mcherry sept2  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

doi: 10.1016/j.jbc.2023.105084

Figure Lengend Snippet: Microtubule-associated SEPT2/6/7 complexes inhibit t he motility of KIF5C, KIF1A, and DDB. A , kymographs show motile ( diagonal lines ) and stationary (vertical lines) KIF5C(1-560)-mCit on an uncoated microtubule ( left ) and a microtubule ( right ) coated with mCherry-SEPT2/6/7 (100 nM). B , mean (±S.D.) landing rates of KIF5C(1-560)-mCit on uncoated microtubules (7.03 ± 1.13 events/μm/min; n = 20 microtubules) and microtubules coated with 100 nM mCherry-SEPT2/6/7 (1.15 ± 0.65 events/μm/min; n = 20 microtubules). ∗∗∗∗ p < 0.0001. C , mean (±S.D.) percentage of KIF5C(1-560)-mCit particles pausing on uncoated microtubules (17.94% ± 16.56%; n = 20 microtubules) and microtubules coated with 100 nM mCherry-SEPT2/6/7 (18.67% ± 16.33%; n = 20 microtubules). n.s., not significant ( p > 0.05). D , mean (±S.D.) velocity of KIF5C(1-560)-mCit particles ( n = 200) on uncoated microtubules (0.78 ± 0.18 μm/s) and microtubules coated with 100 nM of mCherry-SEPT2/6/7 (0.63 ± 0.22 μm/s). ∗∗∗∗ p < 0.0001. E , one-cumulative distribution plot of the run lengths of KIF5C(1-560)-mCit particles ( n = 200) on uncoated microtubules and microtubules with mCherry-SEPT2/6/7 (100 nM). Data were fit to one-phase exponential decay with a decay constant τ (run length), which is shown with the R 2 fit value. The mean (±S.D.) run length values were 1.18 ± 0.52 μm and 0.64 ± 030 μm in the absence and presence of mCherry-SEPT2/6/7, respectively ( p < 0.0001). F , kymographs show motile ( diagonal lines ) and stationary ( vertical lines ) KIF1A(1-393)-GCN4-3XmCit particles on an uncoated microtubule ( left ) and a microtubule ( right ), which was coated with mCherry-SEPT2/6/7 (100 nM). G , mean (±S.D.) landing rates of KIF1A(1-393)-GCN4-3XmCit on uncoated microtubules (5.27 ± 1.62 events/μm/min; n = 20 microtubules) and microtubules coated with 100 nM mCherry-SEPT2/6/7 (1.98 ± 0.79 events/μm/min; n = 20 microtubules). ∗∗∗∗ p < 0.0001. H , mean (±S.D.) percentage of KIF1A(1-393)-GCN4-3XmCit particles pausing on uncoated microtubules (5.99% ± 3.07%; n = 20 microtubules) and microtubules coated with 100 nM mCherry-SEPT2/6/7 (36.19% ± 11.04%; n = 20 microtubules). ∗∗∗∗ p < 0.0001. I , mean (±S.D.) velocity of KIF1A(1-393)-GCN4-3XmCit particles ( n = 200) on uncoated microtubules (1.80 ± 0.34 μm/s) and microtubules coated with mCherry-SEPT2/6/7 (0.80 ± 0.22 μm/s). ∗∗∗∗ p < 0.0001. J , one-cumulative distribution plot of the run lengths of KIF1A(1-393)-GCN4-3XmCit particles ( n = 200) on uncoated microtubules and microtubules with mCherry-SEPT2/6/7 (100 nM). Data were fit to one-phase exponential decay with a decay constant τ (run length), which is shown with the R 2 fit value. The mean (±S.D.) run length values were 6.06 ± 2.85 μm and 2.02 ± 1.13 μm in the absence and presence of mCherry-SEPT2/6/7, respectively ( p < 0.0001). K , kymographs of DDB-GFP motility on an uncoated microtubule and microtubules coated with 20 nM and 50 nM mCherry-SEPT2/6/7. L , mean (±S.D.) landing rates of DDB-GFP particles on uncoated microtubules (0.14 ± 0.09 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) which were coated with 20 nM (0.08 ± 0.06 events/μm/min) and 50 nM mCherry-SEPT2/6/7 (0.02 ± 0.02 events/μm/min). ∗ p = 0.04; ∗∗∗∗ p < 0.0001. M , mean (±S.D.) percentage of DDB-GFP particles that pause on uncoated microtubules (22% ± 27%; n = 20) and microtubules ( n = 20) coated with 20 nM mCherry-SEPT2/6/7 (27% ± 26%). n.s., not significant ( p > 0.05). N , mean (±S.D.) velocity of DDB-GFP particles on uncoated microtubules (0.41 ± 0.27 μm/s; n = 159) and microtubules coated with 20 nM mCherry-SEPT2/6/7 (0.23 ± 0.20 μm/s; n = 150). ∗∗∗∗ p < 0.0001. O , one-cumulative distribution plot of the run lengths of DDB-GFP particles on uncoated microtubules ( n = 159) and microtubules coated with 20 nM mCherry-SEPT2/6/7 ( n = 150). Data were fit to one-phase exponential decay with a decay constant τ (run length) which is shown with the R 2 fit value. The mean (±S.D.) run lengths were 7.38 ± 5.65 μm and 2.57 ± 2.07 μm in the absence and presence of mCherry-SEPT2/6/7, respectively ( p < 0.0001). Statistical analysis of data with normal and non-normal distributions was performed with student's t and Mann-Whitney U tests, respectively. A nonparametric one-way Welch ANOVA test was performed for multiple comparison groups, followed by a post hoc Dunnett T3 test for pairwise comparisons. DDB, dynein-dynactin-bicaudal D.

Article Snippet: The following cotransformations into E. coli BL21 (DE3) (Invitrogen) were performed: His-mCherry-SEPT2 and pnCS SEPT6/7-Strep(+1-57 bp SEPT7 N-term) (SEPT2/6/7), His-mCherry-SEPT5 and SEPT11/7-strep (SEPT5/7/11), and pnEA-vH_His-TEV-SEPT2-mCherry_SEPT6 and pnCS_SEPT7_SEPT9_i1-TEV-Strep (SEPT2/6/7/9).

Techniques: MANN-WHITNEY, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

doi: 10.1016/j.jbc.2023.105084

Figure Lengend Snippet: In microtubule-associated SEPT2/6/7/9 complexes, SEPT9 dampens the i nhibition of KIF1A(1-393) motility by SEPT2/6/7. A , kymographs show motile ( diagonal lines ) and stationary ( vertical lines ) KIF1A(1-393)-GCN4-3XmCit particles on an uncoated microtubule and microtubules which were coated with 10 nM or 100 nM mCherry-SEPT2/6/7/9. B , mean (±S.D.) landing rates of KIF1A(1-393)-GCN4-3XmCit on uncoated microtubules (1.94 ± 0.48 events/μm/min; n = 10 microtubules) and microtubules ( n = 10) coated with 10 nM (1.37 ± 0.35 events/μm/min) or 100 nM mCherry-SEPT2/6/7/9 (0.92 ± 0.37 events/μm/min). ∗∗ p = 0.008; ∗∗∗∗ p < 0.0001. C , mean (±S.D.) percentage of KIF1A(1-393)-GCN4-3XmCit particles that pause on uncoated microtubules (21.09% ± 9.07%; n = 10 microtubules) and microtubules ( n = 10) coated with 10 nM (28.56% ± 12%) or 100 nM mCherry-SEPT2/6/7/9 (48.35% ± 18.82%). n.s., not significant ( p > 0.05); ∗∗∗∗ p < 0.0001. D , mean (±S.D.) velocity of KIF1A(1-393)-GCN4-3XmCit particles ( n = 100) on uncoated microtubules (1.71 ± 0.33 μm/s) and microtubules coated with 10 nM (2.01 ± 0.66 μm/s) or 100 nM mCherry-SEPT2/6/7/9 (1.40 ± 0.37 μm/s). ∗∗ p = 0.002; ∗∗∗∗ p < 0.0001. E , one-cumulative distribution plot of the run lengths of KIF1A(1-393)-GCN4-3XmCit particles ( n = 100) on uncoated microtubules and microtubules coated with 10 nM or 100 nM mCherry-SEPT2/6/7/9. Data were fit to one-phase exponential decay with a decay constant τ (run length) which is shown with the R 2 fit value. The mean (±S.D.) run length value on uncoated microtubules was 7.89 ± 3.88 μm, and the mean (±S.D.) run lengths on microtubules with 10 nM and 100 nM SEPT2/6/7/9 were respectively 5.84 ± 3.18 μm ( p = 0.0003) and 4.44 ± 2.67 μm ( p < 0.0001). Data were statistically analyzed with one-way ANOVA and a post hoc Dunnett test for multiple comparisons ( B and C ) or Kruskal–Wallis test with post hoc Dunn's test for multiple pairwise comparisons ( D and E ). SEPT9, septin 9.

Article Snippet: The following cotransformations into E. coli BL21 (DE3) (Invitrogen) were performed: His-mCherry-SEPT2 and pnCS SEPT6/7-Strep(+1-57 bp SEPT7 N-term) (SEPT2/6/7), His-mCherry-SEPT5 and SEPT11/7-strep (SEPT5/7/11), and pnEA-vH_His-TEV-SEPT2-mCherry_SEPT6 and pnCS_SEPT7_SEPT9_i1-TEV-Strep (SEPT2/6/7/9).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

doi: 10.1016/j.jbc.2023.105084

Figure Lengend Snippet: Microtubule-associated SEPT5/7/11 complexes are permissive to KIF1A motility but inhibit KIF5C and DDB. A , kymographs show motile ( diagonal lines ) and stationary ( vertical lines ) KIF5C(1-560)-mCit on an uncoated microtubule ( left ) and a microtubule ( right ) which was coated with 50 nM mCherry-SEPT5/7/11. Red arrows point to KIF5C(1-560)-mCit motors, which remain immotile with no processive motility prior to dissociation (immotile particles). B , mean (±S.D.) landing rates of KIF5C(1-560)-mCit on uncoated microtubules (2.39 ± 0.53 events/μm/min; n = 20 microtubules) and microtubules coated with 50 nM mCherry-SEPT5/7/11 (1.22 ± 0.33 events/μm/min; n = 20 microtubules). ∗∗∗∗ p < 0.0001. C , mean (±S.D.) percentage of KIF5C(1-560)-mCit particles pausing on uncoated microtubules (6.38% ± 4.00%; n = 20 microtubules) and microtubules coated with 50 nM mCherry-SEPT5/7/11 (11.10% ± 7.50%; n = 20 microtubules). ∗ p = 0.02. D , mean (±S.D.) velocity of KIF5C(1-560)-mCit ( n = 150) on uncoated microtubules (1.19 ± 0.25 μm/s) and microtubules coated with 50 nM of mCherry-SEPT5/7/11 (0.81 ± 0.31 μm/s). ∗∗∗∗ p < 0.0001. E , one-cumulative distribution plot of the run lengths of KIF5C(1-560)-mCit particles ( n = 150) on uncoated microtubules and microtubules with mCherry-SEPT5/7/11 (50 nM). Data were fit to one-phase exponential decay with a decay constant τ (run length), which is shown with the R 2 fit value. The mean (±S.D.) run lengths were 1.58 ± 1 μm and 0.91 ± 0.48 μm in the absence and presence of mCherry-SEPT5/7/11, respectively ( p < 0.0001). F , kymographs show motile ( diagonal lines ) and stationary ( vertical lines ) KIF1A(1-393)-GCN4-3XmCit particles on an uncoated microtubule ( left ) and a microtubule ( right ), which was coated with 50 nM mCherry-SEPT5/7/11. Red arrows point to KIF1A(1-393)-GCN4-3XmCit motors, which associate with a microtubule with no processive motility. G , mean (±S.D.) landing rates of KIF1A(1-393)-GCN4-3XmCit on uncoated microtubules (3.99 ± 1.66 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 50 nM mCherry-SEPT5/7/11 (3.85 ± 1.42 events/μm/min). n.s., not significant ( p > 0.05). H , mean (±S.D.) percentage of KIF1A(1-393)-GCN4-3XmCit particles pausing on uncoated microtubules (13.44% ± 3.69%; n = 20 microtubules) and microtubules ( n = 20) coated with 50 nM mCherry-SEPT5/7/11 (19.33% ± 5.63%; n = 20). ∗∗∗ p = 0.0004. I , mean (±S.D.) velocity of KIF1A(1-393)-GCN4-3XmCit particles ( n = 148) on uncoated microtubules (1.69 ± 0.38 μm/s) and microtubules coated with 50 nM mCherry-SEPT5/7/11 (1.32 ± 0.33 μm/s). ∗∗∗∗ p < 0.0001. J , one-cumulative distribution plot of the run lengths of KIF1A(1-393)-GCN4-3XmCit particles ( n = 148) on uncoated microtubules and microtubules with mCherry-SEPT5/7/11 (50 nM). Data were fit to one-phase exponential decay with a decay constant τ (run length), which is shown with the R 2 fit value. The mean (±S.D.) run length values were 5.25 ± 2.59 μm and 4.68 ± 2.63 μm in the absence and presence of mCherry-SEPT2/6/7, respectively ( p > 0.05). K , kymographs of DDB-GFP on an uncoated microtubule and microtubules which were coated with 10 nM and 50 nM mCherry-SEPT5/7/11. Red arrows point to DDB-GFP particles, which associate with microtubules with no processive motility (immotile particles). L , mean (±S.D.) landing rates of DDB-GFP particles on uncoated microtubules (0.26 ± 0.15 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 10 nM (0.19 ± 0.14 events/μm/min) and 50 nM mCherry-SEPT5/7/11 (0.01 ± 0.02 events/μm/min). n.s., not significant ( p > 0.05); ∗∗∗∗ p < 0.0001. M , mean (±S.D.) percentage of DDB-GFP particles that pause on uncoated microtubules (11.85% ± 14.98%; n = 20 microtubules) and microtubules ( n = 20) which were coated with 10 nM mCherry-SEPT5/7/11 (28.37% ± 24.35%) ∗ p = 0.01. N , mean (±S.D.) velocity of DDB-GFP particles on uncoated microtubules (0.42 ± 0.28 μm/s; n = 150) and microtubules coated with 50 nM mCherry-SEPT5/7/11 (0.28 ± 0.22 μm/s; n = 155). ∗∗∗∗ p < 0.0001. O , one-cumulative distribution plot of the run lengths of DDB-GFP ( n = 150–155) on uncoated microtubules and microtubules coated with mCherry-SEPT5/7/11 (50 nM). Data were fit to one-phase exponential decay with a decay constant τ (run length), which is shown with the R 2 fit value. The mean (±S.D.) run length values were 6.29 ± 4.86 μm ( n = 150) and 3.14 ± 2.80 μm in the absence and presence of mCherry-SEPT5/7/11, respectively ( p < 0.0001). Statistical analysis of data with normal and non-normal distributions was performed with student's t and Mann-Whitney U tests, respectively. A nonparametric one-way ANOVA Kruskal–Wallis test was performed for multiple comparison groups, followed by a post hoc Dunn's test for pairwise comparisons. DDB, dynein-dynactin-bicaudal D.

Article Snippet: The following cotransformations into E. coli BL21 (DE3) (Invitrogen) were performed: His-mCherry-SEPT2 and pnCS SEPT6/7-Strep(+1-57 bp SEPT7 N-term) (SEPT2/6/7), His-mCherry-SEPT5 and SEPT11/7-strep (SEPT5/7/11), and pnEA-vH_His-TEV-SEPT2-mCherry_SEPT6 and pnCS_SEPT7_SEPT9_i1-TEV-Strep (SEPT2/6/7/9).

Techniques: MANN-WHITNEY, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

doi: 10.1016/j.jbc.2023.105084

Figure Lengend Snippet: SEPT5/7/11 promotes DDB and kinesin tethering to microtubules. A , mean (±S.D.) number of immotile DDB-GFP events per micrometer of uncoated microtubules (0.07 ± 0.06 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 10 nM (0.15 ± 0.07 events/μm/min) or 50 nM mCherry-SEPT5/7/11 (0.28 ± 0.12 events/μm/min). ∗∗ p = 0.006; ∗∗∗∗ p < 0.0001. B , mean (±S.D.) number of immotile DDB-GFP events per micrometer of uncoated microtubules (0.05 ± 0.05 events/μm/min; n = 20 microtubules) and microtubules (n = 20) coated with 20 nM (0.07 ± 0.05 events/μm/min) or 50 nM mCherry-SEPT2/6/7 (0.06 ± 0.04 events/μm/min). n.s., not significant ( p > 0.05). C , mean (±S.D.) number of immotile KIF5C(1-560)-mCit particles per micrometer of uncoated microtubules (0.18 ± 0.12 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 50 nM mCherry-SEPT5/7/11 (0.37 ± 0.15 events/μm/min). ∗∗∗∗ p < 0.0001. D , mean (±S.D.) number of immotile KIF5C(1-560)-mCit events per micrometer of uncoated microtubules (1.12 ± 0.41 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 100 nM mCherry-SEPT2/6/7 (1.23 ± 0.49 events/μm/min). n.s., not significant ( p > 0.05). E , mean (±SEM) number of immotile KIF1A(1-393)-GCN4-3XmCit events per micrometer of uncoated microtubule (0.42 ± 0.24 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 50 nM mCherry-SEPT5/7/11 (0.57 ± 0.18 events/μm). ∗ p = 0.03. F , mean (±SEM) number of immotile KIF1A(1-393)-GCN4-3XmCit events per micrometer of uncoated microtubule (0.75 ± 0.39 events/μm/min; n = 20 microtubules) and microtubules ( n = 20) coated with 50 nM mCherry-SEPT2/6/7 (0.67 ± 0.29 events/μm/min). n.s., not significant ( p > 0.05). Statistical analysis of data with normal and non-normal distributions was performed with student's t and Mann-Whitney U tests, respectively. A nonparametric one-way ANOVA Kruskal–Wallis test was performed for multiple comparison groups, followed by a post hoc Dunn's test for pairwise comparisons. DDB, dynein-dynactin-bicaudal D.

Article Snippet: The following cotransformations into E. coli BL21 (DE3) (Invitrogen) were performed: His-mCherry-SEPT2 and pnCS SEPT6/7-Strep(+1-57 bp SEPT7 N-term) (SEPT2/6/7), His-mCherry-SEPT5 and SEPT11/7-strep (SEPT5/7/11), and pnEA-vH_His-TEV-SEPT2-mCherry_SEPT6 and pnCS_SEPT7_SEPT9_i1-TEV-Strep (SEPT2/6/7/9).

Techniques: MANN-WHITNEY, Comparison