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Addgene inc dna for humanmiro1
Dna For Humanmiro1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/myc+miro1/myc-Miro1+(lacking+TM)+(Plasmid+%23127613)/pm40615373-224-1-4
Average 90 stars, based on 7 article reviews

dna for humanmiro1 - by Bioz Stars, 2026-09

90/100 stars

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Generated:

Article Title: Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites
Article Snippet: Our work supports a central role for Miro proteins in coordinating and integrating different mitochondrial functions by organizing and controlling a mitochondrial signalling network that includes the mitochondrial transport pathway, the MIB/MICOS complexes and the ERMCS and that might be the functional equivalent of the ERMIONE in mammalian cells. .. GFP Miro1 and GFP Miro2 were generated as described earlier while Myc Miro1, Myc Miro2 and Myc Miro2ΔTM , , Myc Mfn1 and GFP Su9 were obtained from Addgene. ..

Article Title: Miro clusters regulate ER-mitochondria contact sites and link cristae organization to the mitochondrial transport machinery
Article Snippet: Our work supports a central role for Miro proteins in coordinating and integrating different mitochondrial functions by organizing and controlling a mitochondrial signaling network that includes the mitochondrial transport pathway, the MIB/MICOS complexes and the ERMCS and that might be the functional equivalent of the ERMIONE in mammalian cells. .. GFP Miro1 and GFP Miro2 were generated as described earlier , while myc Miro1, myc Miro2 , , myc Mfn1 , and GFP Su9 were obtained from Addgene. ..

Over Expression:

Article Title: FOXO3A Plays a Role in Wound Healing by Regulating Fibroblast Mitochondrial Dynamics.
Article Snippet: The inserts were first transferred to a pDONR221 donor vector using BP Clonase II Enzyme Mix and then to a CSII-EF-DEST vector (kindly provided by Miyoshi) using LR Clonase II Enzyme Mix. .. In addition, for the overexpression of MIRO1, myc-Miro1 from the pRK5-myc-Miro1 plasmid (a gift from Pontus Aspenström, Addgene plasmid number 47888, http://n2t.net/addgene:47888, Research Resource Identifier: Addgene_47888) was inserted into a pENTR11-IresGFP vector and recombined with the CSII-EF-DEST vector using LR Clonase II Enzyme Mix. .. The resulting plasmids CSII-EF-EmGFP-shRhot1 and CSII-EF-myc-Miro1-IresGFP were mixed with the packaging plas- mids (pCAG-HIVg/p and pCMV-VSVG-RSV-Rev, kindly provided by Miyoshi) and transfected into 293T cells.

Plasmid Preparation:

Article Title: ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner, downstream of ARL2
Article Snippet: .. Cloning and constructs: The following plasmids were generously gifted or commercially obtained and used in this study: MFN1-10xmyc, MFN2-16xmyc, MFN1-3xHA, and MFN2-3xHA in pcDNA3.1 (Dr. David Chan, California Institute of Technology, (Chen et al., 2003)), myc-MIRO1 (Addgene plasmid #47888) and myc-MIRO2 (Addgene plasmid #4789, Dr. Pontus Aspenstrom, Karolinska Institute (Fransson et al., 2003)), human mitoPLD-GFP (Dr. Michael Frohman, Stony Brook University (Huang et al., 2011)), mito-PAGFP (Dr. Richard Youle, NIH, Addgene plasmid #23348, (Karbowski et al., 2004)), mito-DsRed (Dr. James Zheng, Emory), mCherry-Sec61β (Dr. Gia Voeltz, University of Colorado, Addgene plasmid #49155), and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988). ..

Article Title: ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner, downstream of ARL2
Article Snippet: .. The following plasmids were generously given or commercially obtained and used in this study: MFN1-10xmyc, MFN2-16xmyc, MFN1-3xHA, and MFN2-3xHA in pcDNA3.1 (David Chan, California Institute of Technology; Chen et al. , 2003 ); myc-MIRO1 (Addgene plasmid #47888) and myc-MIRO2 (Addgene plasmid #4789, Pontus Aspenstrom, Karolinska Institute; Fransson et al. , 2003 ); human mitoPLD-GFP (Michael Frohman, Stony Brook University; Huang et al. , 2011 ); mito-PAGFP (Richard Youle, National Institutes of Health [NIH]; Addgene plasmid #23348; Karbowski et al. , 2004 ); mito-DsRed (James Zheng, Emory); mCherry-Sec61β (Gia Voeltz, University of Colorado; Addgene plasmid #49155); and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988). ..

Cloning:

Construct:

Article Title: Alpha-Synuclein Delays Mitophagy and Targeting Miro Rescues Neuron Loss in Parkinson’s Models
Article Snippet: .. Constructs The following constructs were used: mito-dsRed [ 19 ]; EGFP [ 41 ]; pA1T7-DMiro [ 41 ], pA1T7-DMiroΔTM [ 17 ], mito-mkeima [ 5 ], Myc-Miro1 [ 14 ], EGFP-SNCA (WT and A53T, Addgene No.: 40822 and 40823) [ 15 ], and HA-SNCA (WT and A53T, Addgene No.: 40824 and 40825). ..

Article Title: Alpha-Synuclein Delays Mitophagy and Targeting Miro Rescues Neuron Loss in Parkinson’s Models
Article Snippet: .. The following constructs were used: mito-dsRed [ 19 ]; EGFP [ 41 ]; pA1T7-DMiro [ 41 ], pA1T7-DMiroΔTM [ 17 ], mito-mkeima [ 5 ], Myc-Miro1 [ 14 ], EGFP-SNCA (WT and A53T, Addgene No.: 40822 and 40823) [ 15 ], and HA-SNCA (WT and A53T, Addgene No.: 40824 and 40825). ..

Article Title: Peroxisomal fission is modulated by the mitochondrial Rho‐GTPases, Miro1 and Miro2
Article Snippet: .. GFP Miro1 and GFP Miro2 were cloned from myc Miro1 and myc Miro2 (described previously ) into pEGFP‐C1; GFP Tom70(1‐70), amino acids 1‐70 of human Tom70, were cloned into pEGFP‐N1; Miro1 truncation constructs were cloned from GFP Miro1: GFP ΔGTP1 (184–618 only), GFP GTP1 (1–177 fused to 562–618) and GFP GTP2 (412–618 only); myc Miro1ΔTM cloned from myc Miro1 (deletion of 593–618), pxDsRed from Addgene (#54503), pxGFP from Addgene (#54501), ER‐DsRed from Addgene (#55836) ; and myc Pex19 mouse Pex19 (NM_023041) cloned into pRK5‐myc vector. .. GFP‐tagged mouse Miro1 splice variants ( GFP v1, GFP v2, GFP v3 and GFP v4) were cloned from NM_021536 (v1), NM_001163354 (v2) and NM_001163354 (v3) (OriGene: MR209606, MR224107 and MR224933, respectively) into pEGFP‐N1.

Clone Assay:

Image Search Results

a Domain diagrams of human MIRO1 and TRAK1, highlighting the conserved regions CR2 and CR4, which contain the MIRO1 binding Site-1 and Site-2, respectively. Secondary structure elements within Site-2 are shown in a cartoon representation below the sequence. Each EF-hand ligand mimic (ELM) domain consists of two EF-hands and a ligand mimic helix, with only the first EF-hand binding Ca 2+ . b Two perpendicular views of the cryo-EM map of the MIRO1-TRAK1 dimer, colored and labeled by domains (as in part a). Bound cofactors (GTP, Mg 2+ , and Ca 2+ ) are highlighted in the ribbon diagram on the right. c Close-up views of specific interactions between MIRO1 and Site-2 of TRAK1, numbered 1 to 6 from N- to C-terminus. MIRO1 is shown as a transparent electrostatic surface. The image in the center shows the cryo-EM map extracted around Site-2 and indicates the location of each close-up view (labeled 1-6).

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a Domain diagrams of human MIRO1 and TRAK1, highlighting the conserved regions CR2 and CR4, which contain the MIRO1 binding Site-1 and Site-2, respectively. Secondary structure elements within Site-2 are shown in a cartoon representation below the sequence. Each EF-hand ligand mimic (ELM) domain consists of two EF-hands and a ligand mimic helix, with only the first EF-hand binding Ca 2+ . b Two perpendicular views of the cryo-EM map of the MIRO1-TRAK1 dimer, colored and labeled by domains (as in part a). Bound cofactors (GTP, Mg 2+ , and Ca 2+ ) are highlighted in the ribbon diagram on the right. c Close-up views of specific interactions between MIRO1 and Site-2 of TRAK1, numbered 1 to 6 from N- to C-terminus. MIRO1 is shown as a transparent electrostatic surface. The image in the center shows the cryo-EM map extracted around Site-2 and indicates the location of each close-up view (labeled 1-6).

Article Snippet: The DNA for human MIRO1 (Addgene, plasmid #127613), human TRAK1 (GenScript, E. coli codon-optimized), and TRAK1 416-446,561-623 (GenScript, E. coli codon-optimized) were used as templates for cloning (primers listed in Source Data file).

Techniques: Binding Assay, Sequencing, Cryo-EM Sample Prep, Labeling

$a Structure of the MIRO1 1-591 -TRAK1 569-623 dimeric complex, with MIRO1 monomers shown in cyan and gray and Site-2 of TRAK1 in red. Red cylinders indicate the likely location of the transmembrane helices as they extend from the structure. b Contact surfaces (yellow) of Site-2 on MIRO1 (1915 Å 2 , top) and MIRO1 on Site-2 (2065 Å 2 , bottom). c MIRO1 dimerization interface shown as a surface representation (cyan, left) and a ribbon diagram (right). ELM2, ELM2-cGTPase linker, and cGTPase are colored orange, magenta, and blue, respectively. Contact surface areas were calculated using the SPPIDER server ( https://sppider.cchmc.org/ ). d Anti-parallel β-sheet formed by TRAK1 residues Q575-V577. e The last observed MIRO1 residue in the structure, L587, is positioned <20 Å from its counterpart in the other dimer subunit, suggesting that the transmembrane helices are closely spaced as they insert into the outer mitochondrial membrane. f Representative mass photometry data for MIRO1 1-591 alone and with TRAK1 569-623 from fraction 16 (see also Supplementary Fig. ). Mass photometry and cryo-EM samples were prepared using GraFix . Data are presented as histograms normalized to the bin with the highest number of counts (bin width = 6.1 kDa). The average mass, standard deviation, and percent of counts are derived from Gaussian fits.$

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a Structure of the MIRO1 1-591 -TRAK1 569-623 dimeric complex, with MIRO1 monomers shown in cyan and gray and Site-2 of TRAK1 in red. Red cylinders indicate the likely location of the transmembrane helices as they extend from the structure. b Contact surfaces (yellow) of Site-2 on MIRO1 (1915 Å 2 , top) and MIRO1 on Site-2 (2065 Å 2 , bottom). c MIRO1 dimerization interface shown as a surface representation (cyan, left) and a ribbon diagram (right). ELM2, ELM2-cGTPase linker, and cGTPase are colored orange, magenta, and blue, respectively. Contact surface areas were calculated using the SPPIDER server ( https://sppider.cchmc.org/ ). d Anti-parallel β-sheet formed by TRAK1 residues Q575-V577. e The last observed MIRO1 residue in the structure, L587, is positioned <20 Å from its counterpart in the other dimer subunit, suggesting that the transmembrane helices are closely spaced as they insert into the outer mitochondrial membrane. f Representative mass photometry data for MIRO1 1-591 alone and with TRAK1 569-623 from fraction 16 (see also Supplementary Fig. ). Mass photometry and cryo-EM samples were prepared using GraFix . Data are presented as histograms normalized to the bin with the highest number of counts (bin width = 6.1 kDa). The average mass, standard deviation, and percent of counts are derived from Gaussian fits.

Techniques: Residue, Membrane, Cryo-EM Sample Prep, Standard Deviation, Derivative Assay

a , b HPLC analysis and quantification of nucleotide bound to MIRO1 1-591 , MBP-MIRO1 1-180 , and MIRO1 177-591 . Proteins were purified and incubated for 15 min, respectively, with 10- and 35-fold molar excesses of nucleotide (GDP, pink trace; GTP, green trace). The normalized maximum absorbance at 256 nm is plotted as a function of retention time. c , e Representative SDS-PAGE analyses of MIRO1 1-591 pulled down by either MBP-TRAK1 569-623 or MBP-TRAK1 416-446,561-623 prepared under GTP or GDP conditions (as described in part a), with either 1 mM CaCl 2 or 5 mM EGTA added during incubation. From left to right, the gel lanes correspond to the MIRO1 1-591 load control and amylose pulldowns of MIRO1 1-591 , TRAK1 constructs, and MIRO1 1-591 + TRAK1 constructs. d , f Densitometric quantification of the pulldowns. Three biological replicates ( N = 3), each comprising three technical replicates, were performed using different MIRO1 1-591 preparations. Data from each biological replicate are represented by distinct symbols (triangles, circles, or squares), with their corresponding averages outlined in black. For each MBP-TRAK1 construct and cofactor condition, replicates are normalized to the average of the CaCl 2 /GTP condition for that replicate. Colored bars (matching the gel contours in parts c , e ) and brackets indicate the mean ± SD for the biological replicates. Statistical analysis was performed using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test, revealing no statistically significant differences among the experiments (p > 0.05). Full gels and quantifications are provided in the file.

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a , b HPLC analysis and quantification of nucleotide bound to MIRO1 1-591 , MBP-MIRO1 1-180 , and MIRO1 177-591 . Proteins were purified and incubated for 15 min, respectively, with 10- and 35-fold molar excesses of nucleotide (GDP, pink trace; GTP, green trace). The normalized maximum absorbance at 256 nm is plotted as a function of retention time. c , e Representative SDS-PAGE analyses of MIRO1 1-591 pulled down by either MBP-TRAK1 569-623 or MBP-TRAK1 416-446,561-623 prepared under GTP or GDP conditions (as described in part a), with either 1 mM CaCl 2 or 5 mM EGTA added during incubation. From left to right, the gel lanes correspond to the MIRO1 1-591 load control and amylose pulldowns of MIRO1 1-591 , TRAK1 constructs, and MIRO1 1-591 + TRAK1 constructs. d , f Densitometric quantification of the pulldowns. Three biological replicates ( N = 3), each comprising three technical replicates, were performed using different MIRO1 1-591 preparations. Data from each biological replicate are represented by distinct symbols (triangles, circles, or squares), with their corresponding averages outlined in black. For each MBP-TRAK1 construct and cofactor condition, replicates are normalized to the average of the CaCl 2 /GTP condition for that replicate. Colored bars (matching the gel contours in parts c , e ) and brackets indicate the mean ± SD for the biological replicates. Statistical analysis was performed using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test, revealing no statistically significant differences among the experiments (p > 0.05). Full gels and quantifications are provided in the file.

Techniques: Purification, Incubation, SDS Page, Control, Construct

a Sequence alignment of a subgroup of TRAK1/2 from 183 vertebrate sequences (Supplementary Fig. ), highlighting conserved region 4 (CR4). Amino acids conserved in 85–97% and 97–100% of the sequences are shaded in light and dark blue, respectively. Residues W589 and L597, which were mutated to aspartate (W589D and L597D), are marked with stars. b , d Representative SDS-PAGE analyses of MIRO1 1-591 pulldown by wild-type (WT), W589D, and L597D variants of MBP-TRAK1 569-623 and MBP-TRAK1 416-446,561-623 (n = 4). Pulldowns were performed in the presence of 1 mM CaCl 2 and 50 µM GTP. From left to right, the gel lanes correspond to the MIRO1 1-591 load control, and amylose pulldowns of MIRO1 1-591 , MBP-TRAK1 constructs, and MIRO1 1-591 + MBP-TRAK1 constructs. c , e Densitometric quantification of the pulldowns. Colored bars (matching the gel contours in parts b and d) and brackets represent the mean ± SD for each pulldown condition. Data are normalized to the average of the corresponding WT MBP-TRAK1 construct. Statistical analyses were performed using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test. P-values: part c (WT vs W589D p < 0.0001, WT vs L597D p < 0.0001, W589D vs L597D p = 0.9754), part e (WT vs W589D p < 0.0001, WT vs L597D p = 0.0019, W589D vs L597D p = 0.0001). P-values are also indicated in the figures, with ns for p > 0.05. Gels and quantifications are provided in the file.

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a Sequence alignment of a subgroup of TRAK1/2 from 183 vertebrate sequences (Supplementary Fig. ), highlighting conserved region 4 (CR4). Amino acids conserved in 85–97% and 97–100% of the sequences are shaded in light and dark blue, respectively. Residues W589 and L597, which were mutated to aspartate (W589D and L597D), are marked with stars. b , d Representative SDS-PAGE analyses of MIRO1 1-591 pulldown by wild-type (WT), W589D, and L597D variants of MBP-TRAK1 569-623 and MBP-TRAK1 416-446,561-623 (n = 4). Pulldowns were performed in the presence of 1 mM CaCl 2 and 50 µM GTP. From left to right, the gel lanes correspond to the MIRO1 1-591 load control, and amylose pulldowns of MIRO1 1-591 , MBP-TRAK1 constructs, and MIRO1 1-591 + MBP-TRAK1 constructs. c , e Densitometric quantification of the pulldowns. Colored bars (matching the gel contours in parts b and d) and brackets represent the mean ± SD for each pulldown condition. Data are normalized to the average of the corresponding WT MBP-TRAK1 construct. Statistical analyses were performed using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test. P-values: part c (WT vs W589D p < 0.0001, WT vs L597D p < 0.0001, W589D vs L597D p = 0.9754), part e (WT vs W589D p < 0.0001, WT vs L597D p = 0.0019, W589D vs L597D p = 0.0001). P-values are also indicated in the figures, with ns for p > 0.05. Gels and quantifications are provided in the file.

Techniques: Sequencing, SDS Page, Control, Construct

a Surface representation of the MIRO1 1-591 -TRAK1 569-623 dimeric complex, highlighting the pocket (yellow) where AlphaFold3 predicts TRAK1 Site-1 (shown in all-atom representation) binds at the interface between ELM2 and cGTPase. The per-residue confidence of this prediction, as indicated by the predicted local distance difference test (pLDDT) score and the predicted aligned error (PAE), is shown in Supplementary Fig. . A close-up view displays details of the interaction using surface and all-atom representations, with MIRO1 residue D344 (mutated to lysine to validate this interaction) shown in red. b , c ITC titrations of MBP-TRAK1 416-431 into MIRO1 177-591 wild-type and mutant D344K in the presence of 1 mM CaCl 2 and 50 µM GTP (left) or 5 mM EGTA and 50 µM GDP (right). The experimental conditions and fitting parameters (stoichiometry, N; dissociation constant, K d ) are indicated with each graph.

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a Surface representation of the MIRO1 1-591 -TRAK1 569-623 dimeric complex, highlighting the pocket (yellow) where AlphaFold3 predicts TRAK1 Site-1 (shown in all-atom representation) binds at the interface between ELM2 and cGTPase. The per-residue confidence of this prediction, as indicated by the predicted local distance difference test (pLDDT) score and the predicted aligned error (PAE), is shown in Supplementary Fig. . A close-up view displays details of the interaction using surface and all-atom representations, with MIRO1 residue D344 (mutated to lysine to validate this interaction) shown in red. b , c ITC titrations of MBP-TRAK1 416-431 into MIRO1 177-591 wild-type and mutant D344K in the presence of 1 mM CaCl 2 and 50 µM GTP (left) or 5 mM EGTA and 50 µM GDP (right). The experimental conditions and fitting parameters (stoichiometry, N; dissociation constant, K d ) are indicated with each graph.

Techniques: Residue, Mutagenesis

a , c Representative maximum-intensity projections of Halo-tagged TRAK1 1-640 wild-type (WT) and Site-2 mutants (W589D, L597D, and W589D + L597D) or Site-1/Site-2 mutants ( 425 IPG 427 ⟶ AAA, W589D + L597D, and 425 IPG 427 ⟶ AAA + W589D + L597D), co-expressed in HeLa cells with Myc-MIRO1 and Mito-DsRed2. Scale bars represent 15 μm. b , d Mitochondrial-to-cytoplasmic intensity ratios of Halo-tagged TRAK1 1-640 WT and the mutants shown in ( a , c ). Data points are color-coded by experimental replicate (N = 3 biological replicates, n = 10 cells), with average values outlined in black. The center line and bars represent the mean ± SD of the three biological replicates. Statistical significance was determined using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test. P-values: b (WT vs W589D p = 0.0696, WT vs L597D p = 0.8494, WT vs W589D + L597D p = 0.0192, W589D vs L597D p = 0.5728, W589D vs W589D + L597D p = 0.9965, L597D vs W589D + L597D p = 0.2345), d (WT vs 425 IPG 427 ⟶AAA p = 0.1111, WT vs W589D + L597D p = 0.0195, WT vs 425 IPG 427 ⟶AAA + W589D + L597D p = 0.0112, 425 IPG 427 ⟶AAA vs W589D + L597D p < 0.0001, 425 IPG 427 ⟶AAA vs 425 IPG 427 ⟶AAA + W589D + L597D p < 0.0001, W589D + L597D vs 425 IPG 427 ⟶AAA + W589D + L597D p > 0.9999). P-values are also indicated in the figures, with ns for p > 0.05. All data points and statistical tests are provided in the file.

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: a , c Representative maximum-intensity projections of Halo-tagged TRAK1 1-640 wild-type (WT) and Site-2 mutants (W589D, L597D, and W589D + L597D) or Site-1/Site-2 mutants ( 425 IPG 427 ⟶ AAA, W589D + L597D, and 425 IPG 427 ⟶ AAA + W589D + L597D), co-expressed in HeLa cells with Myc-MIRO1 and Mito-DsRed2. Scale bars represent 15 μm. b , d Mitochondrial-to-cytoplasmic intensity ratios of Halo-tagged TRAK1 1-640 WT and the mutants shown in ( a , c ). Data points are color-coded by experimental replicate (N = 3 biological replicates, n = 10 cells), with average values outlined in black. The center line and bars represent the mean ± SD of the three biological replicates. Statistical significance was determined using a right-tailed one-way ANOVA with Tukey’s multiple comparisons test. P-values: b (WT vs W589D p = 0.0696, WT vs L597D p = 0.8494, WT vs W589D + L597D p = 0.0192, W589D vs L597D p = 0.5728, W589D vs W589D + L597D p = 0.9965, L597D vs W589D + L597D p = 0.2345), d (WT vs 425 IPG 427 ⟶AAA p = 0.1111, WT vs W589D + L597D p = 0.0195, WT vs 425 IPG 427 ⟶AAA + W589D + L597D p = 0.0112, 425 IPG 427 ⟶AAA vs W589D + L597D p < 0.0001, 425 IPG 427 ⟶AAA vs 425 IPG 427 ⟶AAA + W589D + L597D p < 0.0001, W589D + L597D vs 425 IPG 427 ⟶AAA + W589D + L597D p > 0.9999). P-values are also indicated in the figures, with ns for p > 0.05. All data points and statistical tests are provided in the file.

Techniques:

The MIRO1-TRAK1 complex forms a dimer at the outer mitochondrial membrane, with the dimerization interface involving MIRO1’s ELM2-cGTPase and TRAK1. TRAK1 Site-1 (I425-S428) and Site-2 (L570-R613) bind at the interface of MIRO1’s ELM2-cGTPase and nGTPase-ELM1, respectively. In vitro, both interactions are independent of cofactor conditions (Ca 2+ or nucleotide), though their regulation in cells may involve other factors, such as microtubule-based motors and/or MIRO1 clustering on the mitochondrial membrane.

Journal: Nature Communications

Article Title: Structural-functional characterization of the MIRO1-TRAK1 complex

doi: 10.1038/s41467-025-61174-6

Figure Lengend Snippet: The MIRO1-TRAK1 complex forms a dimer at the outer mitochondrial membrane, with the dimerization interface involving MIRO1’s ELM2-cGTPase and TRAK1. TRAK1 Site-1 (I425-S428) and Site-2 (L570-R613) bind at the interface of MIRO1’s ELM2-cGTPase and nGTPase-ELM1, respectively. In vitro, both interactions are independent of cofactor conditions (Ca 2+ or nucleotide), though their regulation in cells may involve other factors, such as microtubule-based motors and/or MIRO1 clustering on the mitochondrial membrane.

Techniques: Membrane, In Vitro

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

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

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−/−

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−/−

Techniques: Expressing

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

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.

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

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

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

Techniques:

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

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

Techniques: Western Blot, Control

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.

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.

Techniques: Expressing, Incubation

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