plasmid prk5 myc miro2  (Addgene inc)

Journal: Journal of Cell Science

Article Title: Regulation of mitochondrial cristae organization by Myo19, Miro1 and Miro2, and metaxin 3

doi: 10.1242/jcs.263637

Figure Lengend Snippet: Membrane anchor mutants of Miro1/2 rescue Miro DKO. (A) Scheme of Miro1 and Miro2 residues that were exchanged with the corresponding residues of VAMP1B and Bcl-xL, respectively. Residues highlighted in green and blue are part of the transmembrane regions and those in red of the intermembrane space (IMS). Residues in light blue color are derived from VAMP1B and face the cytosol. Dm: Drosophila melanogaster , Hs: Homo sapiens . (B) Western blot analysis of protein levels of Miro, Halo–Miro, Myo19 and β-actin in WT, Miro DKO and Miro DKO cells expressing different Miro constructs as indicated. (C) Quantification of Myo19 protein levels in the different cell lines as indicated (normalized to β-actin levels). Results are mean±s.e.m. ( n =3). * P <0.05; ** P <0.01; *** P <0.001 (two-sample unpaired t -test). (D,E) Confocal images of Miro DKO cells rescued with Halo-tagged Miro constructs (D) and Halo-tagged mutant Miro constructs (E) that were transfected with GFP–Myo19. Mitochondria were stained with Mitotracker Orange. n =20–30 cells per experiment ( n =3 experiments). Scale bars, 10 µm. (F) Miro DKO cells rescued with either Halo–Miro1, Halo–Miro2, Halo–Miro1mutant or Halo–Miro2mutant were lysed. The lysates were subjected to Halo–Trap agarose pull-downs. Western blot analysis of inputs and eluates for Miro, Myo19, Mic60, Sam50, Mtx3 and GAPDH are shown. Images in F are representative of four repeats.

Article Snippet: Similarly, a plasmid encoding for Halo–Miro2 was prepared using human full-length Miro2 cDNA from the plasmid pRK5-myc-Miro2 (Addgene plasmid # 47891 ) ( ).

Techniques: Membrane, Derivative Assay, Western Blot, Expressing, Construct, Mutagenesis, Transfection, Staining

Journal: Journal of Cell Science

Article Title: Regulation of mitochondrial cristae organization by Myo19, Miro1 and Miro2, and metaxin 3

doi: 10.1242/jcs.263637

Figure Lengend Snippet: Ultrastructural analysis of mitochondria in various Miro DKO rescue cells. Representative TEM images are shown of mitochondria in HEK WT (A), Miro DKO (B), Miro DKO with Halo–Miro1 (MiroDKO-HaloMiro1) (C), Miro DKO with Halo–Miro2 (MiroDKO-HaloMiro2) (D), Miro DKO with Halo–Miro1 mutant (MiroDKO-HaloMiro1 mutant) (E) and Miro DKO with Halo–Miro2 mutant (MiroDKO-HaloMiro2 mutant) cells (F). In G, characteristic examples are shown of mitochondria with normal, intermediate and aberrant cristae organization. (H) Percentages of mitochondria with normal, intermediate and aberrant cristae organization in the indicated cell lines. n =200–250 mitochondria for WT and Miro DKO, n =150–175 mitochondria for the rescue Miro DKO cell lines. Scale bars: 500 nm.

Article Snippet: Similarly, a plasmid encoding for Halo–Miro2 was prepared using human full-length Miro2 cDNA from the plasmid pRK5-myc-Miro2 (Addgene plasmid # 47891 ) ( ).

Techniques: Mutagenesis

Journal: Cell reports

Article Title: Modulation of GPR133 (ADGRD1) signaling by its intracellular interaction partner extended synaptotagmin 1

doi: 10.1016/j.celrep.2024.114229

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Myc-Miro2 (RHOT2) , (Fransson et al) , Addgene 47891.

Techniques: Recombinant, Modification, Magnetic Beads, Blocking Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, In Situ, Proximity Ligation Assay, Mutagenesis, Plasmid Preparation

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

doi: 10.1002/cm.21560

Figure Lengend Snippet: MYO19898–970 localizes to mitochondria in the presence of Miro2 and has more rapid exchange kinetics than constructs containing the putative membrane-insertion motif. (a) Mito/cyto ratio was used to quantify the enhancement of mitochondrial localization of MYO19-GFP constructs in the presence of mchr-Miro2. While coexpression of mchr-Miro2 with MYO19898–970-GFP enhanced the GFP-localization on mitochondria, mitochondrial localization of MYO19853–935-GFP and MYO19860–890-GFP were not enhanced by the coexpression of mchr-Miro2 (*p<0.001, t-test between MYO19898–970-GFP and MYO19898–970-GFP coexpressed with mchr-Miro2). (b) Coexpression of mchr-Miro2 decreased the fraction of the population of MYO19898–970-GFP expressing cells displaying a cytosolic staining pattern for GFP, but expression of mchr-Miro2 did not alter the population of MYO19853–935-GFP or MYO19860–890-GFP expressing cells with a cytosolic GFP pattern, further implicating Miro2 in localizing MYO19 to mitochondria (*p < 0.05, compared to MYO19898–970-GFP, Tukey analysis). (c) FRAP recovery of MYO19853–935-GFP and MYO19860–890-GFP expressing cells is relatively slow with a large immobile fraction, when compared to MYO19898–970-GFP in cells expressing mchr-Miro2, indicating that the mechanism of interaction with mitochondria for these two regions of MYO19 differs. (d) Same data as in (c), but plotted on a different time scale to show the complete time course for the longer recovery kinetics. FRAP kinetic parameters are listed in Table 1

Article Snippet: GFP-Cytob 5 RR was a gift from Nica Borgese ( Borgese, Gazzoni, Barberi, Colombo, & Pedrazzini, 2001 ). myc-BioID2-MCS was a gift from Kyle Roux ( Kim et al., 2016 ) (Addgene plasmid # 74223; http://n2t.net/addgene:74223 ; RRID:Addgene_74223). mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068; http://n2t.net/addgene:55068 ; RRID:Addgene_55068). pRK5-myc-Miro2 (Addgene plasmid # 47891; http://n2t.net/addgene:47891 ; RRID:Addgene_47891), pRK5-myc-Miro2 T18N (Addgene plasmid # 47897; http://n2t.net/addgene:47897 ; RRID:Addgene_47897), and pRK5-myc-Miro2 Δ593–618 (Addgene plasmid # 47901; http://n2t.net/addgene:47901 ; RRID:Addgene_47901) were gifts from Pontus Aspenström ( Fransson et al., 2003 , 2006 ).

Techniques: Construct, Expressing, Staining

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

doi: 10.1002/cm.21560

Figure Lengend Snippet: FRAP kinetic analysis for proteins used in this study

Article Snippet: GFP-Cytob 5 RR was a gift from Nica Borgese ( Borgese, Gazzoni, Barberi, Colombo, & Pedrazzini, 2001 ). myc-BioID2-MCS was a gift from Kyle Roux ( Kim et al., 2016 ) (Addgene plasmid # 74223; http://n2t.net/addgene:74223 ; RRID:Addgene_74223). mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068; http://n2t.net/addgene:55068 ; RRID:Addgene_55068). pRK5-myc-Miro2 (Addgene plasmid # 47891; http://n2t.net/addgene:47891 ; RRID:Addgene_47891), pRK5-myc-Miro2 T18N (Addgene plasmid # 47897; http://n2t.net/addgene:47897 ; RRID:Addgene_47897), and pRK5-myc-Miro2 Δ593–618 (Addgene plasmid # 47901; http://n2t.net/addgene:47901 ; RRID:Addgene_47901) were gifts from Pontus Aspenström ( Fransson et al., 2003 , 2006 ).

Techniques:

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

doi: 10.1002/cm.21560

Figure Lengend Snippet: PARF kinetic analysis for proteins used in this study

Article Snippet: GFP-Cytob 5 RR was a gift from Nica Borgese ( Borgese, Gazzoni, Barberi, Colombo, & Pedrazzini, 2001 ). myc-BioID2-MCS was a gift from Kyle Roux ( Kim et al., 2016 ) (Addgene plasmid # 74223; http://n2t.net/addgene:74223 ; RRID:Addgene_74223). mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068; http://n2t.net/addgene:55068 ; RRID:Addgene_55068). pRK5-myc-Miro2 (Addgene plasmid # 47891; http://n2t.net/addgene:47891 ; RRID:Addgene_47891), pRK5-myc-Miro2 T18N (Addgene plasmid # 47897; http://n2t.net/addgene:47897 ; RRID:Addgene_47897), and pRK5-myc-Miro2 Δ593–618 (Addgene plasmid # 47901; http://n2t.net/addgene:47901 ; RRID:Addgene_47901) were gifts from Pontus Aspenström ( Fransson et al., 2003 , 2006 ).

Techniques:

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

doi: 10.1002/cm.21560

Figure Lengend Snippet: A subset of well-conserved, charged residues are essential for Miro2-dependent localization of MYO19898–970. (a) Clustal Omega multiple sequence alignments of the MYO19 MyMOMA domain and the Miro2 N-terminal GTPase domain identified well-conserved, charged residues in each domain, which were mutated to alanine to determine their contribution to the MYO19/Miro2 interaction. (b) Mutating either the basic residues in MYO19898–970-GFP to alanine or the acidic residues in mchr-Miro2 to alanine decreased the localization of MYO19898–970-GFP to mitochondria, as determined by the mito/cyto ratio (*p < 0.05, Dunnett’s test vs. MYO19898–970-GFP wild type coexpressed with mchr-Miro2 wild type). (c) Mutations similarly decreased the fraction of cells displaying a cytosolic staining pattern for GFP, whether those mutations were to MYO19 or Miro2 (*p < 0.05, Dunnett’s test vs. MYO19898–970-GFP wild type coexpressed with mchr-Miro2 wild type). (d) FRAP analysis of MYO19898–970-GFP mutant shows faster exchange kinetics than wild type, consistent with basic residues in the MYO19 mediating interactions with Miro2. The exchange kinetics for wild type MYO19898–970-GFP with the mutant mchr-Miro2 construct were similar to exchange kinetics with the wild type mchr-Miro2 wild type construct. This is likely due to endogenous Miro recruiting MYO19898–970-GFP. Multiple sequence alignments can be found in the Figure S4. Numbers at the base of the bars indicate the number of replicates. Error bars represent SEM. FRAP kinetic parameters are listed in Table 1

Article Snippet: GFP-Cytob 5 RR was a gift from Nica Borgese ( Borgese, Gazzoni, Barberi, Colombo, & Pedrazzini, 2001 ). myc-BioID2-MCS was a gift from Kyle Roux ( Kim et al., 2016 ) (Addgene plasmid # 74223; http://n2t.net/addgene:74223 ; RRID:Addgene_74223). mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068; http://n2t.net/addgene:55068 ; RRID:Addgene_55068). pRK5-myc-Miro2 (Addgene plasmid # 47891; http://n2t.net/addgene:47891 ; RRID:Addgene_47891), pRK5-myc-Miro2 T18N (Addgene plasmid # 47897; http://n2t.net/addgene:47897 ; RRID:Addgene_47897), and pRK5-myc-Miro2 Δ593–618 (Addgene plasmid # 47901; http://n2t.net/addgene:47901 ; RRID:Addgene_47901) were gifts from Pontus Aspenström ( Fransson et al., 2003 , 2006 ).

Techniques: Sequencing, Staining, Mutagenesis, Construct

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes

doi: 10.1002/cm.21560

Figure Lengend Snippet: Miro2-dependent localization of MYO19898–970 may depend on the nucleotide state of Miro2. (a) Point mutations made to the N-terminal domain of Miro2 are thought to result in the GTPase being in the GTP-bound (A13V) or GDP-bound (T18N) state. (b) Expressing the putative GDP-bound mchr-Miro2 T18N mutant decreased the localization of MYO19898–970-GFP to mitochondria while the putative GTP-bound A13V mutant did not, as determined by the mito/cyto ratio (*p < 0.05, Dunnett’s test vs. MYO19898–970-GFP coexpressed with mchr-Miro2 wild type). (c) Expression of mchr-Miro2 T18N increased the fraction of cells displaying a cytosolic staining pattern for GFP (*p < 0.05, Dunnett’s test vs. MYO19898–970-GFP coexpressed with mchr-Miro2 wild type). (d) FRAP analysis of MYO19898–970-GFP with wild type or GTP-state mutants shows similar exchange kinetics of MYO19898–970-GFP for all three conditions. For the mchr-Miro2 T18N, this is likely due to endogenous Miro recruiting MYO19898–970-GFP. Numbers at the base of the bars indicate the number of replicates. Error bars represent SEM. FRAP kinetic parameters are listed in Table 1

Article Snippet: GFP-Cytob 5 RR was a gift from Nica Borgese ( Borgese, Gazzoni, Barberi, Colombo, & Pedrazzini, 2001 ). myc-BioID2-MCS was a gift from Kyle Roux ( Kim et al., 2016 ) (Addgene plasmid # 74223; http://n2t.net/addgene:74223 ; RRID:Addgene_74223). mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid # 55068; http://n2t.net/addgene:55068 ; RRID:Addgene_55068). pRK5-myc-Miro2 (Addgene plasmid # 47891; http://n2t.net/addgene:47891 ; RRID:Addgene_47891), pRK5-myc-Miro2 T18N (Addgene plasmid # 47897; http://n2t.net/addgene:47897 ; RRID:Addgene_47897), and pRK5-myc-Miro2 Δ593–618 (Addgene plasmid # 47901; http://n2t.net/addgene:47901 ; RRID:Addgene_47901) were gifts from Pontus Aspenström ( Fransson et al., 2003 , 2006 ).

Techniques: Expressing, Mutagenesis, Staining