Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 1. MIRO1 is required for proliferation in vitro and wound healing in vivo. (A) Schematic de- picting the genetic strategy used to generate fibroblast-specific MIRO1−/−mice. Miro1fl/fl mice were crossed with mice expressing tamoxifen-inducible, fibroblast-specific Cre recombinase, Col1a2CreERT. Tamoxifen was administered (80 mg/kg/day) for a total of 10 days to induce fibroblast-restricted Cre expression. (B) Representative immunoblot for MIRO1 in mitochondrial fractions of lysates from the skin of WT and Miro1−/−mice after wound closure. The quantification of MIRO1 protein is adjusted to COX IV; n = 5 mice per group. (C) Cell counts of skin fibroblasts explanted from WT and Miro1−/−mice incubated in media containing 10% FBS with and without PDGF for 72 h (20 ng/mL); n = 10 independent experiments. (D) Representative FACS analysis for DNA content in synchronized/growth-arrested WT and MIRO1−/−skin fibroblasts at 0 h and after release from arrest with 10% FBS for 24 h and 48 h. (E) Cell cycle phase distribution (% of cells) of skin fibroblasts in the G1, S, and G2/M phases; n = 4–6 independent experiments. (F) Representative images of wounds after intrascapular skin punch at days 0 (immediately after punch), 3, and 6 in WT and Miro1-/- mice. The scale depicted below the images represents 1 mm. (G) Quantification of wound areas. Data were normalized to the wound area at day 0; n = 14 mice per genotype. Data are shown as the mean ± SEM. Analyses were performed using the Mann–Whitney test (B), one-way ANOVA (C), two-way ANOVA (or mixed model) (E), or two-way ANOVA (G).

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques: In Vitro, In Vivo, Expressing, Western Blot, Incubation, MANN-WHITNEY

Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 2. MIRO1 regulates the number of mitochondria–ER contacts during the cell cycle. (A) Rep- resentative images of VSMCs expressing a split-GFP-based contact-site sensor for wide juxtaposi- tion (40–50 nm) between the ER and mitochondria (SPLICSL, green) colocalized with mitochondria (MitoTracker, blue) in WT and MIRO1−/−cells. Scale bar = 20 µm, ×63. (B) Quantification of SPLICSL in (A); n = 7–11 independent experiments. (C) Representative images of VSMCs expressing a split-GFP-based contact-site sensor for narrow juxtaposition (8–10 nm) between the ER and mito- chondria (SPLICSS; green) colocalized with mitochondria (MitoTracker, blue) in WT and MIRO1−/−

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing

Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 3. MIRO1 resides at MAM interfaces and interacts with Ca2+-transfer MERCS proteins. (A) Representative immunoblots for MERCS proteins in fractions of purified mitochondria (PM) and of mitochondria-associated membranes (MAMs) isolated from WT and MIRO1−/−skin fibrob- lasts following synchronization in serum-free medium (0 h) and at 24 h after release from growth arrest in medium containing 10% FBS. PM: purified mitochondria, MAM: mitochondria-associated membrane. Markers for MAMs and ER (FACL4) and mitochondria (cytochrome c oxidase (COX IV)) were also examined. VDAC1 was used as a loading control. (B–F) Quantification of the immunoblot experiments as in (A). (B) MIRO1, (C) IP3R, (D) GRP75, (E) FACL4, and (F) VAPB levels, adjusted to VDAC1; n = 3–7 independent experiments. (G) Coimmunoprecipitation (co-IP) analysis of MIRO1 WT, MIRO1 KK, MIRO1 dnC, and MIRO1 ∆TM with MERCS proteins. c-Myc-tagged MIRO1 con- structs were expressed in HEK cells for 24 h, and cell lysis and pull-down assays were performed. (H–J) Quantification of the co-IP experiments shown in (G). (H) MIRO1 expression in MIRO1 KK, MIRO1 dnC, and MIRO1 ∆TM adjusted to MIRO1 WT. (I) GRP75 and (J) MCU levels, adjusted for immunoprecipitated c-Myc-tagged MIRO1; n = 6 independent experiments. Data are shown as the mean ± SEM. Analyses were performed using the Kruskal–Wallis test.

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques: Western Blot, Purification, Isolation, Membrane, Control, Co-Immunoprecipitation Assay, Lysis, Expressing, Immunoprecipitation

Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 4. MIRO1 regulates changes in subcellular Ca2+ distribution during the cell cycle. (A) PDGF-induced ER Ca2+ release as assessed with CEPIA1er in synchronized/growth-arrested WT and MIRO1-/- VSMCs at 0 h and after release from arrest with 10% FBS for 24 h and 48 h. Arrows indicate the addition of PDGF (20 ng/mL). (B) Quantification of the peak amplitude of CEPIA1er recordings shown in (A); n = 8 independent experiments. (C) PDGF-induced cytosolic Ca2+ tran- sients as assessed with Fura 2-AM in synchronized/growth-arrested WT and MIRO1−/−VSMCs at 0 h and after release from arrest with 10% FBS for 24 h and 48 h. Arrows indicate the addition of PDGF

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques:

Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 5. Loss of MIRO1 attenuates mitochondrial and cytosolic ATP levels. (A) Representative immunoblots of phosphorylated (inactive) pyruvate dehydrogenase (p-PDH) and total pyruvate dehydrogenase (t-PDH) in whole-cell lysates of WT and MIRO1−/−VSMCs at 0 h and after release from arrest with 10% FBS for 24 h. (B) Quantification of p-PDH (α1-ser293), adjusted to t-PDH. COX IV was used as a loading control; n = 4 independent experiments. (C) Quantification of mitochondrial ATP levels in synchronized/growth-arrested WT and MIRO1−/−VSMCs at 0 h and

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques: Western Blot, Control

Journal: Cells

Article Title: MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

doi: 10.3390/cells14070482

Figure Lengend Snippet: Figure 6. MIRO1 EF hands and transmembrane domain is required for MERCS formation, increased ATPlevels and cell proliferation in skin fibroblasts. (A) Representative images of WT skin fibroblasts and MIRO1−/−skin fibroblasts expressing MIRO1 KK, MIRO1 dnC, or MIRO1 ∆TM and a split- GFP-based contact-site sensor for wide juxtaposition (40–50 nm) between the ER and mitochondria (SPLICSL; green) colocalized with mitochondria (MitoTracker; blue) after release from arrest with 10% FBS for 24 h. Scale bar = 20 µm, ×63. (B) Quantification of the SPLICSL shown in (A); n = 30 to 65 cells for each group from 5 independent experiments. (C) Quantification of mitochondrial ATP levels at 24 h in WT skin fibroblasts and MIRO1−/−skin fibroblasts expressing MIRO1 KK, MIRO1 dnC, or MIRO1 ∆TM after release from growth arrest with 10% FBS; n = 12 independent experiments. (D) Cell counts of WT skin fibroblasts and MIRO1−/−skin fibroblasts incubated in media containing 10% FBS with and without PDGF for 72 h (20 ng/mL); n = 10 independent experiments. (E) Cell counts of MIRO1−/−skin fibroblasts expressing MIRO1 WT, MIRO1 KK, MIRO1 dnC, or MIRO1 ∆TM incubated in media containing 10% FBS with and without PDGF for 72 h (20 ng/mL); n = 10 independent experiments. Data are shown as the mean ± SEM. Analyses were performed using Kruskal–Wallis (B,D,E) and one-way ANOVA (C) tests.

Article Snippet: • Construction and transduction of MIRO1 cDNA-expressing adenoviruses The MIRO1 plasmid constructs pRK5-myc-Miro1 (# 47888), pRK5-myc-Miro1 E208K/ E328K (# $7894), pRK5-myc-Miro1 S432N (# 47893), and pRK5-myc-Miro1 ∆593–618 (# 47895) were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing, Incubation