Limits...
Orientation and structure of the Ndc80 complex on the microtubule lattice.

Wilson-Kubalek EM, Cheeseman IM, Yoshioka C, Desai A, Milligan RA - J. Cell Biol. (2008)

Bottom Line: The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules.Such a binding mode, in which the Ndc80 complex interacts with sequential alpha/beta-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules.Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential alpha/beta-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.

Show MeSH

Related in: MedlinePlus

Orientation of the Ndc80/Nuf2 dimer relative to the polarity of the microtubule lattice. Negatively stained centrosome-nucleated microtubule asters decorated with the Ndc80/Nuf2 dimer (A and C). Boxed areas are magnified in B and D. + and − signs indicate microtubule polarity. Bars, 50 nm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2542468&req=5

fig1: Orientation of the Ndc80/Nuf2 dimer relative to the polarity of the microtubule lattice. Negatively stained centrosome-nucleated microtubule asters decorated with the Ndc80/Nuf2 dimer (A and C). Boxed areas are magnified in B and D. + and − signs indicate microtubule polarity. Bars, 50 nm.

Mentions: In the experiments described here, we used bacterially expressed Caenorhabditis elegans NDC-80/Nuf2HIM-10 heterodimers (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200804170/DC1; Cheeseman et al., 2006). We first analyzed the binding of Ndc80/Nuf2 relative to the polarity of the microtubule lattice. We grew microtubules of known polarity from centrosomes attached to EM grids and decorated these with the Ndc80/Nuf2 heterodimer. Examination of the negatively stained asters revealed that the arrowheads formed by the Ndc80/Nuf2 dimer point toward the centrosome (Fig. 1). Thus, the coiled-coil region of the heterodimer projects toward the plus end of the microtubule. In the heterotetrameric complex, this geometry of binding would force the Spc24/25 subunits to extend away from the microtubule plus end, where they likely bind to the kinetochore scaffold protein KNL-1 via their globular domains. This result is consistent with experiments comparing Spc24 and Ndc80/Hec1 localization at kinetochores in vertebrate cells using high resolution light microscopy (DeLuca et al., 2006). It is noteworthy that, given the length of the Ndc80 complex (∼550 Å), this binding geometry provides room at the microtubule plus end for polymerization and depolymerization reactions that are known to accompany chromosome movement in vivo.


Orientation and structure of the Ndc80 complex on the microtubule lattice.

Wilson-Kubalek EM, Cheeseman IM, Yoshioka C, Desai A, Milligan RA - J. Cell Biol. (2008)

Orientation of the Ndc80/Nuf2 dimer relative to the polarity of the microtubule lattice. Negatively stained centrosome-nucleated microtubule asters decorated with the Ndc80/Nuf2 dimer (A and C). Boxed areas are magnified in B and D. + and − signs indicate microtubule polarity. Bars, 50 nm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542468&req=5

fig1: Orientation of the Ndc80/Nuf2 dimer relative to the polarity of the microtubule lattice. Negatively stained centrosome-nucleated microtubule asters decorated with the Ndc80/Nuf2 dimer (A and C). Boxed areas are magnified in B and D. + and − signs indicate microtubule polarity. Bars, 50 nm.
Mentions: In the experiments described here, we used bacterially expressed Caenorhabditis elegans NDC-80/Nuf2HIM-10 heterodimers (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200804170/DC1; Cheeseman et al., 2006). We first analyzed the binding of Ndc80/Nuf2 relative to the polarity of the microtubule lattice. We grew microtubules of known polarity from centrosomes attached to EM grids and decorated these with the Ndc80/Nuf2 heterodimer. Examination of the negatively stained asters revealed that the arrowheads formed by the Ndc80/Nuf2 dimer point toward the centrosome (Fig. 1). Thus, the coiled-coil region of the heterodimer projects toward the plus end of the microtubule. In the heterotetrameric complex, this geometry of binding would force the Spc24/25 subunits to extend away from the microtubule plus end, where they likely bind to the kinetochore scaffold protein KNL-1 via their globular domains. This result is consistent with experiments comparing Spc24 and Ndc80/Hec1 localization at kinetochores in vertebrate cells using high resolution light microscopy (DeLuca et al., 2006). It is noteworthy that, given the length of the Ndc80 complex (∼550 Å), this binding geometry provides room at the microtubule plus end for polymerization and depolymerization reactions that are known to accompany chromosome movement in vivo.

Bottom Line: The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules.Such a binding mode, in which the Ndc80 complex interacts with sequential alpha/beta-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules.Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential alpha/beta-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.

Show MeSH
Related in: MedlinePlus