Article Figures & Tables
Figures
- Fig. 1.
Design of a reporter of mitochondria–ER contacts (MERCs). (A) spGFP1-10 was fused to the N-terminus of UBE2J2 fragment (spGFP1-10–ERt) and spGFP11 was fused to the C-terminus of the first 59 amino acids of TOMM70 (Mitot–spGFP11). Linkers with different lengths (∼3 nm or ∼10 nm) were inserted between spGFP11 and TOMM70 fragments to ensure that spGFP11 and spGFP1-10 could reach each other. When the distance between mitochondria and the ER is about 10–30 nm, spGFP1-10 and spGFP11 will refold and emit green fluorescence. (B) U2OS cells stably transfected with spGFP1-10–ERt and Mitot–2×spGFP11constructs (MERC reporter) were transfected with ER marker Myc–UBE2J2, fixed and stained with anti-Myc and anti-HSP60 antibodies. ER–mitochondria contact reporter signals (green, arrows) localized only to the junctions of the ER (indicated by Myc–UBE2J2, red) and mitochondria (indicated by HSP60 staining, blue). (C) Green channel of B. (D–H) U2OS cells with MERC reporter expressed were fixed and stained with anti-TOM20 antibody. 3D SIM images show that the contact reporter (green) forms thin lines or rings on the mitochondria, indicated by TOM20 staining (red). (E) Zoom of the green channel in the boxed region in D. (F) Red channel of D. (G) Green channel of D. (H) U2OS cells with MERC reporter (green) stably expressed were transfected with Mito–RFP (red). 3D SIM images were collected using GE DeltaVision OMX. The 3D rendering was made using Imaris NIS 4.3. MERCs are indicated by the green signals and mitochondria by the red signals. (I,J) TEM analysis of control U2OS cells (Ctrl) and U2OS cells with MERC reporter stably expressed (stable line). Yellow arrows indicate the contact regions (distance between ER and mitochondrion is less than 30 nm). (K) The contact sizes were normalized by dividing contact length with the circumference of the mitochondrion. The statistical analysis of the size of the contact shows that there is no dramatic difference between control and stable line; n=61 mitochondria were analyzed. The experiments shown were replicated twice. NS, no significance (two-tailed t-test). Data are presented as means±s.d. (see also Fig. S1).
- Fig. 2.
Characterization of the MERC reporter. U2OS cells with MERC reporter stably expressed were transfected with an artificial ER–mitochondria tether (B–B″,C), or PTPIP51–Myc and VAPB–RFP expression vectors (E–E″,F), or PTPIP51 and VAPB siRNAs (H–H″,I), respectively. The cells then were fixed and stained with anti-TOM20 antibodies to label mitochondria. Expression of the tether and VAPB are indicated by RFP fluorescence and expression of PTPIP51 by anti-Myc antibody staining. The expression of ER–mitochondria tether (tether, red in B) (A–C) or the overexpression of PTPIP51 (blue in E) and VAPB (red in E) (D–F) enhanced the size and intensity of the reporter. The RNAi of PTPIP51 and VAPB decreased the size and intensity of the MERC reporter (G–I). The middle panels of the fluorescence images (A′,B′,D′,E′,G′,H′) indicate the green channel. The right panels of the fluorescence images (A″,B″,D″,E″,G″,H″) show the heat map of the green channel. (C,F,I) Quantification of the fluorescence images. The sizes of the contacts were calculated by using the ratio between the areas of contacts to the areas of the mitochondria. n=50 cells were analyzed. The experiments shown were replicated three times. ***P<0.001 (two tailed t-test). Data are presented as means±s.d. (see also Figs S2 and S3).
- Fig. 3.
MERCs are dynamic structures. (A) U2OS cells with MERC reporter stably expressed were transfected with Mito–RFP. Time-lapse movies recording the cells were collected with a Yokogawa spinning-disk confocal microscope and analyzed using the ImageJ plugin particle tracker. A typical frame is shown. The trajectories of each contact are indicated by lines in different colors. (B) The statistics of proportions of the MERCs with different durations (short lived <60 s, medium 60–250 s, long lived >250 s). n=6 cells were analyzed. Data are presented as means±s.d. (C) A typical time-lapse series to show MERCs with different durations (white arrow: a contact lasted less than 60 s; pink arrow: a contact lasted more than 60 s but less than 250 s; green arrow: a contact lasted longer than 250 s). (D) Mitochondrial fission sites are associated with MERCs (white arrow). Mitochondria are marked by Mito–RFP (red), MERCs by GFP (green) (see also Movie 1).
- Fig. 4.
Changes of MERCs during the cell cycle. U2OS cells stably expressing MERC reporter were synchronized and different cell cycle phases were judged by DAPI, anti-PCNA and anti-Aurora B antibody staining. MERCs were indicated by GFP signals (green) and mitochondria were marked by anti-TOM20 staining (red). (A) At G1–S phase, MERCs covered less surface area of mitochondria than they did at other cell cycle stages. PCNA and Aurora B staining were used to distinguish G1–S and G2 phases. Other cell cycle stages were distinguished by DAPI staining. (B) Quantification of the size of contact in cells at different cell cycle phases. n=50 cells were analyzed. The experiments shown were replicated three times. **P<0.01, ***P<0.001 (two tailed t-test). Data are presented as means±s.d.
- Fig. 5.
Mitochondrial defects affect MERCs. U2OS cells with MERC reporter stably expressed were treated with DMSO (Ctrl), CCCP (10 μM, 4 h), mdivi-1 (50 μM, 4 h) and oligomycin A1 (10 μg/ml, 4 h), respectively. The cells were fixed and stained with anti-TOM20 antibodies (red). MERCs are indicated by the green signals. (A) CCCP and oligomycin A treatment increased MERCs and mdivi-1 treatment reduced MERCs. Mitochondria were labeled with TOM20 staining. (B) Quantification of the size of contacts in cells treated with different drugs. n=50 cells were analyzed. The experiments were replicated three times. *P<0.05,**P<0.01 (two tailed t-test). Data are presented as means±s.d. (see also Figs S3 and S4).
- Fig. 6.
ER morphology changes and ER stress do not change MERCs significantly. U2OS cells with MERC reporter stably expressed were transfected with sec61β–RFP, ALT1–HA or ALT1-K80A–HA expression vectors. The cells were fixed and stained with anti-HA (red) and anti-TOM20 (blue) antibodies. MERCs are indicated by green signal. ALT1 or ALT1-K80A overexpression changed the ER morphology but did not affect the size and intensity of MERCs. Sec61β–RFP overexpression served as a control. (B) U2OS cells with MERC reporter stably expressed were treated with DMSO or tunicamycin (0.5 μg/ml, 4 h). The cells were fixed and stained with anti-TOM20 (red) antibodies and DAPI (blue). MERCs are indicated by green signal. Tunicamycin treatment does not change MERCs significantly. (C) Quantification of the size of MERCs in the cells with indicated treatments. n=50 cells were analyzed. The experiments shown were replicated three times. NS, not significant (two tailed t-test). Data are presented as means±s.d. (see also Fig. S5).
- Fig. 7.
Starvation and induction of apoptosis, but not lipid feeding and mtUPR lead to MERC remodeling. U2OS cells with MERC reporter stably expressed were used for all the treatments. (A) For OA treatment, cells were incubated 200 mM oleic acid for 24 h or control medium (Ctrl). Cells were then fixed and stained with anti-TOM20 antibody (blue) to label mitochondria and 10 ng/ml Nile Red (red) to indicate the lipid droplets. MERCs are indicated by the green signal. OA feeding increased cytosolic lipid droplets, but did not change the size of MERCs. For starvation treatment, cells were incubated in HBSS for 20 h. For STS treatment, cells were incubated with 10 μM STS for 1 h. Cells were then fixed and stained with anti-TOM20 antibody (blue) to label mitochondria. MERCs are indicated by the green signal. Starvation and STS treatment could increase MERCs. To induce mtUPR, Myc-tagged OTC-D was transiently expressed in the cells. As a control, Myc-tagged OTC was also transiently expressed. Cells were then fixed and stained with anti-TOM20 (blue) and anti-Myc (red) antibodies. MERCs are indicated by the green signals. The mtUPR induced by OTC-D overexpression did not change MERCs. (B) Quantifications of the size of contacts in the cells with indicated treatments. n=50 cells were analyzed. The experiments shown were replicated three times. ***P<0.001; NS, no significance (two tailed t-test). Data are presented as means±s.d. (see also Figs S3 and S6).
Article navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Subject collections
Other journals from The Company of Biologists
Introducing FocalPlane’s new Community Manager, Esperanza Agullo-Pascual
We are pleased to welcome Esperanza to the Journal of Cell Science team. The new Community Manager for FocalPlane, Esperanza is joining us from the Microscopy Core at Mount Sinai School of Medicine. Find out more about Esperanza in her introductory post over on FocalPlane.
New funding scheme supports sustainable events
As part of our Sustainable Conferencing Initiative, we are pleased to announce funding for organisers that seek to reduce the environmental footprint of their event. The next deadline to apply for a Scientific Meeting grant is 26 March 2021.
Read & Publish participation continues to grow
"Alongside pre-printing for early documentation of work, such mechanisms are particularly helpful for early-career researchers like me.”
Dr Chris MacDonald (University of York) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 150 institutions in 15 countries and four library consortia taking part – find out more and view our full list of participating institutions.
Cell scientist to watch: Romain Levayer
In an interview, Romain Levayer talks about starting his own lab, his love for preprints and his experience of balancing parenting with his research goals.
Live lactating mammary tissue
In a stunning video, Stewart et al. demonstrate warping of the alveolar unit due to basal cell-generated force as part of their recent work investigating roles for mechanically activated ion channels in lactation and involution.
Visit our YouTube channel to watch more videos from JCS, our sister journals and the Company.
JCS and COVID-19
For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.
If you have any questions or concerns, please do not hestiate to contact the Editorial Office.