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Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation.

Umbreit NT, Miller MP, Tien JF, Ortolá JC, Gui L, Lee KK, Biggins S, Asbury CL, Davis TN - Nat Commun (2014)

Bottom Line: Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear.An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro.We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Kinetochores assemble on centromeric DNA and present arrays of proteins that attach directly to the dynamic ends of microtubules. Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear. Here, using a combination of biophysical assays and live-cell imaging, we find that oligomerization of the Dam1 complex is required for its ability to form microtubule attachments that are robust against tension in vitro and in vivo. An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro. In cells, the oligomerization-deficient Dam1 complex is unable to support stable bipolar alignment of sister chromatids, indicating failure of kinetochore-microtubule attachments under tension. We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.

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Equal bipolar distribution of kinetochores requires oligomerization of the Dam1 complexTime-lapse images of spindle poles (Spc110-mCherry, top row), kinetochores (Nuf2-GFP, middle row), and merged channels (bottom row) in control (a) and hsk3-degron (b) cells. Cells were imaged immediately following release from α-factor (G1) arrest; time stamps show time after release. Scale bars are 5 μm. At right in both (a) and (b) are spindle profiles of Nuf2-GFP intensity from corresponding images taken at 135 minutes after release. The integrated intensity in each half-spindle is printed on the plot. (c) Average spindle intensity profiles of Nuf2-GFP in control (black curve) and hsk3-degron (red curve) cells. Markers indicate average ± s.e.m. (d) Box and whisker plots show the distribution of kinetochore intensity ratios measured for control and hsk3-degron cells. Whiskers show the full range of the distribution, while the box shows the 75th, 50th, and 25th percentiles. (e) Plot of kinetochore intensity ratio versus spindle length shows that asymmetric kinetochore alignment in hsk3-degron cells is independent of spindle length. See Methods for additional information about the measurements presented in this figure.
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Figure 6: Equal bipolar distribution of kinetochores requires oligomerization of the Dam1 complexTime-lapse images of spindle poles (Spc110-mCherry, top row), kinetochores (Nuf2-GFP, middle row), and merged channels (bottom row) in control (a) and hsk3-degron (b) cells. Cells were imaged immediately following release from α-factor (G1) arrest; time stamps show time after release. Scale bars are 5 μm. At right in both (a) and (b) are spindle profiles of Nuf2-GFP intensity from corresponding images taken at 135 minutes after release. The integrated intensity in each half-spindle is printed on the plot. (c) Average spindle intensity profiles of Nuf2-GFP in control (black curve) and hsk3-degron (red curve) cells. Markers indicate average ± s.e.m. (d) Box and whisker plots show the distribution of kinetochore intensity ratios measured for control and hsk3-degron cells. Whiskers show the full range of the distribution, while the box shows the 75th, 50th, and 25th percentiles. (e) Plot of kinetochore intensity ratio versus spindle length shows that asymmetric kinetochore alignment in hsk3-degron cells is independent of spindle length. See Methods for additional information about the measurements presented in this figure.

Mentions: To ask if oligomerization is required for normal kinetochore-microtubule attachment, cells were depleted of Hsk3 and imaged as they entered mitosis. Upon separation of the duplicated spindle pole bodies, kinetochores (marked by Nuf2-GFP) in control cells aligned symmetrically between the two spindle poles, while in Hsk3-depleted cells, kinetochores often aligned asymmetrically towards one pole (Fig. 6a–c). Hsk3-depleted cells additionally exhibited abnormal spindle lengths (e.g., Fig. 6b shows spindle hyper-extension), consistent with the high incidence of spindle breakage observed in an extended degron-induced arrest (Supplementary Fig. 6). Asymmetric kinetochore clustering towards one pole is inconsistent with a lack of kinetochore-microtubule attachment, which causes unclustering of kinetochores in the nucleus23, 24, 25, 26. Instead, this asymmetric kinetochore clustering phenotype suggests a defect in sister kinetochore biorientation3, 27. To quantify asymmetry in the kinetochore distribution, we developed a “kinetochore intensity ratio” metric. Nuf2-GFP fluorescence intensity was integrated across the two halves of each spindle (Fig. 6a–b), and a ratio was taken of the brighter side over the dimmer side (and is thus always 1). For control cells, this ratio was narrowly distributed around the average of 1.1 ± 0.02 (s.e.m.), indicating accurate bipolar segregation of kinetochores with high fidelity (Fig. 6d–e). By contrast, kinetochore intensity ratios for Hsk3-depleted cells varied widely (from 1.0 to 14) and averaged 3.3 ± 0.6, reflecting a strong bias towards monopolar alignment that was evident across all observed spindle lengths (Fig. 6d–e). Therefore, the equal partitioning of kinetochores during mitosis requires oligomerization of the Dam1 complex.


Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation.

Umbreit NT, Miller MP, Tien JF, Ortolá JC, Gui L, Lee KK, Biggins S, Asbury CL, Davis TN - Nat Commun (2014)

Equal bipolar distribution of kinetochores requires oligomerization of the Dam1 complexTime-lapse images of spindle poles (Spc110-mCherry, top row), kinetochores (Nuf2-GFP, middle row), and merged channels (bottom row) in control (a) and hsk3-degron (b) cells. Cells were imaged immediately following release from α-factor (G1) arrest; time stamps show time after release. Scale bars are 5 μm. At right in both (a) and (b) are spindle profiles of Nuf2-GFP intensity from corresponding images taken at 135 minutes after release. The integrated intensity in each half-spindle is printed on the plot. (c) Average spindle intensity profiles of Nuf2-GFP in control (black curve) and hsk3-degron (red curve) cells. Markers indicate average ± s.e.m. (d) Box and whisker plots show the distribution of kinetochore intensity ratios measured for control and hsk3-degron cells. Whiskers show the full range of the distribution, while the box shows the 75th, 50th, and 25th percentiles. (e) Plot of kinetochore intensity ratio versus spindle length shows that asymmetric kinetochore alignment in hsk3-degron cells is independent of spindle length. See Methods for additional information about the measurements presented in this figure.
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Related In: Results  -  Collection

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Figure 6: Equal bipolar distribution of kinetochores requires oligomerization of the Dam1 complexTime-lapse images of spindle poles (Spc110-mCherry, top row), kinetochores (Nuf2-GFP, middle row), and merged channels (bottom row) in control (a) and hsk3-degron (b) cells. Cells were imaged immediately following release from α-factor (G1) arrest; time stamps show time after release. Scale bars are 5 μm. At right in both (a) and (b) are spindle profiles of Nuf2-GFP intensity from corresponding images taken at 135 minutes after release. The integrated intensity in each half-spindle is printed on the plot. (c) Average spindle intensity profiles of Nuf2-GFP in control (black curve) and hsk3-degron (red curve) cells. Markers indicate average ± s.e.m. (d) Box and whisker plots show the distribution of kinetochore intensity ratios measured for control and hsk3-degron cells. Whiskers show the full range of the distribution, while the box shows the 75th, 50th, and 25th percentiles. (e) Plot of kinetochore intensity ratio versus spindle length shows that asymmetric kinetochore alignment in hsk3-degron cells is independent of spindle length. See Methods for additional information about the measurements presented in this figure.
Mentions: To ask if oligomerization is required for normal kinetochore-microtubule attachment, cells were depleted of Hsk3 and imaged as they entered mitosis. Upon separation of the duplicated spindle pole bodies, kinetochores (marked by Nuf2-GFP) in control cells aligned symmetrically between the two spindle poles, while in Hsk3-depleted cells, kinetochores often aligned asymmetrically towards one pole (Fig. 6a–c). Hsk3-depleted cells additionally exhibited abnormal spindle lengths (e.g., Fig. 6b shows spindle hyper-extension), consistent with the high incidence of spindle breakage observed in an extended degron-induced arrest (Supplementary Fig. 6). Asymmetric kinetochore clustering towards one pole is inconsistent with a lack of kinetochore-microtubule attachment, which causes unclustering of kinetochores in the nucleus23, 24, 25, 26. Instead, this asymmetric kinetochore clustering phenotype suggests a defect in sister kinetochore biorientation3, 27. To quantify asymmetry in the kinetochore distribution, we developed a “kinetochore intensity ratio” metric. Nuf2-GFP fluorescence intensity was integrated across the two halves of each spindle (Fig. 6a–b), and a ratio was taken of the brighter side over the dimmer side (and is thus always 1). For control cells, this ratio was narrowly distributed around the average of 1.1 ± 0.02 (s.e.m.), indicating accurate bipolar segregation of kinetochores with high fidelity (Fig. 6d–e). By contrast, kinetochore intensity ratios for Hsk3-depleted cells varied widely (from 1.0 to 14) and averaged 3.3 ± 0.6, reflecting a strong bias towards monopolar alignment that was evident across all observed spindle lengths (Fig. 6d–e). Therefore, the equal partitioning of kinetochores during mitosis requires oligomerization of the Dam1 complex.

Bottom Line: Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear.An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro.We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Kinetochores assemble on centromeric DNA and present arrays of proteins that attach directly to the dynamic ends of microtubules. Kinetochore proteins coordinate at the microtubule interface through oligomerization, but how oligomerization contributes to kinetochore function has remained unclear. Here, using a combination of biophysical assays and live-cell imaging, we find that oligomerization of the Dam1 complex is required for its ability to form microtubule attachments that are robust against tension in vitro and in vivo. An oligomerization-deficient Dam1 complex that retains wild-type microtubule binding activity is primarily defective in coupling to disassembling microtubule ends under mechanical loads applied by a laser trap in vitro. In cells, the oligomerization-deficient Dam1 complex is unable to support stable bipolar alignment of sister chromatids, indicating failure of kinetochore-microtubule attachments under tension. We propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis.

Show MeSH
Related in: MedlinePlus