracks). f Examples of single-particle trajectories (collected at 100-ms intervals) for an immobile punctum (i) or mobile puncta moving by diffusion (ii) or directionally (iii). g Distribution of EGFP-IP 3 R1 puncta between mobility states. Trajectories of 1,509 puncta from five cells were analyzed by TraJClassifier (Supplementary Fig. ). h Wide-field images, typical of nine cells, show EGFP-IP 3 R1 puncta, microtubules (mCherry-tubulin) and their co-localization. Final panel shows an enlargement of the boxed area in the overlay. Scale bar = 10 µm (2 µm in enlargement)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Diffusion-based Assay, Single-particle Tracking, Single Particle

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Ca 2+ puffs evoked by photolysis of caged-IP 3 occur at immobile IP 3 Rs. a TIRFM image of a single cell loaded with EGTA and Cal-590 shows immobile IP 3 Rs (white, see Supplementary Fig. ) and Ca 2+ puffs (red) evoked by photolysis of caged-IP 3 . Boxed area of the overlay (iii) is shown enlarged in (iv). Scale bars = 10 µm. b Co-localization of Ca 2+ release and immobile IP 3 R puncta shown by their fluorescence intensity profiles along the dashed line in a iv. EGFP profiles were captured immediately before and ~30 s after recording Cal-590 fluorescence. c Enlargements of four puff sites highlighted in a show co-localization of each Ca 2+ release event with an immobile IP 3 R punctum. d Enlargements of site 2 (from a ) show four successive puffs generated at the same immobile punctum. Scale bars = 2 μm ( c , d ). e Temporal profiles of Cal-590 fluorescence changes (Δ F / F 0 ) show Ca 2+ puffs last ~200 ms. f Summary shows the fraction of Ca 2+ puffs evoked by histamine (432 puffs in two cells, mean ± range) or photorelease of IP 3 (871 puffs in three cells, mean ± SD) occurring at immobile IP 3 R puncta. Events and puncta were considered to occur at the same site if the centre of mass of the peak change in Cal-590 fluorescence was within 6 pixels (0.96 µm) of the punctum. g Comparison of fluorescence intensity distributions of all detected EGFP-IP 3 R1 puncta, and puncta associated with Ca 2+ puffs evoked by photolysis of caged-IP 3 . Results are from three cells. Dashed lines show 50th and 95th percentiles for the intensities of all puncta

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence, Generated

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Depletion of ER Ca 2+ stores causes native STIM1 to accumulate at functional ER-PM junctions adjacent to immobile IP 3 R puncta. a , b Representative TIRFM images of EGFP-IP 3 R1 HeLa cells fixed and immunostained for STIM1 before ( a ) or after treatment with thapsigargin (Tg, 1 µM, 15 min) to deplete the ER of Ca 2+ ( b ). Overlaid images of Tg-treated cells show no significant co-localization of STIM1 and IP 3 R puncta (Pearson’s coefficient with Costes’ automatic threshold: 0.331 ± 0.026, n = 7 cells). c Distribution of mobile (green and magenta) and immobile (white) IP 3 R puncta in Tg-treated cell. Scale bars ( a – c ) = 10 µm. d Enlargements of the boxed regions in b show that immobile IP 3 R puncta (identified before fixation ( c ), with all shown by arrowheads) abut STIM1 puncta without coinciding with them. Scale bars = 2 µm. e Fluorescence intensity profiles for EGFP-IP 3 R1 and STIM1 across the lines shown in d . Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown. f Co-localization of CFP-STIM1 (pseudocoloured green) and mCherry-Orai1 (red) puncta in a Tg-treated HeLa cell. We used tagged proteins because available antibodies do not reliably detect endogenous Orai1. Scale bar = 10 µm (2 μm in enlargement)

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Functional Assay, Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: STIM1 translocates to ER-PM junctions adjacent to immobile IP 3 R puncta. a , b TIRFM images show that thapsigargin (Tg, 1 µM, 5 min) causes formation of STIM1-mCherry puncta at ER-PM junctions (i). Immobile IP 3 Rs (ii, white, identified from overlays of pseudocoloured EGFP-IP 3 R distribution at 30-s intervals) abut STIM1 puncta (iii). Enlarged image of the boxed area shows all immobile EGFP-IP 3 R1 (arrows) and STIM1-mCherry without pseudocolours for clarity (iv). Scale bars = 10 µm (i–iii), 2 µm (iv). c Similar overlay images from three additional Tg-treated cells, with all immobile IP 3 R punta identified with arrows. Scale bars = 2 µm. d Fluorescence intensity profiles for EGFP-IP 3 R1 measured at 30-s intervals (green and magenta) and STIM1-mCherry for transects drawn across Tg-treated cells. Results show that immobile IP 3 Rs (white, where green and magenta coincide) and STIM1 after store depletion are juxtaposed, but not perfectly aligned. Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown; 84 ± 9% (mean ± SD, n = 3 cells) of the centroids of immobile EGFP-IP 3 R1 puncta were within twice their radius (2 r = 0.64–0.96 μm) of a STIM1 punctum. e STIM1-mCherry fluorescence was measured before ( F 0 ) and after Tg treatment ( F Tg ) at ROI with twice the radius of underlying mobile or immobile EGFP-IP 3 R1 puncta. Summary results show the change in mCherry fluorescence (Δ F = ( F Tg − F 0 )/ F 0 , mean ± SEM, n = 3 cells, 29–33 puncta). * P < 0.05, Student’s t -test. f , g Density of STIM1-mCherry and EGFP-IP 3 R1 puncta from randomly selected peripheral regions of cells (where puncta are most clearly separated) ( f ) and the separation between any STIM1 puncta identified within the randomly selected regions and the nearest STIM1 or immobile EGFP-IP 3 R1 punctum ( g ). h Frequency distributions for the separations of STIM1 puncta from mobile and immobile EGFP-IP 3 R1 puncta. Whereas 28% of mobile IP 3 R puncta were within 300 nm of a STIM1 punctum, only 10% of immobile IP 3 R puncta fell within this distance. Results ( f – h ) are from four cells. ** P < 0.01, Student’s t -test

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Fluorescence

Journal: Nature Communications

Article Title: Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions

doi: 10.1038/s41467-017-01644-8

Figure Lengend Snippet: Ca 2+ signals evoked by SOCE occur alongside immobile IP 3 Rs. Clustered IP 3 Rs evoke Ca 2+ puffs when they bind IP 3 and then respond to Ca 2+ released by a neighbouring IP 3 R. Only a small fraction of all IP 3 Rs, namely immobile clusters close to the PM, are licensed to respond (green). The licensed IP 3 Rs are adjacent to the junctions where STIM1 and Orai interact to mediate SOCE, which is itself locally regulated by Ca 2+ passing through Orai (red lines). We suggest that the juxtaposition of responsive IP 3 Rs and the SOCE machinery allows local depletion of ER Ca 2+ stores to activate SOCE, while sparing it from inhibition by the large Ca 2+ fluxes through IP 3 Rs

Article Snippet: An EGFP-IP 3 R1 construct that replicates the linker produced by gene editing of endogenous IP 3 R1 in HeLa cells (Supplementary Fig. ) was transiently expressed in HEK cells in which all endogenous IP 3 R genes had been disrupted (Kerafast, Boston, MA, USA) .

Techniques: Inhibition