far-red dye jf646  (Promega)

Journal: Cell Reports

Article Title: Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

doi: 10.1016/j.celrep.2022.110610

Figure Lengend Snippet: CH-STED time-lapse imaging of control, HAUS6-, and Ndc80-depleted IM fibroblasts stably expressing 2xGFP-CENP-A (magenta) and EB3-Halotag, conjugated with a far-red dye JF646 (green). Cells were treated for 30 min with MG-132 and imaged every 8 sec for 2 min. Scale bar: 1 μm.

Article Snippet: To select pLVx-EB3-Halotag positive cells, 20 nM of the far-red dye JF646 (Promega) was added 5–10 min before FACS sorting.

Techniques:

Journal: Cell Reports

Article Title: Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

doi: 10.1016/j.celrep.2022.110610

Figure Lengend Snippet: Live CH-STED time-lapses of control, HAUS6-, and Ndc80-depleted IM fibroblasts stably expressing 2xGFP-CENP-A (magenta) and EB3-Halotag, conjugated with a far-red dye JF646 (green), show MT polymerization events within one k-fiber. Time-lapse was 750 ms. Time is displayed in seconds. Scale bar: 1 μm.

Article Snippet: To select pLVx-EB3-Halotag positive cells, 20 nM of the far-red dye JF646 (Promega) was added 5–10 min before FACS sorting.

Techniques:

Journal: Cell Reports

Article Title: Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

doi: 10.1016/j.celrep.2022.110610

Figure Lengend Snippet: Augmin promotes kinetochore microtubule turnover and poleward flux (A–K) (A) Pre-recording snapshots of control, HAUS6-, and Ndc80-depleted IM fibroblasts stably expressing 2xGFP-CENP-A (magenta) and EB3-Halo tag conjugated with JF646 (green), imaged by confocal microscopy. Scale bar: 5 μm. (A′) Collapsed kymographs of live CH-STED recordings (time lapse: 8 s; pixel size, 40 nm). Graphical sketches on the right highlight chromosome movement over time (tracing); P, pole (green); KT, kinetochore (magenta); EB3 accumulation at KT is shown in green. Quantitative analysis of chromosome anti-poleward (B) and poleward (C) velocities, chromosome oscillatory amplitude (D), and period (E). Fraction of EB3 accumulation at KT per minute (approximately one period) was measured from track data in (F), and KT-to-pole distance determined in (G). Horizontal bar, 1 μm; vertical bar, 10 s (n = 8 control cells, n = 9 siHAUS6 cells, and n = 8 siNdc80 cells). (H) Examples of control and partial HAUS6-depleted metaphase cells displaying photoactivatable PA-GFP-α-tubulin (inverted grayscale), GFP-Centrin-1 (inverted grayscale), and labeled with 50 nM SiR-DNA to visualize chromosomes (inverted grayscale). Pre-PA, frame immediately before photoactivation; PA, frame immediately after photoactivation. Scale bar: 5 μm. (I) Normalized fluorescence dissipation after photoactivation (FDAPA) curves of control and partial HAUS6-depleted cells. Whole lines show double exponential curve fittings (R 2 > 0.98), and error bars show 95% confidence interval for each time point. (J) Table showing the calculated MT percentages and turnover values for control and partial HAUS6-depleted cells (n = 20 control cells; n = 11 siHAUS6 cells). (K) MT flux velocity (n = 21 control cells; n = 16 siHAUS6 cells). Each data point represents one measurement; data pooled from at least three independent experiments and analyzed using a Mann-Whitney test (B, C, F, G) or an unpaired t test (D, J, K); Error bars indicate mean ± SD; ns, not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: To select pLVx-EB3-Halotag positive cells, 20 nM of the far-red dye JF646 (Promega) was added 5–10 min before FACS sorting.

Techniques: Control, Stable Transfection, Expressing, Confocal Microscopy, Labeling, Fluorescence, MANN-WHITNEY

Journal: Cell Reports

Article Title: Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

doi: 10.1016/j.celrep.2022.110610

Figure Lengend Snippet: Microtubule growth within individual k-fibers shows a wide angular dispersion and requires augmin (A) CH-STED images of control, HAUS6-, and Ndc80-depleted cells stained with α-tubulin, ACA (cyan), and DAPI (white; opacity 15%). Temporal color code tool on Fiji was used to match each α-tubulin z plane to a different color. Scale bar: 5 μm. Insets show the maximum-intensity projection of relevant z planes highlighting the presence/absence of k-fibers (α-tubulin, inverted grayscale). Scale bar: 1 μm. (B) IM fibroblasts stably expressing 2xGFP-CENP-A (magenta) and EB3-Halo tag conjugated with JF646 (green) were used to track MT polymerization events within one k-fiber by live CH-STED microscopy (time lapse, 750 ms; pixel size, 40 nm). Images on the top show a pre-recording snapshot (confocal) of control, HAUS6-, and Ndc80-depleted cells. Images below show chromo-projections of the time-lapse movie of fluorescently labeled EB3 over time and CENP-A contours. A limited time-window of 12 s (five frames) was selected, allowing a fine time-discrimination of MT growing events within a k-fiber. Scale bar: 500 nm. (C) Frequency count of EB3 comets’ angular dispersion relative to the k-fiber axis in control cells (n = 17 cells). (D) Corresponding collapsed kymographs of control, HAUS6-, and Ndc80-depleted cells from (B). Graphical sketches on the right highlight detected EB3 comets’ trajectories; KT, kinetochore (magenta); kMTs, green; non-kMTs, light brown. Vertical bar, 10 s; horizontal bar, 1 μm. (E) Number of EB3 growing events per KT (control, n = 46 kMT comets/n = 21 non-kMT comets/n = 12 cells; siHAUS6, n = 38 kMT comets/n = 41 non-kMT comets/n = 15 cells; siNdc80, n = 23 kMT comets/n = 41 non-kMT comets/n = 10 cells). Error bars indicate mean ± SD, ns, not significant; ∗∗ p ≤ 0.01; ∗∗∗ p < 0.001. (F) Distance between KT pairs upon stable expression of 2xGFP-CENP-A (control n = 34 cells; siHAUS6 n = 36 cells; siNdc80 n = 28 cells). Data pooled from at least three independent experiments and analyzed using an unpaired t test (E, F); Error bars indicate mean ± SD; ns, not significant; ∗ p ≤ 0.05.

Article Snippet: To select pLVx-EB3-Halotag positive cells, 20 nM of the far-red dye JF646 (Promega) was added 5–10 min before FACS sorting.

Techniques: Dispersion, Control, Staining, Stable Transfection, Expressing, Microscopy, Labeling

Journal: Cell Reports

Article Title: Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

doi: 10.1016/j.celrep.2022.110610

Figure Lengend Snippet:

Article Snippet: To select pLVx-EB3-Halotag positive cells, 20 nM of the far-red dye JF646 (Promega) was added 5–10 min before FACS sorting.

Techniques: Recombinant, Software