Journal: Cells

Article Title: Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level

doi: 10.3390/cells11111827

Figure Lengend Snippet: Detection of GFP-tagged long and short OGA isoforms in HEK-293T cells. HEK-293T cells were transfected with mitochondrial-targeted mCherry and either GFP-tagged short or long OGA isoforms, fixed, stained with DAPI for visualization of the nuclei (blue), and analyzed by confocal microscopy in the GFP (green) and mCherry (red) channels. Merge images are shown. ( A ) The long OGA isoform was detected in the cytosol and the nucleus but poorly co-localized with mito-cherry labeled mitochondria. ( B ) Co-localization of the short OGA isoform with mito-mCherry in a cell expressing GFP-tagged short OGA. ( C , D ) Structured illumination microscopy (SIM) confirmed localization of S-OGA in the mitochondria. ( C ) Separated SIM and merge images of this cell in the GFP, mCherry, and Dapi channels are shown. ( D ) Densitometric analysis of the different signals detected along the arrow shown on the upper panel indicates co-localization of S-OGA-GFP with mito-mCherry (green: S-OGA-GFP, red: mito-mCherry, blue: Dapi staining of the nucleus, white bar scale: 5 μm). Representative images are shown.

Article Snippet: HEK-293T cells plated on polylysine-coated coverslips were transfected with cDNA (100 ng/40,000 cells) coding for mitochondrial-targeted mCherry (mCherry-Mito-7, a gift from Michael Davidson (Addgene plasmid # 55102) [ ]) and either GFP-tagged long or short OGA isoforms (gifts from John A. Hanover).

Techniques: Transfection, Staining, Confocal Microscopy, Labeling, Expressing, Microscopy

Journal: Nature

Article Title: Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway

doi: 10.1038/s41586-020-2078-2

Figure Lengend Snippet: (a) Schematic of screen. (b) Comparison of gene scores (defined in Methods) from the screen in untreated and oligomycin-treated conditions. (c) Knockdown of DELE1 quantified by qPCR ( n = 3 technical replicates). (d) Reporter activation in cells expressing a non-targeting control sgRNA (NTC) or DELE1 sgRNAs (mean ± s.d., n = 3 culture wells). (e) Immunoblot of endogenous ATF4. Cells were treated with 1.25 ng/mL of oligomycin for 16 hrs (Oli) or untreated (Un). Left , representative blot; Right , quantification of n = 2 blots. (f) ATF4 reporter activation in cells transiently overexpressing (OE) DELE1-mClover and expressing sgRNA to knockdown (KD) HRI where indicated. (mean ± s.d., n = 3 culture wells). (g) ATF4 reporter activation in cells transiently overexpressing (OE) HRI and expressing sgRNA to knockdown (KD) DELE1 where indicated. (mean ± s.d., n = 3 culture wells). (h) DELE1-mClover expression from the AAVS1 safe-harbor locus is not sufficient to induce the ATF4 reporter in the absence of stress, but complements DELE1 knockdown upon oligomycin treatment (mean ± s.d., n = 3 culture wells). (i) Co-localization of DELE1-mClover with the mitochondrial-targeted mRuby (Mito7-mRuby). Scale bar, 7 μm. Similar results for n = 2 culture wells.

Article Snippet: HEK293T cells and derived cell lines were seeded in the 8-well chamber slide (Ibidi #80824) for 24 h and transfected with DELE1 truncated constructs and/or plasmid mito7-mRuby (a gift from Michael Davidson, Addgene plasmid # 55874) as indicated, using TransIT-Lenti Transfection Reagent (Mirus #MIR 6600).

Techniques: Comparison, Knockdown, Activation Assay, Expressing, Control, Western Blot

Journal: Nature

Article Title: Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway

doi: 10.1038/s41586-020-2078-2

Figure Lengend Snippet: ( a-b ) Zoom-out views for two-color 3D-STORM super-resolution images of DELE1-mClover vs. TOM20 and Hsp60. Two-color DELE1-mClover (magenta) vs. (a) TOM20 (green) or (b) Hsp60 (green), followed by the two separated color channels. Scale bars: 1 μm. The boxed regions correspond to , . Similar results were obtained in n = 3 independent experiments. ( c ) Biochemical fractionation indicates that DELE1 associates with mitochondrial membranes. Cells stably expressing DELE1-mClover were fractionated into cytosol and mitochondria. Mitochondria were incubated in either isotonic buffer (10 mM Tris HCl, pH 6.7, 0.15 mM MgCl 2 0.25mM sucrose,1 mM DTT, protease inhibitor cocktail (Sigma #5892970001)) or H 2 O (extreme hypotonic condition) for 5 min, followed by centrifugation (10000 xg for 10 min) to separate the supernatant and pellet. The pellet was either dissolved with RIPA buffer or incubated with 0.1M NaCO 3 (pH=11.4) for 30 min at 4°C. Supernatant and pellet from NaCO 3 -treated samples were collected for WB. Unlike soluble matrix protein LonP1 and HSPD1, which can be extracted with H 2 O incubation, only small proportion of DELE1 is present in the supernatant. NaCO 3 can extract the majority of the DELE1 S but not DELE1 L , which is similar to the pattern of mitochondrial membrane protein VDAC, suggesting that DELE1 L is more likely a membrane associated protein. n = 2 independent experiments. For gel source data, see . ( d ) The indicated DELE1-mClover constructs were transiently overexpressed in reporter cells, and reporter induction was quantified by flow cytometry (mean ± s.d., n = 3 culture wells). Subcellular localization was evaluated by microscopy in cells also expressing mitochondrial-targeted mRuby. ( e ) Lack of co-localization of transiently expressed DELE1ΔN100-mClover and DELE1ΔN148-mClover (green) with the mitochondrial stain Mitotracker (red). Scale bar, 7 μm. Similar results obtained in n = 2 culture wells. ( f ) Increased detection of DELE1-mClover outside the mitochondria upon oligomycin treatment. 3D-STORM super-resolution images of stably expressed DELE1-mClover (colors indicating depth in the z dimension) in untreated cells ( left ) and cells treated with 1.25 ng/mL oligomycin for 16 h ( right ). Areas boxed in red in the top panels are shown in higher magnification in the bottom panels. Similar results were obtained in n = 3 independent experiments. ( g ) Co-localization of transiently expressed HRI-mClover and DELE1-mClover with the mitochondrial-targeted mRuby (Mito7-mRuby). Scale bar, 7 μm. n = 1 culture well.

Article Snippet: HEK293T cells and derived cell lines were seeded in the 8-well chamber slide (Ibidi #80824) for 24 h and transfected with DELE1 truncated constructs and/or plasmid mito7-mRuby (a gift from Michael Davidson, Addgene plasmid # 55874) as indicated, using TransIT-Lenti Transfection Reagent (Mirus #MIR 6600).

Techniques: Fractionation, Stable Transfection, Expressing, Incubation, Protease Inhibitor, Centrifugation, Membrane, Construct, Flow Cytometry, Microscopy, Staining

Journal: Frontiers in Neuroscience

Article Title: Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons

doi: 10.3389/fnins.2018.00402

Figure Lengend Snippet: Altered mitochondrial trafficking in M 1 R overexpressing neurons was rescued by antagonist treatment. (A) Representative images (top panels) of the first frame of a series of live cell time-lapse images showing the expression of DsRed2Mito7 in the mitochondria of GFP/M 1 R–GFP expressing neurons. The circles represent mitochondria identified by MTrackJ plugin, which then tracked migration through time in a series of time lapse images and calculated velocity of specific mitochondria. White/blue line represents neurite trace. The bottom panel represents Kymographs generated from live cell time-lapse images. The Kymograph was generated using ImageJ Kymograph plugin. The X -axis represents the physical location of mitochondria on the neurite, and the Y -axis represents the location of mitochondria in time. Streak of particles traversing the kymograph from left to right in angular lines indicates retrograde/anterograde mitochondrial motion. (B) Whisker plot showing mitochondrial velocity. DRG neurons were cultured in LGF media supplemented with 100 nM MT7 or 1 μM pirenzepine for 48 h following transfection. N = 40 from three independent experiments, p -value by one-way ANOVA followed by Dunnett’s multiple comparisons test. (C) Binning of the entire data set presented in (B) . (D,E) Immunofluorescence images showing mito7-RFP and β-tubulin-III staining in the GFP/GFP-M 1 R expressing neurites. White arrows indicate continuous/discontinuous tubulin cytoskeleton in GFP/GFP-M 1 R expressing neurites, respectively. Scale bar: 5 μm.

Article Snippet: Mitochondrial trafficking in GFP or GFP-M 1 R overexpressing neurons was monitored using LSM510 confocal live cell imaging and involved co-transfection of sensory neurons with GFP/GFP-M 1 R and DsRed2Mito7 plasmids (Addgene plasmid #55838, a gift from Dr. Michael W. Davidson, Florida State University), respectively.

Techniques: Expressing, Migration, Generated, Whisker Assay, Cell Culture, Transfection, Immunofluorescence, Staining

Journal: Light, Science & Applications

Article Title: Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

doi: 10.1038/s41377-021-00536-3

Figure Lengend Snippet: a , b Color-coded Mito-pHRed absolute pH maps of live HeLa cells after the application of 20 µM CCCP for 6 h, for cells without ( a ) and with ( b ) the co-expression of fluorescently untagged Parkin. c Distribution of the measured pH values in each mitochondrion for a , b . d Excitation spectra on our setup for the five fluorescent species unmixed to enable absolute pH imaging with three additional FP markers. e Unmixed images of color-coded Mito-pHRed absolute pH map, mOrange2-Parkin, PhiYFP-LC3, and LAMP1-Clover for two Parkin-expressing live HeLa cells after the application of 20 µM CCCP for 4 h. f Overlay of the images in e . g Zoom-in of the white box in f , as well as its separation into different channels. Cyan, magenta, and red arrowheads point to individual mitochondria only marked by Parkin, LC3, and LAMP1, with respective matrix pH values quantified from the enclosed Mito-pHRed signals indicated. Orange arrowhead points to a mitochondrion marked by both Parkin and LC3. h Distribution of the Mito-pHRed-quantified matrix pH of individual mitochondria in the sample labeled by the different markers (color diamonds), compared to that in untreated HeLa cells (black circles). Shaded band: typical range of cytoplasmic pH. Experiments were performed with 8-wavelength excitation at 10 fps (0.8 s acquisition time for each spectral image). Scale bars: 5 µm ( a , b ); 10 µm ( e , f ); 2 µm ( g )

Article Snippet: For the expression of untagged Parkin, pCMV-Parkin was constructed by inserting Parkin (cloned from Addgene #89299, a gift from Michael Lazarou) into an N1 cloning vector (cut from Addgene #54642, a gift from Michael Davidson). mOrange2-Parkin was constructed by first inserting mCherry-Parkin (cloned from Addgene #59419, a gift from Richard Youle) into an N1 cloning vector (cut from Addgene #54642, a gift from Michael Davidson), and then replacing mCherry with mOrange2 (cloned from Addgene #57969, a gift from Michael Davidson) between the BmtI and BspEI sites.

Techniques: Expressing, Imaging, Labeling