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