Augmin prevents merotelic attachments by promoting proper arrangement of bridging and kinetochore fibers
- Division of Molecular Biology, Ruđer Bošković Institute, Croatia
Figures
Figure 1 with 1 supplement
Augmin ensures the proper formation of entities consisting of bridging fibers that connect two sister k-fibers.
(A) STED superresolution images of microtubules immunostained for α-tubulin (gray) in control (left), HAUS6- (middle), and HAUS8-depleted (right) RPE1 cells stably expressing CENP-A-GFP (rainbow, …
Figure 1—figure supplement 1
Augmin is necessary for the formation of uniformly arranged microtubule bundles in the spindle.
Panels with whole spindles: STED superresolution images of microtubules immunostained for α-tubulin (gray) in control (top), HAUS6- (middle), and HAUS8-depleted (bottom) RPE1 cells stably expressing …
Figure 2 with 1 supplement
Augmin-nucleated midplane-crossing microtubules prevent kinetochore tilt and thus merotelic attachments.
(A) Time-lapse images of RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP (rainbow, confocal) in Mad2-depleted cells (left) and Mad2/HAUS6-codepleted cells (right). Yellow arrows represent …
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Figure 2—source data 1
Immunoblot analysis of HAUS6 siRNA treatment efficiency in RPE1 cells stably expressing CENP-A-GFP and Centrin1-GFP.
Full unedited blots of all three independent experiments are shown.
- https://cdn.elifesciences.org/articles/83287/elife-83287-fig2-data1-v2.pdf
Figure 2—figure supplement 1
Augmin is required for accurate chromosome segregation during anaphase.
(A) Fixed control, HAUS6 and HAUS8 siRNA-treated HeLa cells stably expressing PRC1-GFP (yellow) and immunostained for HAUS6 (red, left panels) or HAUS8 (red, right panels). Chromosomes were stained …
Figure 3 with 1 supplement
Augmin is crucial for the nucleation of bridging microtubules.
(A) The insets of kinetochore pairs in RPE1 cells stably expressing CENP-A-GFP (not shown) immunostained for α-tubulin (gray, STED) in control cells (left) and after HAUS6 (middle) or HAUS8 (right) …
Figure 3—figure supplement 1
Augmin ensures proper architecture and dynamics of the metaphase spindle.
(A) The fractions of kinetochore pairs with bridging fibers (dark gray) and with undetectable bridging fibers (light gray). The fractions are obtained from the tubulin intensity measured in STED …
Figure 4 with 1 supplement
Augmin-depleted spindles have fewer bridging fibers, which have larger overlap length and are located at the spindle periphery.
(A) The four columns on the left represent live images of metaphase spindles in untreated HeLa or MG-132-treated RPE1 cells stably expressing PRC1-GFP (yellow) and stained with SiR-DNA (blue) in …
Figure 4—figure supplement 1
Spindles without augmin are wider with fewer overlaps, which occupy a larger portion of the spindle length and accumulate at the spindle periphery.
(A) Four columns represent live images of metaphase spindles in HeLa cells stably expressing PRC1-GFP (yellow) and stained with SiR-DNA (blue) in control (top) and after HAUS6 (middle) or HAUS8 …
Figure 5 with 1 supplement
The reduction of the interkinetochore distance after augmin depletion is related to the impairment of bridging fibers.
(A) STED superresolution images of microtubules immunostained for α-tubulin (gray) in control (left), HAUS6- (middle) and HAUS-8 depleted (right) RPE1 cells stably expressing CENP-A-GFP (rainbow, …
Figure 5—figure supplement 1
Augmin-generated bridging fibers have a distinct role in the regulation of kinetochore tension.
(A) Univariate scatter plot of the interkinetochore distance in control (gray), HAUS6- (yellow) and HAUS8-depleted (orange) RPE1 cells stably expressing CENP-A-GFP and immunostained for α-tubulin …
Figure 6
A model of augmin-dependent nucleation of bridging microtubules with their contribution to mitotic fidelity.
(A) Bridging microtubules are to a large extent formed by augmin-dependent nucleation. They ensure the alignment of sister kinetochores parallel to the spindle axis and the interkinetochore tension. …
Videos
Video 1
RPE1 cell stably expressing CENP-A-GFP and Centrin1-GFP (16-colors) following Mad2 depletion.
Kinetochores are color-coded for depth from blue to red with the 16 Colors LUT and noise was processed with the Despeckle function in ImageJ. Scale bar, 2 µm.
Video 2
RPE1 cell stably expressing CENP-A-GFP and Centrin1-GFP (16-colors) following Mad2/HAUS6 codepletion.
Kinetochores are color-coded for depth from blue to red with the 16 Colors LUT and noise was processed with the Despeckle function in ImageJ. Scale bar, 2 µm.
