Journal: The Journal of Cell Biology

Article Title: Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology

doi: 10.1083/jcb.201911122

Figure Lengend Snippet: Mitofusins form puncta that localize to ER tubules. (A) Representative image of a U-2 OS cell expressing mCh-Sec61β (ER, green) and immunostained with antibody against Mfn1/2 (magenta) and Tom20 (gray). Magnified merged image of insets show punctate distribution of Mfn1/2 (magenta) relative to mitochondria and ER (in right panels). (B) Graph of the MCC measured for Mfn1/2 relative to the ER, mitochondria, or a 90° rotated Mfn1/2 relative to the ER. Error bars represent SEM. ***, P < 0.0001 by two-tailed paired t test; normality was determined by Shapiro–Wilk test; n = 12 regions. (C) Representative live-cell image of a cell expressing GFP-Mfn1 (magenta), mCherry-Sec61β (green), and mito-BFP (gray). Magnified merged images of inset show GFP-Mfn1 puncta relative to mitochondria and ER (right panels). (D) Graph of the MCC of GFP-Mfn1 relative to ER, mitochondria, or a 90° rotated MFN1 relative to the ER. Significance determined as in B; ***, P = 0.0007, n = 10 regions. (E) Time-lapse images of live cells as in C reveals tracking of GFP-Mfn1 (magenta) puncta with ER tubules (green) over time. (F) Graph shows that the vast majority of Mfn1 puncta (84%) maintain their association with ER tubules during the entire 2-min video ( n = 50 puncta from six cells). (G) Representative images of U-2 OS cells, GFP-Mfn1 or GFP-Mfn1-E209A. Insets and dashed lines correspond to the location of the linescans graphed below. (H) Linescan of Mfn1 signal on mitochondria showing the mean of five cells with seven linescans (as in ) from each cell. Error bars represent SEM. Cartoon describes where measurements are taken and how signal is distributed on mitochondria. (I) Graph showing the difference in ratio of maximum punctate signal over the mean diffuse mitochondrial signal from GFP-Mfn1 and GFP-Mfn1-E209A. **, P = 0.0026 by two-tailed t test. Scale bars for whole cell image = 5 µm; insets = 1 µm. Error bars represent SEM.

Article Snippet: RA was amplified from RA-nuclear export sequence (RA-NES) plasmid (Addgene 61019), and RA-C1 vector was derived from pAcGFP-C1 as above; Sec61β was inserted into the XhoI/KpnI sites.

Techniques: Expressing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology

doi: 10.1083/jcb.201911122

Figure Lengend Snippet: Mfn1 puncta and fusion events localize to bona fide ER contact sites. (A) Cartoon demonstrating the ddFP system: the monomers are targeted to the mitochondria (B) and the ER (RA), and an increase in fluorescent signal is indicative of dimerization at MCSs. (B) Histogram of ddFP spot values taken from regions of ER alone (monomer) and regions where ER crossed mitochondria (dimer) validates signal increase upon dimerization ( n = 120 spots). (C) Representative image of cells expressing GA-MFF (green dimerization partner), B-MFF, and mito-mScarlet (magenta) shows that B-MFF is not punctate without an ER partner. (D) Representative image of a cell expressing the ddFP system (RA-Sec61β + B-MFF) with a marker for ER (green) and mitochondria (mito-BFP; gray). Note that a bright ddFP signal (magenta) accumulates at a position where an ER tubule crosses the mitochondria. Linescan analysis of dashed line along the mitochondria shows relative FI of ddFP domains relative to ER tubule crossing. (E) Most but not all ER tubule crossings (75%) label positive for ddFP dimerization, indicating that ∼75% of ER tubule crossings are bona fide MCSs. (F) Representative image of cells expressing the ddFP system (magenta), GFP-Mfn1 (green), and mito-BFP (gray). (G) Magnified merged images and linescan analysis of F show the position of ddFP-positive domains relative to GFP-Mfn1 puncta along mitochondria. (H) Graph of MCC for GFP-Mfn1 puncta relative to ddFP-positive domains, mitochondria, or 90° rotated GFP-Mfn1 puncta shows that Mfn1 puncta and ddFP ER MCSs strongly overlap. (I) Representative merged time-lapse images of a ddFP-positive fusion event in a cell expressing the ddFP system (magenta) and mito-BFP (gray). (J) Linescan analysis of dashed line in I shows the relative FI of ddFP-positive domains along the length of a mitochondrion at the location of fusion (white arrow). (K) Percentage of fusion events that are ddFP positive for ER contact (90%) as in I relative to the mean coverage of ddFP (13%) on mitochondria. Scale bar: 5 µm in whole cell images; insets, 1 µm.

Article Snippet: RA was amplified from RA-nuclear export sequence (RA-NES) plasmid (Addgene 61019), and RA-C1 vector was derived from pAcGFP-C1 as above; Sec61β was inserted into the XhoI/KpnI sites.

Techniques: Expressing, Marker

Journal: The Journal of Cell Biology

Article Title: Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology

doi: 10.1083/jcb.201911122

Figure Lengend Snippet: Depolarized mitochondria are rescued by ER-associated fusion. (A) Representative image of a U-2 OS cell expressing GFP-Mfn1 (yellow), mito-BFP (blue), and SNAP-Sec61β (green) and loaded with a dye that accumulates within the matrix of mitochondria in proportion to membrane potential [30 nm] TMRE (red). Note the occurrence of an Mfn1 punctum at an ER tubule crossing at the border of electrically uncoupled mitochondrial segments. (B) Another example as in A. (C) Table of mitochondria imaged as in A and B shows that ER MCSs and Mfn1 puncta mark the boundaries between the vast majority of electrically uncoupled segments. (D) Cells expressing an OMM marker (SNAP-OMP25) were loaded with [30 nM] TMRE to indicate membrane polarization. Individual mitochondria were irradiated with 405 nm (in dashed boxes) to photobleach the OMM marker and depolarize the membrane. Depolarized mitochondria were imaged for 8 min following irradiation. In this example, a photobleached and depolarized mitochondrion fuses ∼382 s after irradiation, and this fusion leads to an increase in OMM FI and a subsequent increase in TMRE signal (indicating rescue of membrane potential). (E) Plot of relative FI of SNAP-OMP25 and TMRE signal for experiment in D. (F) Representative time-lapse image series of irradiated mitochondria in cells expressing GFP-Drp1 (to label ER MCSs), SNAP-OMP25, and loaded with [30 nM] TMRE. Dashed box indicates irradiated area. Arrows indicate location of fusion event. (G) Relative FI plots of SNAP-OMP25 and TMRE signal from the time-lapse in F. Note that the TMRE signal recovers shortly after the time of OMM FI increase by fusion (arrow). (H) Two groups of mitochondria in (D; control and irradiated) were imaged over time following irradiation and were assigned to three categories: fission first, fusion first, and no event. The graph shows the distribution of categories between control and irradiated. *, P < 0.05 by χ 2 analysis ( n = 78 mitochondria for control, n = 81 mitochondria for irradiated). (I) As in H except for the addition of transfected Drp1 ( n = 91 mitochondria for control, n = 88 mitochondria for irradiated). *, P < 0.05 by χ 2 analysis. (J) Fusion rescues mitochondrial membrane potential. The mean normalized TMRE FI was measured for the 10 frames directly preceding fusion and the 10 frames immediately after fusion for individual irradiated mitochondria. The two measurements were connected by lines. Red lines indicate an increase in TMRE intensity after fusion (mean FI increase = 65%), and black lines indicate no change or a decrease in TMRE intensity. Events are categorized from irradiation experiments in U-2 OS cells expressing SNAP-OMP25 alone and SNAP-OMP25 with GFP-Drp1. Scale bars, 1 µm.

Article Snippet: RA was amplified from RA-nuclear export sequence (RA-NES) plasmid (Addgene 61019), and RA-C1 vector was derived from pAcGFP-C1 as above; Sec61β was inserted into the XhoI/KpnI sites.

Techniques: Expressing, Membrane, Marker, Irradiation, Control, Transfection

Journal: bioRxiv

Article Title: Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor

doi: 10.1101/2024.05.31.596797

Figure Lengend Snippet: Example micrographs of HCT116 cells co-expressing emerin, LAP2β or BAF construct tagged with FKBP-GFP at the N-terminus (green), with Stargazin-mCherry-FRB, pMito-mCherry-FRB ( C ) or a control with no FRB construct expressed (as indicated), stained with DAPI (blue). All samples were treated with rapamycin (200 nM) for 30 min prior to fixation. Shown are single slices from z-stacks of an interphase and metaphase cell. Scale bars, 10 µm; insets, 3× expansion of ROI.

Article Snippet: The following plasmids were available from Addgene or previous work: pEBFP2-N1 (Addgene #54595); EGFP-BAF (Addgene #101772); Emerin pEGFP-C1 (637) (Addgene #61993); FKBP-alpha(740-977)-GFP (Addgene #100731); FKBP-GFP-Sec61β (Addgene #172442); FRB-mCherry-Giantin (Addgene #186575); GFP-EEA1 (Addgene #42307); Lamp1-mCherry-FRB (Addgene #186576); LAP2 Full I pAcGFP-N1 monomeric GFP (1317) (Addgene #62044); LBR pEGFP-N2 (646) (Addgene #61996); pFKBP-GFP-C1 ( ); pMaCTag-P05 (Addgene #120016); pMito-mCherry-FRB (Addgene #59352); pMito-dCherry-FRB (Addgene #186573); pmScarlet-Giantin-C1 (Addgene #85048); SH4-FRB-mRFP (Addgene #100741); Stargazin-dCherry-FRB (Addgene #172444); Stargazin-GFP-LOVpep (Addgene #80406); Stargazin-mCherry-FRB (Addgene #172443) ( ; ; ).

Techniques: Expressing, Construct, Control, Staining