Journal: Cell Cycle

Article Title: The novel actin/focal adhesion-associated protein MISP is involved in mitotic spindle positioning in human cells

doi: 10.4161/cc.24602

Figure Lengend Snippet: Figure 9. MISP interacts with p150 glued and EB1. ( A ) Immunoblot of a co-immunoprecipitation showing that EB1 co-precipitates with MISP (MISP-IP, bound). ( B ) Immunoblot of a GFP-trap from GFP-MISP-U2OS cells showing a weak interaction of GFP-MISP with p150 glued (GFP-trap, bound). ( C ) Co-immunoprecipitation demonstrating that endogenous MISP co-precipitates with p150 glued (p150 glued -IP, bound). ( D ) Co-immunostaining of UPCI:SCC114 cells with antibodies to either MISP (C118, green) and the Golgi component GM130 (red, upper panel) or α-tubulin (green) and GM130 (red, lower panel) 48 h after transfection with indicated siRNAs demonstrating that the Golgi complex is scattered in MISP-depleted cells. Cell boundaries are depicted as white lines. Scale bar, 10 µm.

Article Snippet: Salisbury), cyclin B1 (Santa Cruz, sc-245), cyclin E (Santa Cruz, sc-198), GAPDH (Santa Cruz, sc-47724), GFP (Santa Cruz, sc-9996) and Hec-1 (Novus Biologicals, NB100-338) as well as rabbit polyclonal antibodies to actin (Santa Cruz, sc-1616-R), α-tubulin (Abcam, ab52866), FAK (Cell Signaling, 3285), FAKY397 (Abcam, ab4803), GM130 (Abcam, ab52649), paxillin (Sigma-Aldrich, SAB4300592), pan-cadherin (Abcam, ab16505), pericentrin (Abcam, ab4448) and p150 glued (Bethyl Laboratories, A303-072A-1).

Techniques: Western Blot, Immunoprecipitation, Immunostaining, Transfection

Journal: bioRxiv

Article Title: Parental genome unification is highly erroneous in mammalian embryos

doi: 10.1101/2020.08.27.269779

Figure Lengend Snippet: (A) Representative image of a zygote expressing mClover3-KASH5-DN (green) and H2B-mScarlet (DNA, magenta). (B-G) Representative images and quantification of zygotes in indicated groups displaying, upon KASH5-DN treatment, detached centrosomes (B and C), pronuclear migration defects (D and E) or separate spindles at anaphase onset (F and G). Gray, microtubules (mClover3-MAP4-MTBD). Magenta, DNA (H2B-mScarlet). Arrows indicate detached centrosomes and dashed lines indicate pronuclear envelopes. Z-projection, respectively 4, 7, 5, 8, 4, and 10 sections every 2.50 µm. (H) Nuclear and surface occupancy indices in zygotes expressing GST or KASH5-DN. (I) Zygotes treated with DMSO or nocodazole that displayed or not pronuclear migration defects. (J) Nuclear and surface occupancy indices in zygotes treated with DMSO or nocodazole. (K) Nuclear and surface occupancy indices in zygotes injected with BSA or P150-CC1. Data are from four (C, E, G, H, K) and six (I, J) independent experiments. The number of analyzed zygotes (C, E, G, I) and pronuclei (H, K, L) is specified in italics. Indicated p -values calculated using Fisher’s exact test (C, E, G, I) and unpaired two-tailed Student’s t-test (H, J, K). Scale bars, 10 µm.

Article Snippet: His-P150-CC1 ( ) was expressed in and purified from BL21(DE3)pLysS competent cells (Promega) as previously described ( ) with some modifications.

Techniques: Expressing, Migration, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Parental genome unification is highly erroneous in mammalian embryos

doi: 10.1101/2020.08.27.269779

Figure Lengend Snippet: (A) Representative images of zygotes before nuclear envelope breakdown expressing GST or KASH5-DN. Gray, DNA (H2B-mScarlet). Dashed lines mark pronuclei. Z-projections, 9 sections every 3.08 µm. (B) Max polarity index (see (C)) in zygotes expressing GST or KASH5-DN. (C) Schematics of the max polarity index. (D) Representative immunofluorescence images of a zygote with microtubules wrapped around the pronuclei. Gray, microtubules (α-tubulin). Magenta, DNA (DAPI). Z-projection, 35 sections every 0.1 µm. (E-H) Representative images and max polarity indices in zygotes treated with DMSO or nocodazole (E, F) or injected with BSA or P150-CC1 (G, H). The last time point before nuclear envelope breakdown is shown. Gray, DNA (H2B-mScarlet). Dashed lines mark pronuclei. Z-projections, respectively 8 and 12 sections every 2.50 µm. Data are from four (B, H) or six (F) independent experiments. The number of analyzed pronuclei is specified in italics. Indicated p -values calculated using unpaired two-tailed Student’s t-test. Scale bars, 10 µm.

Article Snippet: His-P150-CC1 ( ) was expressed in and purified from BL21(DE3)pLysS competent cells (Promega) as previously described ( ) with some modifications.

Techniques: Expressing, Immunofluorescence, Injection, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: NuMA localization, stability, and function in spindle orientation involve 4.1 and Cdk1 interactions

doi: 10.1091/mbc.E13-05-0277

Figure Lengend Snippet: NuMA recruits dynein/dynactin to the cell cortex of keratinocytes. (A–D) Immunofluorescence analysis of endogenous NuMA, p150 glued , and DIC localization in wild-type and NuMA-knockdown mouse keratinocytes, as indicated. (E) Quantitation of cells with cortical NuMA, p150-glued, and DIC localization. n = 50 cells for each, p < 0.0001 for each. (F–I) Immunofluorescence analysis of endogenous NuMA and DIC localization in untransfected and dynamitin-GFP–transfected cells. (J) Quantitation of cells with cortical NuMA and DIC localization. n = 25 cells for each, p = 1 for NuMA, p < 0.0001 for DIC. Scale bars, 10 μm.

Article Snippet: Primary antibodies used in this study include chicken α-GFP (Abcam), rabbit α-NuMA (Abcam), mouse α-p150 glued (BD Biosciences), mouse α-DIC (Millipore), and guinea pig α-LGN.

Techniques: Immunofluorescence, Quantitation Assay, Transfection

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) In COS-7 cells, expressed PEX3-6×His-mRFP (PEX3-C) or PEX3-6×His-mRFP-Miro1 (PEXMiro1) (green) co-localizes with the mTurquoise-SRL peroxisomal marker (magenta). Scale bar: 10 μm. Inset scale bar: 2 μm. (B) Schematic of the PEX3Miro1 construct recruiting TRAK1/2 and KIF5C to the surface of a peroxisome. (C) Expression of mTurquoise-SRL (magenta) with either PEX3-C or PEX3Miro1 and mCitrine-KIF5C (green) with or without the co-expression of myc-TRAK1 in COS-7 cells. The presence of PEX3-C and PEXMiro1 on peroxisomes was confirmed in each cell by imaging their RFP tags. Scale bar: 10 μm. Inset scale bar: 2 μm. (D) Quantification of the amount of KIF5C enriched on peroxisomes that carry PEX3-C control or PEX3Miro1 with or without the overexpression of myc-TRAK1. The quantifications here and subsequently are represented as “box-and-whisker” plots with the median value indicated. Outliers are represented as individual dots and are considered in statistical calculations. Here and in subsequent figures, P -values are indicated and were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. (E) Distribution of peroxisomes marked with mTurquoise-SRL in the COS-7 cells analyzed in (D). Distance from the center of the cell in concentric shells was quantified using the DoveSonoPro FIJI macro (linked in the Materials and Methods section). N = 15 cells over three biological replicates. Error bars represent the SD. (F) PEX3-C or PEX3Miro1 was expressed in HEK293T cells along with myc-TRAK1 and mCitrine-KIF5C and co-immunoprecipitated using antibodies to the RFP tag on the PEX3-C and PEX3Miro1 constructs. Western blots were stained using anti-His, myc, and KIF5 antibodies. Data are from >3 biological replicates throughout. N = 15 for panels (A, C, D, E). The corner inserts show the enlargement of the boxed regions.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Marker, Construct, Expressing, Imaging, Control, Over Expression, Whisker Assay, Immunoprecipitation, Western Blot, Staining

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) COS-7 cells were co-transfected with PEX3-C, PEX3Miro1, or PEX3Miro2 together with the peroxisomal marker mCitrine-PTS1 (green) and the mitochondrial marker Mito-mTurquoise (blue). The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 1 μm. (B) From cells as in (A), the quantification of the RFP tags on PEX3-C and PEX3Miro that co-localized with the peroxisomal and mitochondrial markers. The data are represented as “box-and-whisker” plots with the median value indicated. The indicated P -values were obtained from one way-ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. (C) PEX3Miro1 (green) localized appropriately to peroxisomes (magenta) despite mutations in the GTPase domain. The cells shown are the same as those in . The localization of KIF5C in is identical to that of PEX3Miro1 except in cells expressing PEX3Miro1 GDP-WT, where the KIF5C no longer co-localizes with the Miro construct and the peroxisomal marker.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Marker, Whisker Assay, Expressing, Construct

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Expression in COS-7 cells of mTurquoise-SRL (magenta), myc-TRAK1, and mCitrine-KIF5C (green) with either PEX3Miro1 wild-type or PEX3Miro1 carrying a mutation of either the N- or C-GTPase domains: N-GDP-state T18N, N-GTP-state P13V, C-GDP-state S432N, and C-GTP-state K427N. The presence of the PEXMiro1 constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. The corner inserts show the enlargement of the boxed regions. Here and in subsequent figures, the status of the two GTPase domains is abbreviated as either WT, GTP, or GDP for first the N-domain and then the C-domain. Scale bar: 10 μm. Inset scale bar: 2 μm. (B) Quantification of the amount of KIF5C enriched on peroxisomes carrying PEX3-C and the constructs shown in (A). For clarity, the mutations of the N-GTPase domain (above) are plotted separate from those of the C-GTPase (below) although transfected and imaged in the same experiments with the same controls. The quantification is represented as “box-and-whisker” plots with the median value indicated. Outliers are represented as single plot points and are included in all statistical calculations. P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. (C) Quantification of peroxisomal distribution in concentric shells radiating from the center of the cell in the same cells analyzed in (B), with separate plots for mutations of the N-GTPase (left) and C-GTPase (right) for clarity, as in (B). N = 15 cells over three biological replicates. Error bars represent the SD. The data for the negative control (PEX3C) and the positive control (PEX3Miro1-WT-WT) are repeated from for clarity and are from experiments conducted at the same time.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Mutagenesis, Construct, Imaging, Transfection, Whisker Assay, Negative Control, Positive Control

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Expression of PEX3Miro1 N-GTPase and C-GTPase double mutants in which both domains were in either the GTP or the GDP state and its consequences for mCitrine-KIF5C (green) recruitment to peroxisomes (mTurquoise-SRL, magenta) with the co-expression of myc-TRAK1. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 1 μm. (B) Expression of PEX3Miro1 N-GTPase and C-GTPase double mutants in which the two domains were in opposite states and its consequences for mCitrine-KIF5C (green) recruitment to peroxisomes (mTurquoise-SRL, magenta) with the co-expression of myc-TRAK1. In (A, B), the presence of the PEXMiro1 constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. (C) Quantification of the amount of KIF5C enriched on cells transfected as in (A, B). If the N-GTPase is in the GDP-bound state (upper graph) or in the GTP-bound state (lower graph), the state of the C-GTPase has little or no effect, except for a modest enhancement of KIF5C recruitment when both domains are GTP-bound rather than only the N-GTPase. The quantification is represented as “box-and-whisker” plots with the median value indicated. Outliers are represented as single plot points and are included in all statistical calculations. Indicated P -values are determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. (D) Quantification of the peroxisomal distribution from the cells imaged for (C). Error bars represent the SD. The data for the negative control (PEX3C) and the positive control (PEX3Miro1-WT-WT) are repeated from for clarity and are from experiments conducted at the same time.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Construct, Imaging, Transfection, Whisker Assay, Negative Control, Positive Control

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) PEX3-C, PEX3Miro1, or PEX3Miro1 with mutations of either the N- or C-GTPase domains and with or without TRAK (as indicated) were expressed in COS-7 cells with mCitrine-P135 (green) and the peroxisomal marker mTurquoise-SRL (magenta). The presence of the PEXMiro1 constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 1 μm. (B, C, D) P135 co-localization with peroxisomes quantified from cells transfected as in (A) and represented as “box-and-whisker” plots with the median value indicated. Outliers are plotted as single points and are included in all statistical calculations. P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. The data for the negative control (PEX3C) and the positive control (PEX3Miro1-WT-WT) are repeated in each graph for clarity and are from experiments conducted at the same time. (B) Quantification of the amount of P135 enriched on PEX3-C control or PEX3Miro1 peroxisomes with or without the expression of myc-TRAK1. (C) Quantification of the amount of P135 enriched on peroxisomes bearing PEX3Miro1 wild-type or the PEX3Miro1 N-GTPase mutants co-expressed with myc-TRAK1. (D) Quantification of the amount of P135 enriched on peroxisomes bearing PEX3Miro1 wild-type or the PEX3Miro1 C-GTPase mutants co-expressed with myc-TRAK1.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Marker, Construct, Imaging, Transfection, Whisker Assay, Negative Control, Positive Control, Control, Expressing

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) PEX3-C, PEX3Miro1, or PEX3Miro1 with N-GTPase GDP- and GTP-state mutants was expressed in COS-7 cells with mCitrine-TRAK1 (green) and mTurquoise-SRL (magenta). The presence of the PEXMiro1 and PEX3-C constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. Scale bar: 10 μm. Inset scale bar: 1 μm. (B) From cells as in (A), the quantification of the amount of TRAK1 enriched on peroxisomes bearing PEX3-C, PEX3Miro1, and PEX3Miro1 N-GTPase mutants. (C) PEX3-C, PEX3Miro1, or PEX3Miro1 with N-GTPase GDP- and GTP-state mutants was expressed in COS-7 cells with mCitrine-TRAK2 (green) and mTurquoise-SRL (magenta). The presence of the PEXMiro1 constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. Scale bar: 10 μm. Inset scale bar: 2 μm. (D) From cells as in (C), the quantification of the amount of TRAK2 enriched on peroxisomes bearing PEX3-C, PEX3Miro1, and PEX3Miro1 N-GTPase mutants. In (B, D), TRAK co-localization with peroxisomes is represented as “box-and-whisker” plots with the median value indicated. Outliers are plotted as single points and are included in all statistical calculations. The indicated P -values are from analysis with one-way ANOVA. N = 15 cells over three biological replicates. N = 15 for all panels. The corner inserts show the enlargement of the boxed regions.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Construct, Imaging, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) PEX3Miro1, with the indicated mutations of the N- and C-GTPase domains, was co-expressed with mCitrine-TRAK1 (green) and mTurquoise-SRL (magenta) in COS-7 cells. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: marked as either 1 or 2 μm. ( B) Quantification of the amount of TRAK1 enriched on peroxisomes transfected as in (A). The data are represented as “box-and-whisker” plots with the median value indicated. Outliers are represented as individual dots and are considered in all statistical calculations. The indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. All experiments were conducted and imaged in parallel except the GDP-GTP and GTP-GDP forms of PEXMiro1; they were performed together with controls that were consistent with the values of the other data shown.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) PEX3Miro1, with the indicated mutations of the N- and C-GTPase domains, was co-expressed with mCitrine-TRAK2 (green) and mTurquoise-SRL (magenta) in COS-7 cells. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 2 μm. (B) Quantification of the amount of TRAK2 enriched on peroxisomes transfected as in (A). The data are represented as “box-and-whisker” plots with the median value indicated. Outliers are represented as individual dots and are considered in all statistical calculations. The indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates. All experiments were conducted and imaged in parallel except the GDP-GTP and GTP-GDP forms of PEXMiro1; they were performed together with controls that were consistent with the values of the other data shown.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Representative images of COS-7 cells transfected with plasmids expressing mCitrine-TRAK1 (green) and mito-dsRED (magenta), and with or without a plasmid expressing VopE. Scale bar: 10 μm. (B) For each cell as in (A), the amount of TRAK1 present on mitochondria was quantified. The quantifications are represented as “Tukey’s box-and-whisker” plots with the median value indicated. The indicated P -value was derived from a two-tailed t test with Welsh’s correction. N = 100 cells from four independent experiments.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Expressing, Plasmid Preparation, Whisker Assay, Derivative Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Either myc-TRAK1 or myc-TRAK2 was expressed in COS-7 cells together with mCitrine-KIF5C (green), mTurquoise-SRL (magenta), and PEX3-C or PEX3Miro1. The presence of the PEX3Miro1 or PEX3-C constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 1 μm. (B) From cells transfected as in (A), quantification of the amount of KIF5C enriched on peroxisomes with either myc-TRAK1 or myc-TRAK2. (C) Either mCitrine-TRAK1 or mCitrine-TRAK2 (green) was expressed in COS-7 cells together with mTurquoise-SRL (magenta) and PEX3-C or PEX3Miro1. The presence of the PEX3Miro1 and PEX3-C constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. Scale bar: 10 μm. Inset scale bar: 1 μm. (D) From cells transfected as in (C), the quantification of the amount of mCitrine-TRAK1 or mCitrine-TRAK2 enriched on peroxisomes. In (B, D), co-localization with peroxisomes is represented as “box-and-whisker” plots with the median value indicated. The indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Construct, Imaging, Transfection, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Either wild-type or N-GTPase mutations of PEX3Miro were expressed in COS-7 cells together with myc-TRAK2, mCitrine-KIF5C (green), and mTurquoise-SRL (magenta). Expression of PEX3Miro constructs on peroxisomes was confirmed by visualization of their RFP tags. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 2 μm. (B) From cells as in (A), the quantification of mCitrine-KIF5C on peroxisomes. The data are represented as “box-and-whisker” plots with the median value indicated. The indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. Outliers are represented as single plot points and are included in all statistical calculations. N = 15 cells over three biological replicates.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Construct, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: COS-7 cells were transfected with mTurquoise-KIF5C, PEX3Miro1, and either mCitrine-TRAK1 or mCitrine-TRAK2. The amount of mTurquoise on peroxisomes was normalized to the intensity of mCitrine on peroxisomes to determine the relative efficacy of the two TRAKs isoforms for recruiting KIF5C. The data are represented as “box-and-whisker” plots with the median value indicated. The indicated P -value was obtained from the two-tailed unpaired t test with Welch’s correction. N = 15 cells over three biological replicates.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) PEX3Miro2 (green) localized appropriately to peroxisomes (magenta). The cells shown are the same as those expressing PEX3Miro2 in . Scale bar: 10 μm. Inset scale bar: 2 μm. (B, C) Quantification of the amount of mCitrine-TRAK1 (B) or mCitrine-TRAK2 (C) enriched on peroxisomes carrying PEX3-C, PEX3Miro1, PEX3Miro2, or the PEX3Miro2 GTPase mutant constructs as indicated on the x-axis. Peroxisomes were marked with mTurquoise-SRL. The quantification is represented as “box-and-whisker” plots with the median value indicated. Outliers have been removed from this data set using the ROUT method (Q = 1%) and are not included in statistical calculations. Indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Mutagenesis, Construct, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Expression in COS-7 cells of mTurquoise-SRL (magenta), myc-TRAK1, and mCitrine-KIF5C (green) with either PEX3-C, PEX3Miro1, PEX3Miro2, or PEX3Miro2 carrying a mutation of either the N- or C-GTPase domains: N-GDP-state T18N, N-GTP-state A13V, C-GDP-state S430N, and C-GTP-state A425V. The presence of the PEX3Miro2 constructs on peroxisomes was confirmed in each cell by imaging their RFP tags. The corner inserts show the enlargement of the boxed regions. Scale bar: 10 μm. Inset scale bar: 2 μm. (B) Quantification of the amount of KIF5C enriched on peroxisomes carrying the constructs shown in (A). For clarity, the mutations to GDP state and GTP state are plotted separately. The quantification is represented as “box-and-whisker” plots with the median value indicated. Outliers have been removed from this data set using the ROUT method (Q = 1%) and are not included in statistical calculations. Indicated P -values were determined by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. N = 15 cells over three biological replicates.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Mutagenesis, Construct, Imaging, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: (A) Expression of mTurquoise-SRL peroxisomal marker (magenta) with either PEX3 control, wild-type PEX3Miro1, or PEX3Miro1 N-GTPase mutants together with myc-TRAK1 (signal not shown) and mCitrine-KIF5C (signal not shown) in soma and growth cone of rat hippocampal neurons. Cells were fixed at DIV5. Scale bar: for soma panel, 10 μm; and for growth cone panel, 5 μm. (B) Quantification of the distribution of peroxisomes from neurons as in (A). For each neuron, peroxisomes within a single representative growth cone or soma were counted. All data points are plotted, N = 9 neurons from three independent experiments. For each data set, the line indicates mean, and the whisker indicates SD. The indicated P -values were obtained by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons. (C) Quantification of the distribution of peroxisomes from neurons as in (A). For each neuron, peroxisomes within a single representative growth cone were counted and then expressed as a ratio relative to the number of peroxisomes in the soma of that neuron. All data points are plotted, N = 9 neurons from three independent experiments. The indicated P -values were obtained by one-way ANOVA with Dunnett’s T3 correction for multiple comparisons.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Expressing, Marker, Control, Whisker Assay

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: Neurons were transfected as in , that is, with a PEX3-C or a PEX3Miro1 construct, along with mTurquoise-SRL, myc-TRAK1, and mCitrine-KIF5. The mTurquoise-SRL marker was used to visualize the distribution of peroxisomes throughout the neurons and enabled the peroxisomal counts in the soma and growth cone in . The mCitrine-KIF5C signal was used to get a low-resolution image of the entire cell. Representative neurons expressing PEX3-C and PEX3Miro1-WT-WT are shown. Upper panels show a full neuron. The soma and growth cone whose peroxisomal signals appear below are marked (dotted lines). Scale bar: 20 μm.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Construct, Marker, Expressing

Journal: Life Science Alliance

Article Title: Miro GTPase domains regulate the assembly of the mitochondrial motor–adaptor complex

doi: 10.26508/lsa.202201406

Figure Lengend Snippet: Representative images of neurons transfected with the indicated form of PEX3Miro1, myc-TRAK1, mCitrine-KIF5C, and mTurquoise-SRL (peroxisomal marker). These neurons were among those whose peroxisomal distribution was quantified in , and consistent with the redistribution of peroxisomes in that figure, the WT and GTP-locked forms of the N-GTPase recruited KIF5C to peroxisomes, but the GDP-bound form did not. Scale bar: 50 μm. Inset scale bar: 10 μm.

Article Snippet: mCitrine-P135 was subcloned by VectorBuilder from mCitrine-P150 Glued .

Techniques: Transfection, Marker