Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Gene-silencing Reticulon 4 or ERLIN2 inhibits mtDNA replication in primary human fibroblasts. Cells were transfected with and without silencer RNA Tm targeting RTN4 or ERLIN2 (Thermo Fisher). Isolated protein from fibroblasts was immunoblotted for Reticulon 4 ( A ) or ERLIN2 ( D ) after fractionation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and quantified relative to stained total protein (No-stain™) ( n = 3–4 independent experiments). Representative images of cells treated without and with silencer RNAs targeting RTN4 ( B ) or ERLIN2 ( E ) pulse-labelled with bromo-deoxyuridine (BrdU) for 8 h and stained with the anti-BrdU antibody (green), and anti-TOMM20 antibody (red), the latter to highlight the mitochondrial network. Scale bar = 30 µm. Quantification of the cytoplasmic BrdU foci (C, F). Data represent the pooled results of three independent experiments (Figs and , and ). Data in panel (C) are derived from an analysis of 316 non-target transfected cells (NT) versus 247 RTN4 silenced cells ( n = 10 independent experiments, P < .001). Data in panel ( F ) are derived from an analysis of 308 NT cells versus 229 ERLIN2 silenced cells ( n = 10 independent experiments, P < .001). ( G ) Interpretation of the relationship between ERMCS and mtDNA replication. The mtDNA forms nucleoprotein complexes, or nucleoids, which are tightly associated with the IMM (inner mitochondrial membrane). MtDNA replicates (detected as BrdU incorporation in newly synthesized DNA—green circles) where the tubular ER (TER) forms connections with the mitochondria that include lipid rafts associated with the outer mitochondrial membrane (OMM). Without Reticulon 4 TER cannot form, while the absence ERLIN2 compromises the lipid microdomains; hence, the absence of RTN4 and ERLIN2 inhibit mtDNA synthesis (pink nucleoids containing non-replicating mtDNA).

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Transfection, Isolation, Fractionation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Derivative Assay, Membrane, BrdU Incorporation Assay, Synthesized

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques:

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Depletion of ER calcium stores and inhibition of MCU repress mtDNA replication, and ERMCS disruption lowers MCU abundance. ( A ) Fibroblasts were treated without (Ctrl) or with 200 nM thapsigargin (Tg) for 8 h, in the presence of BrdU for the final 8 h. To the left, representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red) antibodies. Scale bar = 30 µm. To the right, quantification of the cytoplasmic BrdU foci in 140 control cells versus 110 Tg treated cells ( n = 6 independent experiments, P < 0.001). ( B ) Left: Representative images of cells treated without and with MCU-i4 and pulse-labelled with BrdU prior to staining with anti-BrdU antibody (green) and anti-TOMM20 antibody (red). Scale bar = 30 µm. Right: Quantification of the cytoplasmic BrdU foci per cell in 164 control (Ctrl) cells versus 124 MCU-i4 treated cells ( n = 5 independent experiments, P = .008). ( C ) Primary human fibroblasts were treated with or without siRNAs targeting RTN4 or GRP75 ( n = 6 independent experiments, P = .002 and n = 4 independent experiments, P = .005, respectively) and extracted proteins analysed by immunoblotting of MCU.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition, Disruption, Control, Staining, Western Blot

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Manganese supplementation rescues mtDNA replication after ERLIN2 or RTN4 silencing or inhibition of MCU. ( A ) Primary human fibroblasts were cultured with (+) or without (−) 50 µM manganese (Mn 2+ ) for 16 h and labelled with BrdU for 8 h, after ERLIN2 or RTN4 silencing. Representative images of cells immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 30 µm. ( B ) Quantification of the cytoplasmic BrdU foci per cell in 145 NT and 154 NT + Mn 2+ cells, P = .69; 99 si ERLIN2 and 138 si ERLIN2 + Mn 2+ cells P = .03; 92 si RTN4 and 119 si RTN4 + Mn 2+ cells P = .03, 98 MCU-i4 and 89 MCU-i4 + Mn 2+ cells P = 0.11 ( n = 4 independent experiments). ( C ) Interpretive model of the relationship between ERMCS and mtDNA replication with and without Mn 2+ . Without ERMCS, mtDNA replication is inhibited, while the supplementation of Mn 2+ rescues mtDNA replication in the absence of ERMCS.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition, Cell Culture

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: Gene-silencing of Superoxide dismutase 2 inhibits mtDNA replication and is rescued by manganese. ( A ) si SOD2 or non-target (NT) silenced human fibroblasts were labelled with BrdU for 8 h and immunostained with anti-BrdU (green) and anti-TOMM20 (red). Scale bar = 50 µm. ( B ) Isolated protein from fibroblasts was immunoblotted for SOD2 after fractionation by SDS–PAGE, and quantified relative to VCL ( n = 4 independent experiments). ( C ) Quantification of the cytoplasmic BrdU foci per cell in 131 control (Ctrl) cells and 131 control cells supplemented with 50 µM MnCl 2 for 16 h, P = .89. 131 Ctrl versus 119 si SOD2 cells P = .03; and 119 si SOD2 cells versus 137 si SOD2 + Mn 2+ cells P = .03 ( n = 4 independent experiments). ( D ) EdU pulse-chase in primary human fibroblasts after transfection with siNT, si RTN4 , or si SOD2 . The EdU pulse was 26 h, and EdU incorporation in cytoplasmic foci was measured after chases of 0, 4, and 24 h. The chart represents the quantification of the EdU foci per cell in 26 non-target (NT) cells, 31 si RTN4 and 28 si SOD2 cells after a EdU pulse of 26 h (no chase); 46 NT cells, 57 si RTN4 , and 52 si SOD2 cells after a 4-h chase; and 41 NT, 36 si RTN4 , and 30 si SOD2 cells after a 24-h chase ( n = 3 independent experiments).

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Isolation, Fractionation, SDS Page, Control, Pulse Chase, Transfection

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: ERMCS regulate mtDNA replication via manganese. ( A ) Reticulon 4 (RTN4) is a component of tubular ER (TER), which is required to form connections with the mitochondria (ERMCS), and RTN4 abundance is proportional to mtDNA synthesis (Fig. and ). ERLIN2 is a fundamental component of ER lipid rafts, on which ERMCS depend (Fig. ), and so it too supports mtDNA synthesis (Fig. ). Equally, because GRP75 is a bridging component of ERMCS , its repression inhibits mtDNA replication . The abundance of the MCU correlates with that of RTN4 and GRP75 (Fig. ), and MCU inhibition represses mtDNA synthesis (Fig. ). Hence, mtDNA synthesis depends on ERMCS and MCU. Manganese is inferred to be the ER signalling factor that stimulates mtDNA replication, as it restores mtDNA synthesis after silencing of ERMCS factors and is a recognized substrate of MCU (Fig. ). SOD2 silencing also inhibits mtDNA replication and is rescued by manganese (Fig. ), suggesting it relays manganese entering via MCU to the mitochondrial nucleoid, where one proposed role of manganese is: ( B ) to shift the equilibrium slightly from long polycistronic transcripts to shorter RNAs that can serve as primers for DNA replication, based on manganese’s effects on the properties of the mitochondrial RNA polymerase, POLRMT (Fig. ). IMM and OMM: inner and outer mitochondrial membranes, respectively; VDAC: voltage dependent anion channel, aka porin, POLG: mtDNA polymerase γ, LSP: light strand promoter, TER: transcription termination site, OriR: origin of replication.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques: Inhibition

Journal: Nucleic Acids Research

Article Title: Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial calcium uniporter, and manganese

doi: 10.1093/nar/gkag233

Figure Lengend Snippet: ERLIN2 silencing depletes ER-mitochondrial junctions. ( A ) Antibodies to ER-mitochondrial junction components IP3R1 and GRP75 individually label the ER and the mitochondria, respectively, verified by KDEL labelling the ER and TOMM20 the mitochondria. Scale bars = 30 µm. ( B ) RTN4 and ERLIN2 silencing diminished the GRP75/IP3R1 PLA signal, quantified in 121 non-target (NT) cells versus 91 RTN4 and 61 ERLIN2 silenced cells ( n = 3–6 independent experiments, P = .002). Scale bars = 50 µm.

Article Snippet: Anti-DNA (Progen, #690014S, 1:250), BrdU (Abcam, #Ab6326, 1:250), TOMM20 (Proteintech, #11802-1-AP, 1:250), TOMM20 (Santa Cruz, #Sc-17764, 1:250), GRP75 (Santa Cruz, #Sc-133137, 1:200), IP3R1 (Novus, #NB120-5908, 1:200), ERLIN2 (Sigma–Aldrich, #HPA002025, 1:200), RTN4 (Atlas, #HPA023977, only ICC, 1:250), KDEL (Novus, #NBP1-97469, 1:700), GRP78/BiP (Abcam, #ab21685, 1:1000).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: The P23H Rhodopsin Mouse Model Reveals a Novel Interaction Between the Endoplasmic Reticulum and Connecting Cilium Rootlet Within Photoreceptors

doi: 10.1167/iovs.67.3.57

Figure Lengend Snippet: ER association with the rootlet and mitochondria . ER associations demonstrated by immunofluorescence and immunoelectron microscopy staining in 6-month-old control mouse retina. ( A ) Schematic diagram showing the inner segment region imaged in ( B , C ) between the outer segments (OS) and outer nuclear layer (ONL). B Immunoreactivity for the ER marker protein disulfide-isomerase (PDI) was observed running through the inner segment between the mitochondria stained with mitofusin 1 (Mfn1). C ER stained with anti-KDEL shows a close association with the rootlet marker rootletin. Green arrowheads point to the rootletin staining running through the photoreceptor inner segments. ( D , E ) The association of KDEL and the rootlet (Root) was also shown by immuno-EM labeling of KDEL. D Longitudinal and E transverse orientated inner segments show KDEL labeling on the membrane surrounding the rootlet. ( F ) The concentration of KDEL labeling is much higher within 100 nm of the rootlet compared with the entire cross-sectionally orientated inner segments. Plot shows the mean and statistical significance determine using a paired, two-tailed Student's t -tests: **** P ≤ 0.0001. Scale bars = B and C = 2.5 µm and D and E = 100 nm.

Article Snippet: Single labeling with anti-KDEL (1:10, NBP1-97469, Novus Biologicals) and double labeling using two anti-Rhodopsin antibodies RET-P1 (1:100, Abcam) and 1D4 (1:250) was performed using methods previously described.

Techniques: Immunofluorescence, Immuno-Electron Microscopy, Staining, Control, Marker, Labeling, Membrane, Concentration Assay, Two Tailed Test