Article Figures & Data
Figures
- Figure 1. Rational design of the MERLIN system and subcellular localization of its components.
(A) Scheme illustrating the structure of the BRET biosensors. The mitochondrial part of the biosensor is targeted to the MOM by the alpha-helical C-terminal domain of Bcl-xL (B33C). For ER targeting, a truncated nonfunctional variant of calnexin (sCal) is used. A fully synthetic linker system which can be combined in different ways to span a distance of up to 24-nm connects the membrane domain to the proteins of the BRET pair. (B) Confocal image of an individual Cos1 cell expressing the mitochondrial biosensor mVen-L1-B33C (green). Mitochondria were stained with MitoTracker Red (magenta). (C) Confocal image of an individual Cos1 cell expressing the ER biosensor sCal-L1-mVen (green). ER was immunostained with anti-Grp78 antibody (magenta). Scale bar 10 μM.
- Figure S1. MERLIN expression does not alter cell viability.
Top: representative images of cells expressing different-size MERLIN in the absence (left) and presence (right) of staurosporine (STS) in HCT116 cells. Bottom: quantification of cell viability upon MERLIN expression by measuring Smac-mCherry release in the presence/absence of STS and relativized to mVenus transfected cells.
- Figure 2. Systematic optimization of MERLIN.
(A, B) Scheme and saturation curve for MERLIN based on the 12-nm linker with (A) the donor targeted to the ER and the acceptor targeted to mitochondria and (B) the donor targeted to mitochondria and the acceptor targeted to the ER. (C) Maximum BRET signals for the different linker lengths and organelle localizations of the MERLIN components. (D) BRET signal for the negative controls sCal-L1-RLuc (3-nm donor) and mVen-ER5 (luminal ER protein) or mVen-H2B6 (nucleus). (E) BRET signal of the positive control mVen-L1-RLuc compared with the 3- and 6-nm linker lengths.
- Figure S2. Titration curves of quantitative BRET for different MERLIN combinations.
(A, B, C) Scheme and saturation curve for (A) 0-, (B) 6-, and (C) 24-nm linker length with the donor targeted to the ER and the acceptor targeted to the mitochondria. (D, E, F) Like (A, B, C), but with the donor targeted to mitochondria and the acceptor targeted to the ER.
- Figure S3. Characterization of stable cell lines expressing MERLIN and controls of MERLIN activity.
(A) Synthetic tether alone is not capable of energy transfer to Scal-mVenus in the presence of coelenterazine. Break from 105 to 106 a.u. (B) Transfection with increasing amounts of DNA do not affect the % of transfected cells but increases linearly the mVenus fluorescence signal per cell. (C) Validation of MERLIN with an alternative method to quantify MERCs. Quantification of MERCs by EM in HCT116 cells and MERLIN-containing HCT116 cells untreated and in presence of tunicamycin or starvation. (D, E, F) Localization of the donor and acceptor to the mitochondria and ER, respectively. Scale bar 10 μm. Stable expression of MERLIN does not affect viability nor sensitivity to apoptosis induced by treatment with 1 μM STS for 4 h, raw data and quantification, respectively. Scale bar 5 μm. (G) Left: starvation, hypoxia, or treatment with bortezomib, Taxol, or tunicamycin does not affect luciferase activity. Right: hypoxia effects in HCT116 wt cells measured by BODIPY. Scale bar 100 μm. (H) coelenterazine H concentration does not affect the BRET ratio.
- Figure 3. Validation of MERLIN.
(A, B, C, D) PDZD8 modulates ER–mitochondria distance.(A, B, C) Representative Western blot of the PDZD8 levels when transiently transfected and (B) upon silencing with siRNA_PDZD8 in HCT116 cells, whose quantification is shown in (C) (n = 3). (D) BRET signal in cells co-expressing Rluc-L1-B33C and Scal-L1-mVenus biosensor combination, in the presence of overexpressed PDZD8, the synthetic tether mTagBFP2 and PDZD8 knockdown in HCT116 cells. (**P < 0.025, ***P ≤ 0.001). t test, data are expressed as mean ± SD. (E, F) The BRET signal of MERLIN is increased in apoptotic cells. (E) Confocal images of Cos1 cells transfected with sCal-L1-mVen (green) and RLuc-L1-B33C (magenta) under healthy condition and upon apoptosis induction with 1 μM STS at different times. Scale bar 10 μM. (F) Scheme and graph showing the change of the BRET signal in apoptotic cells over time for the 12-nm linker MERLIN. Black lines represent four individual measurements and the grey line the control measurement without induction of apoptosis. Apoptosis was induced at time point 0 h by addition of 1 μM STS. (N = 4). (G) MERLIN detects a NAC-induced decrease in MERCs (**P < 0.025) t test, data are expressed as mean ± SD.
- Figure S4. BRET signal changes in apoptotic cells.
(A, B, C) Scheme and graph showing the change of the BRET signal in apoptotic cells over time for the (A) 0-, (B) 6-, and (C) 24-nm linker length. Black lines represent four individual measurements and the grey line the control measurement without induction of apoptosis. Apoptosis was induced at time point 0 h by addition of 1 μM STS. (N = 4).
- Figure 4. MERC plasticity characterized by MERLIN in stable HCT116 cells and use of MERLIN in neuroprogenitors and dopaminergic neurones.
(A) Measurement of BRET signal of MERLIN as a function of time in HCT116 cells exposed to stress: starvation (green), bortezomib (purple), Taxol (orange), staurosporine (dark blue), tunicamycin (cyan), and hypoxia (grey). Control shown in black. BRET was quantified before treatment (−4 h), after 4 h of stress (0 h) and upon recovery at 4 and 16 h. (B) Localization of the donor and acceptor to the mitochondria and ER, respectively in neuroprogenitor cells. Scale bar 5 μm. (C) Representative image of a differentiated dopaminergic (top) and embryonic mice primary neurons (bottom). Scale bar 100 and 20 μm, respectively. (D) Quantification of BRET signal in neuroprogenitor cell (magenta) and dopaminergic neurons (grey) in the presence of absence of PDZD8. (**P < 0.025 and ***P < 0.001). T test, data are expressed as mean ± SD.
- Figure 5. FLIM-FRET of MERLIN.
(A) Upper plane shows a representative Cos1 cell transfected with mCer-L1-B33C (blue) and sCal-L1-mVen (yellow). Scale bar 25 μM. The area in the white rectangle was used for FLIM-FRET measurement. Lower plane shows the zoom in in this area. Scale bar 5 μM. (B) The fluorescence lifetime is shown for the donor fluorophore with the 6-nm linker MERLIN, the negative and the positive control as well as the donor only control. Graph shows three biological replicates with n = 10, Error bars SD.
- Figure 6. siRNA knockdown of proteins involved in mitochondrial dynamics alters the BRET signal of MERLIN.
(A) Confocal images of Cos1 cells after Mfn1, Mfn2, Drp1 knockdown, or scramble (Ctr) siRNA transfection. Scale bar 10 μM. (B) Changes in percentage of BRET signal in cells co-expressing the 12-nm linker MERLIN sCal-L2-RLuc and mVen-L2-B33C after knockdown with Mfn1, Mfn2, Drp1, or scramble (Ctr) siRNA normalized to cells without knockdown. n = 3–4, Error bars SD.
- Figure S5. Western blots verifying the knockdown of Mfn1, Mfn2, and Drp1.
Western Blots verifying siRNA knockdown of Mfn1, Mfn2, and Drp1 compared with wild-type cells. Ponceau staining is shown as loading control.
- Figure S6. Mitochondrial morphology and localization of the mitochondrial part of MERLIN upon knockdown of Mfn1, Mfn2, and Drp1.
Representative microscopy images showing MERLIN (RLuc-L1-B33C) localization and mitochondrial morphology after siRNA knockdown of Mfn1, Mfn2, or Drp1. Scale bar 10 μm.
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