crl 2992  (ATCC)

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression levels of MFN1 and MFN2 in wt, Mfn2-null, and Mfn1-null MEFs. ( B, C ) Spread area ( B ) and circularity ( C ) of wt, Mfn1-null, and Mfn2-null MEFs after overnight culture. The individual points represent individual MEF cells. ( D–G ) representative images with individual tracks ( D ), Wind–Rose plots ( E ), quantification of velocity ( F ), and directionality ( G ) of wt, Mfn1-null, and Mfn2-null MEFs cells during random migration. ( H, I ) Quantification of cell circularity ( H ) and representative images ( I ) of indicated MEFs during cell spreading at indicated time points. Data are presented as mean ± SD in ( F ) and were pooled from a total of 18 cells in three independent experiments. Bars represent arithmetic means ± SD. One representative result of three biological repeats is shown in ( A, D, E, I ). Data are pooled from three independent experiments in ( B, C, F, G ). n = 50 cells are tracked and counted in ( B, C ). N = 30 cells are quantified in ( D ). ****p<0.0001 (one-way ANOVA). Scale bars: 50 µm. Figure 1—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Immunofluorescence of F-actin (phalloidin), α-tubulin, and mitochondria (Mito Tracker) in wt , Mfn1 -null, and Mfn2 -null MEFs. ( B–D ) Cre-induced Mfn2 disruption in MEFs from Mfn2 flox/flox mice displays similar cell morphology as Mfn2 -null MEFs ( B ). ( E, F ) Cell spread area ( E ) and circularity ( F ) of indicated cells in . The individual points represent the circularity or spread area of individual MEF cells in ( C–F ). ( G ) Cells were transfected with mitochondria probes. Mfn2 -null MEFs and Mfn2 -null MEFs overexpressing MFN1 display large and fragmented mitochondria, while MFN2 re-expression in Mfn2 -null MEFs restored mitochondria tubules. One representative result of three biological repeats is shown in ( A, B ). n = 25 cells in each group are quantified in ( C–-F ). ***p≤0.001, ****p<0.0001 (one-way ANOVA). Scale bars: 10 µm in ( A, B ), 20 µm in ( G ).

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Immunofluorescence, Disruption, Transfection, Expressing

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression level of MFN1 and MFN2 in indicated MEF cells. Percentages of knockdown or re-expression were calculated by normalizing the intensity to vinculin first, then normalizing to the wt group. ( B–D ) Representative images with individual tracks ( B ), quantification of velocity ( C ), and Wind–Rose plots ( D ) of indicated MEF cells during random migration. ( E ) Quantification of cell circularity of wt and Mfn2-null MEFs with vec, MFN1, or MFN2 re-expressed during spreading at indicated time points. Data are presented as mean ± SD in ( E ) (n = 5). ( F, G ) Cell circularity ( F ) and cell spreading area ( G ) of indicated MEFs measured after overnight culture. ( H, I ) Percentage of Actin abundance in the cell border region ( H ) and peripheral actin band (PAB) cell percentage in each view was quantified using our custom algorism (see ). ( J ) Representative images of wt, Mfn2-null with doxycycline-induced MEF2 (DIn-MFN2) MEF cells treated with or without doxycycline for 48 hr. The cells are immunostained with phalloidin and MFN2. One representative result of three biological repeats is shown in ( A, B, D, H ). Data are pooled from three independent experiments in ( C, F–I ). n = 30 cells are tracked and counted in ( C ); n = 35 cells are quantified in ( F–H ). Five different views from three biological repeats are quantified in ( I ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA in C, E, F , unpaired t -test in H, I ). Scale bars: 50 µm in ( B ), 10 µm in ( J ). Figure 2—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Knockdown, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) Western blot ( A ) and quantification ( B ) of the amount of pMLCII and total MLCII in wt , Mfn1 -null, and Mfn2 -null MEFs. ( C, D ) Increased pMLCII in Mfn2 -null MEFs can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( C ) Western blot and ( D ) quantification determining the amount of pMLCII and total MLCII protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expressed, or with an artificial ER-mitochondria tether. ( E ) Representative images of wt and Mfn2 -null MEFs immunostained for F-actin (phalloidin), pMLCII, and DAPI. ( F ) Western blot determining the expression levels of MLCK or ROCK in Mfn2 -null MEFs with shMLCK or shROCK . ( G ) Western blot of pMLCII and total MLCII Mfn2 -null MEFs with shMLCK or shROCK . ( H ) Representative images of Mfn2 -null MEFs with shMLCK or shROCK immunostained for F-actin (green) and paxillin (red). ( I, J ) Cellular spread area and circularity of wt , Mfn2 -null MEFs with vec, shMLCK, or shROCK were measured after overnight culture. ( K ) Percentage of actin abundance in the cell border region in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . ( L ) Percentage of PAB cells identified by a custom algorithm in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . The individual points stand for the size or circularity of individual MEF cells. One representative result of three biological repeats is shown in ( A, B, F, G ). Four biological repeats were done in ( C, D ). Data are pooled from three independent experiments in ( I, J ). n = 30 cells are quantified in ( I, K ). Five different views from three biological repeats are quantified in ( L ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA, comparing each group to the average of Mfn2 -/- vec group in I, K ). Scale bars: 20 µm in ( H ), 10 µm in ( E ). Figure 7—source data 1. Original blots and figures with the bands labeled for . Figure 7—source data 2. Original blots and figures with the bands labeled for . Figure 7—source data 3. Original blots and figures with the bands labeled for . Figure 7—source data 4. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null, and Mfn1 -null MEFs in the µ-slide 15 min after plating. Time-lapse images were taken every 10 min for 16 hr and 40 min. Individual MEFs were tracked for velocity quantification. Scale bar: 50 m.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt and Mfn2 -null MEFs with vec , MFN1, or MFN2 re-expressed in the µ-slide. Time-lapse images were taken every 10 min for 14 hr and 50 min. Individual MEFs were tracked for velocity quantification. Scale bar: 50 m.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt and Mfn2 -null MEFs treated with DMSO or BAPTA-AM (20 μM) in the μ-slide. Time-lapse images were taken every 10 min for 14 hr and 30 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null MEFs with vec or synthetic tether construct in the µ-slide. Time-lapse images were taken every 10 min for 17 hr and 50 min. Individual MEFs were tracked for velocity quantification. Scale bar: 50 m.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt and Mfn2 -null MEFs treated with DMSO or AIP (40 μM) in the μ-slide. Time-lapse images were taken every 10 min for 17 hr and 50 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null MEFs with vec , CaMKII-WT, or CaMKII-DN in the μ-slide. Time-lapse images were taken every 10 min for 17 hr and 50 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt treated with DMSO and Mfn2 -null MEFs treated with DMSO, RhoA inhibitor-I (0.1 µg/ml), ML-7 (2 µM), Y29632 (5 µM), or Blebbistatin (4 µM) in the μ-slide. Time-lapse images were taken every 10 min for 14 hr and 30 min. MEFs were tracked for velocity quantification. Scale bar: 50 μm.

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression levels of MFN1 and MFN2 in wt, Mfn2-null, and Mfn1-null MEFs. ( B, C ) Spread area ( B ) and circularity ( C ) of wt, Mfn1-null, and Mfn2-null MEFs after overnight culture. The individual points represent individual MEF cells. ( D–G ) representative images with individual tracks ( D ), Wind–Rose plots ( E ), quantification of velocity ( F ), and directionality ( G ) of wt, Mfn1-null, and Mfn2-null MEFs cells during random migration. ( H, I ) Quantification of cell circularity ( H ) and representative images ( I ) of indicated MEFs during cell spreading at indicated time points. Data are presented as mean ± SD in ( F ) and were pooled from a total of 18 cells in three independent experiments. Bars represent arithmetic means ± SD. One representative result of three biological repeats is shown in ( A, D, E, I ). Data are pooled from three independent experiments in ( B, C, F, G ). n = 50 cells are tracked and counted in ( B, C ). N = 30 cells are quantified in ( D ). ****p<0.0001 (one-way ANOVA). Scale bars: 50 µm. Figure 1—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Expressing, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Immunofluorescence of F-actin (phalloidin), α-tubulin, and mitochondria (Mito Tracker) in wt , Mfn1 -null, and Mfn2 -null MEFs. ( B–D ) Cre-induced Mfn2 disruption in MEFs from Mfn2 flox/flox mice displays similar cell morphology as Mfn2 -null MEFs ( B ). ( E, F ) Cell spread area ( E ) and circularity ( F ) of indicated cells in . The individual points represent the circularity or spread area of individual MEF cells in ( C–F ). ( G ) Cells were transfected with mitochondria probes. Mfn2 -null MEFs and Mfn2 -null MEFs overexpressing MFN1 display large and fragmented mitochondria, while MFN2 re-expression in Mfn2 -null MEFs restored mitochondria tubules. One representative result of three biological repeats is shown in ( A, B ). n = 25 cells in each group are quantified in ( C–-F ). ***p≤0.001, ****p<0.0001 (one-way ANOVA). Scale bars: 10 µm in ( A, B ), 20 µm in ( G ).

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Immunofluorescence, Disruption, Transfection, Expressing

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression level of MFN1 and MFN2 in indicated MEF cells. Percentages of knockdown or re-expression were calculated by normalizing the intensity to vinculin first, then normalizing to the wt group. ( B–D ) Representative images with individual tracks ( B ), quantification of velocity ( C ), and Wind–Rose plots ( D ) of indicated MEF cells during random migration. ( E ) Quantification of cell circularity of wt and Mfn2-null MEFs with vec, MFN1, or MFN2 re-expressed during spreading at indicated time points. Data are presented as mean ± SD in ( E ) (n = 5). ( F, G ) Cell circularity ( F ) and cell spreading area ( G ) of indicated MEFs measured after overnight culture. ( H, I ) Percentage of Actin abundance in the cell border region ( H ) and peripheral actin band (PAB) cell percentage in each view was quantified using our custom algorism (see ). ( J ) Representative images of wt, Mfn2-null with doxycycline-induced MEF2 (DIn-MFN2) MEF cells treated with or without doxycycline for 48 hr. The cells are immunostained with phalloidin and MFN2. One representative result of three biological repeats is shown in ( A, B, D, H ). Data are pooled from three independent experiments in ( C, F–I ). n = 30 cells are tracked and counted in ( C ); n = 35 cells are quantified in ( F–H ). Five different views from three biological repeats are quantified in ( I ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA in C, E, F , unpaired t -test in H, I ). Scale bars: 50 µm in ( B ), 10 µm in ( J ). Figure 2—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Expressing, Knockdown, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) RhoA pulldown activation assay demonstrates increased RhoA-GTP in Mfn2 -null MEFs, which can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( A ) Western blot and ( B ) quantification determining the amount of RhoA-GTP and total RhoA protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expression, or with an artificial ER-mitochondria tether; the indicated cell lines were treated with 25 ng/ml PDGF-BB for 0 or 4 min. ( C ) Immunofluorescence of F-actin (phalloidin) and Paxillin in wt , Mfn1 -null, and Mfn2 -null MEFs after overnight culture. ( D, E ) Mfn2 -null MEFs display slightly decreased FA numbers ( D ) but significantly larger FA sizes ( E ). ( F, G ) RhoA pulldown activation assay demonstrates cytosolic Ca 2+ inhibition corrects RhoA-GTP level in Mfn2 -null MEFs. ( F ) Western blot and ( G ) quantification showing the amount of RhoA-GTP protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs treated with BAPTA, or Mfn2 -null MEFs with doxycycline (DOX)-induced MFN2 re-expression for 48 hr; the indicated cells were treated with 25 ng/ml PDGF-BB for the indicated time. RhoA-GTP/total RhoA ratios at 4 min were normalized to 0 min to show the fold changes in ( B, G ). n = 30 cells were quantified in ( D, E ). One representative result of two biological repeats is shown in ( A, B, F, G ). *p≤0.05 (one-way ANOVA comparing each group with the average of the wt group). Scale bar: 50 µm. Figure 5—source data 1. Original blots and figures with the bands labeled for . Figure 5—source data 2. Original blots and figures with the bands labeled for .

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Inhibition, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) Western blot ( A ) and quantification ( B ) of the amount of pMLCII and total MLCII in wt , Mfn1 -null, and Mfn2 -null MEFs. ( C, D ) Increased pMLCII in Mfn2 -null MEFs can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( C ) Western blot and ( D ) quantification determining the amount of pMLCII and total MLCII protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expressed, or with an artificial ER-mitochondria tether. ( E ) Representative images of wt and Mfn2 -null MEFs immunostained for F-actin (phalloidin), pMLCII, and DAPI. ( F ) Western blot determining the expression levels of MLCK or ROCK in Mfn2 -null MEFs with shMLCK or shROCK . ( G ) Western blot of pMLCII and total MLCII Mfn2 -null MEFs with shMLCK or shROCK . ( H ) Representative images of Mfn2 -null MEFs with shMLCK or shROCK immunostained for F-actin (green) and paxillin (red). ( I, J ) Cellular spread area and circularity of wt , Mfn2 -null MEFs with vec, shMLCK, or shROCK were measured after overnight culture. ( K ) Percentage of actin abundance in the cell border region in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . ( L ) Percentage of PAB cells identified by a custom algorithm in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . The individual points stand for the size or circularity of individual MEF cells. One representative result of three biological repeats is shown in ( A, B, F, G ). Four biological repeats were done in ( C, D ). Data are pooled from three independent experiments in ( I, J ). n = 30 cells are quantified in ( I, K ). Five different views from three biological repeats are quantified in ( L ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA, comparing each group to the average of Mfn2 -/- vec group in I, K ). Scale bars: 20 µm in ( H ), 10 µm in ( E ). Figure 7—source data 1. Original blots and figures with the bands labeled for . Figure 7—source data 2. Original blots and figures with the bands labeled for . Figure 7—source data 3. Original blots and figures with the bands labeled for . Figure 7—source data 4. Original blots and figures with the bands labeled for .

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Expressing, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: HEK293T (CRL-11268), wild-type (CRL-2991), Mfn2 -null (CRL-2993), and Mfn1 -null (CRL-2992) MEFs were from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt , Mfn2 -null, and Mfn1 -null MEFs in the µ-slide 15 min after plating. Time-lapse images were taken every 10 min for 16 hr and 40 min. Individual MEFs were tracked for velocity quantification. Scale bar: 50 m.

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: Cell spreading and random migration of wt and Mfn2 -null MEFs with vec , MFN1, or MFN2 re-expressed in the µ-slide. Time-lapse images were taken every 10 min for 14 hr and 50 min. Individual MEFs were tracked for velocity quantification. Scale bar: 50 m.

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques:

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression levels of MFN1 and MFN2 in wt, Mfn2-null, and Mfn1-null MEFs. ( B, C ) Spread area ( B ) and circularity ( C ) of wt, Mfn1-null, and Mfn2-null MEFs after overnight culture. The individual points represent individual MEF cells. ( D–G ) representative images with individual tracks ( D ), Wind–Rose plots ( E ), quantification of velocity ( F ), and directionality ( G ) of wt, Mfn1-null, and Mfn2-null MEFs cells during random migration. ( H, I ) Quantification of cell circularity ( H ) and representative images ( I ) of indicated MEFs during cell spreading at indicated time points. Data are presented as mean ± SD in ( F ) and were pooled from a total of 18 cells in three independent experiments. Bars represent arithmetic means ± SD. One representative result of three biological repeats is shown in ( A, D, E, I ). Data are pooled from three independent experiments in ( B, C, F, G ). n = 50 cells are tracked and counted in ( B, C ). N = 30 cells are quantified in ( D ). ****p<0.0001 (one-way ANOVA). Scale bars: 50 µm. Figure 1—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Immunofluorescence of F-actin (phalloidin), α-tubulin, and mitochondria (Mito Tracker) in wt , Mfn1 -null, and Mfn2 -null MEFs. ( B–D ) Cre-induced Mfn2 disruption in MEFs from Mfn2 flox/flox mice displays similar cell morphology as Mfn2 -null MEFs ( B ). ( E, F ) Cell spread area ( E ) and circularity ( F ) of indicated cells in . The individual points represent the circularity or spread area of individual MEF cells in ( C–F ). ( G ) Cells were transfected with mitochondria probes. Mfn2 -null MEFs and Mfn2 -null MEFs overexpressing MFN1 display large and fragmented mitochondria, while MFN2 re-expression in Mfn2 -null MEFs restored mitochondria tubules. One representative result of three biological repeats is shown in ( A, B ). n = 25 cells in each group are quantified in ( C–-F ). ***p≤0.001, ****p<0.0001 (one-way ANOVA). Scale bars: 10 µm in ( A, B ), 20 µm in ( G ).

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Immunofluorescence, Disruption, Transfection, Expressing

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A ) Western blot determining the expression level of MFN1 and MFN2 in indicated MEF cells. Percentages of knockdown or re-expression were calculated by normalizing the intensity to vinculin first, then normalizing to the wt group. ( B–D ) Representative images with individual tracks ( B ), quantification of velocity ( C ), and Wind–Rose plots ( D ) of indicated MEF cells during random migration. ( E ) Quantification of cell circularity of wt and Mfn2-null MEFs with vec, MFN1, or MFN2 re-expressed during spreading at indicated time points. Data are presented as mean ± SD in ( E ) (n = 5). ( F, G ) Cell circularity ( F ) and cell spreading area ( G ) of indicated MEFs measured after overnight culture. ( H, I ) Percentage of Actin abundance in the cell border region ( H ) and peripheral actin band (PAB) cell percentage in each view was quantified using our custom algorism (see ). ( J ) Representative images of wt, Mfn2-null with doxycycline-induced MEF2 (DIn-MFN2) MEF cells treated with or without doxycycline for 48 hr. The cells are immunostained with phalloidin and MFN2. One representative result of three biological repeats is shown in ( A, B, D, H ). Data are pooled from three independent experiments in ( C, F–I ). n = 30 cells are tracked and counted in ( C ); n = 35 cells are quantified in ( F–H ). Five different views from three biological repeats are quantified in ( I ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA in C, E, F , unpaired t -test in H, I ). Scale bars: 50 µm in ( B ), 10 µm in ( J ). Figure 2—source data 1. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Knockdown, Migration, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) RhoA pulldown activation assay demonstrates increased RhoA-GTP in Mfn2 -null MEFs, which can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( A ) Western blot and ( B ) quantification determining the amount of RhoA-GTP and total RhoA protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expression, or with an artificial ER-mitochondria tether; the indicated cell lines were treated with 25 ng/ml PDGF-BB for 0 or 4 min. ( C ) Immunofluorescence of F-actin (phalloidin) and Paxillin in wt , Mfn1 -null, and Mfn2 -null MEFs after overnight culture. ( D, E ) Mfn2 -null MEFs display slightly decreased FA numbers ( D ) but significantly larger FA sizes ( E ). ( F, G ) RhoA pulldown activation assay demonstrates cytosolic Ca 2+ inhibition corrects RhoA-GTP level in Mfn2 -null MEFs. ( F ) Western blot and ( G ) quantification showing the amount of RhoA-GTP protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs treated with BAPTA, or Mfn2 -null MEFs with doxycycline (DOX)-induced MFN2 re-expression for 48 hr; the indicated cells were treated with 25 ng/ml PDGF-BB for the indicated time. RhoA-GTP/total RhoA ratios at 4 min were normalized to 0 min to show the fold changes in ( B, G ). n = 30 cells were quantified in ( D, E ). One representative result of two biological repeats is shown in ( A, B, F, G ). *p≤0.05 (one-way ANOVA comparing each group with the average of the wt group). Scale bar: 50 µm. Figure 5—source data 1. Original blots and figures with the bands labeled for . Figure 5—source data 2. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Inhibition, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet: ( A, B ) Western blot ( A ) and quantification ( B ) of the amount of pMLCII and total MLCII in wt , Mfn1 -null, and Mfn2 -null MEFs. ( C, D ) Increased pMLCII in Mfn2 -null MEFs can be corrected by re-expressing MFN2 or inducing a mitochondria-endoplasmic reticulum (ER) tether. ( C ) Western blot and ( D ) quantification determining the amount of pMLCII and total MLCII protein in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with MFN2 re-expressed, or with an artificial ER-mitochondria tether. ( E ) Representative images of wt and Mfn2 -null MEFs immunostained for F-actin (phalloidin), pMLCII, and DAPI. ( F ) Western blot determining the expression levels of MLCK or ROCK in Mfn2 -null MEFs with shMLCK or shROCK . ( G ) Western blot of pMLCII and total MLCII Mfn2 -null MEFs with shMLCK or shROCK . ( H ) Representative images of Mfn2 -null MEFs with shMLCK or shROCK immunostained for F-actin (green) and paxillin (red). ( I, J ) Cellular spread area and circularity of wt , Mfn2 -null MEFs with vec, shMLCK, or shROCK were measured after overnight culture. ( K ) Percentage of actin abundance in the cell border region in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . ( L ) Percentage of PAB cells identified by a custom algorithm in wt , Mfn2 -null MEFs, Mfn2 -null MEFs with shMLCK or shROCK . The individual points stand for the size or circularity of individual MEF cells. One representative result of three biological repeats is shown in ( A, B, F, G ). Four biological repeats were done in ( C, D ). Data are pooled from three independent experiments in ( I, J ). n = 30 cells are quantified in ( I, K ). Five different views from three biological repeats are quantified in ( L ). *p≤0.05, **p≤0.01, ***p≤0.001, ****p<0.0001 (one-way ANOVA, comparing each group to the average of Mfn2 -/- vec group in I, K ). Scale bars: 20 µm in ( H ), 10 µm in ( E ). Figure 7—source data 1. Original blots and figures with the bands labeled for . Figure 7—source data 2. Original blots and figures with the bands labeled for . Figure 7—source data 3. Original blots and figures with the bands labeled for . Figure 7—source data 4. Original blots and figures with the bands labeled for .

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Western Blot, Expressing, Labeling

Journal: eLife

Article Title: Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells

doi: 10.7554/eLife.88828

Figure Lengend Snippet:

Article Snippet: Cell line ( M. musculus ) , Mfn1 -null MEF , ATCC , CRL-2992 , .

Techniques: Transfection, Construct, Over Expression, Expressing, Dominant Negative Mutation, Recombinant, Plasmid Preparation, Control, Knockdown, Sequencing, Staining, Activation Assay, Imaging, Cloning, Software, Microscopy