Microtubule-Kinetochore Interactions

We are studying the yeast Dam1 kinetochore complex (or DASH). Our initial studies, in collaboration with the Drubin and Barnes labs (UC Berkeley), showed that this complex assembles into rings around microtubules and that the rings move processively with microtubule ends, coupling microtubule depolymerization to directional movement.


Towards a mechanistic understanding of Dam1 essential role in yeast mitosis, have used EM and image reconstruction to produce the only existing structures of the Dam1 complex and of its self-assembly around microtubules. We have also defined the subunit organization of Dam1 and characterize important structural elements for interaction with tubulin.  Our interest in chromosome segregation has led us to study the highly conserved KMN kinetochore network. We are charcterizing the Ndc80 kinetochore complex, the microtubule interacting component of the KMN. We visualized the full-length yeast Ndc80 complex and found a dramatic kink within the 560-Å complex localized to a conserved break in the coiled-coil and proposed its important in kinetochore geometry and likely as part of a tension sensing mechanism (Wang et al, JMB 2008). Using a minimal bonsai construct of the huma Ndc80 complex, we obtained a subnanometer structure of Ndc80 bound to the microtubule (Alushin et al., Nature 2010). The complex binds with a monomeric tubulin repeat, both at intra- and inter-dimer interfaces, using a minimal “toe-print” that reads highly conserved sequences in tubulin. Binding to the microtubule is coupled to a self-interaction among Ndc80 complexes along protofilaments that explains its cooperativity. The location of the interaction site on the microtubule makes it an excellent candidate to “probe” the conformational state of tubulin, leading us to propose a variation on the Hill model where directionality of diffusion by loss of affinity in one direction is coupled to the conformational change into curved protofilaments

Text Box: (a)         (b)    (c)   					       NDC80 complex structure and binding to microtubules. (a) The full-length yeast NDC80 (almost 600 Å long) shows a conserved kink in the coiled-coil region. (b,c) Cryo-EM reconstruction of human NDC80 bonsai complex on microtubules: tubulin (green), Ndc80 (blue) and Nuf2 (yellow). The C-terminal tail of tubulin (red) interacts with the NDC80 in the adjacent protofilament via the CH domain of NUF2 and the N-terminal NDC80 tail (purple).

More recently we have dissected the role of the unstructured N-terminus of Ndc80, a major site of Aurora B phosphorylation, leading to a model of how Ndc80’s interaction with MT ends is tuned by the phosphorylation state of its tail. In the process, we have obtained the only existing structure of the C-terminal tail of tubulin, as it engages the Ndc80 complex in an adjacent protofilament (Alushin et al, NSMB 20012). Budding Yeast

We have extended our studies to other kinetochore complexes (Mist12 complex, CENP-C), as well as to MT binding proteins with key roles in mitosis (CENP-E, CENP-F. Our work, in the context of additional in vivo studies, has led us to propose models for the organization of both the yeast and the metazoan kinetochore.



See also:

Stefan Westermann, Agustin Avila-Sakar, Hong-Wei Wang, Hanspeter Niederstrasser, Jonathan Wong, David G. Drubin, Eva Nogales, and Georjana Barnes. Formation of a dynamic kinetochore-microtubule interface through assembly of the Dam1 ring complex. (2005) Mol Cell. 17, 277-290.

S. Westermann, H.-W. Wang, A. Avila-Sakar, D.G. Drubin, E. Nogales and G. Barnes. The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends. (2006) Nature. 440, 565-569.

Wang, H.-W., Ramey, V.H., Westermann, S., Leschziner, A., Welburn, J.P.I., Nakajima, Y., Drubin, D.G., Barnes, G., and Nogales, E. (2007)Architecture of the Dam1 kinetochore ring complex: implications for microtubule-driven assembly and force-coupling mechanisms. Nat Struct Mol Biol.14, 721-726.

Alushin, G., Ramey, V.H., Pasqualato, S., Ball, D., Grigorieff, N., Musacchio, A. and Nogales, E. (2010) The NDC80 complex forms oligomeric arrays along microtubules. Nature,467, 805-810. Leading Edge article in Cell (Nov. 24).

Ramey VH, Wong A, Fang J, Howes S, Barnes G, Nogales E. (2011) Subunit organization in the Dam1 kinetochore complex and its ring around microtubules.  Mol Biol Cell. 22, 4335-42

Screpanti, E., De Antoni, A., Alushin, G.M., Petrovic, A., Nogales, E., and Musacchio, A. (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore.  Current Biology , 391-8

Homung, P., Maier, M., Alushin, G.M., Lander, G.C., Nogales, E. and Westermann, S. (2011) Molecular architecture and connectivity of the budding yeast Mtw1 kinetochore complex.  JMB 405, 548-559

Alushin, G. and Nogales, E. (2011) Visualizing kinetochore architecture. Current Opinion in Struct. Biol. ,21, 661-669.

Alushin, G. M., Musinipally, V., Matson, D., Tooley, J., Stukenberg P.T. and Nogales, E. (2012) Multimodal microtubule binding by the Ndc80 kinetochore complex. Nature Struct. Mol. Biol, 19 , 1161-7.

Lampert, F., Mieck, C., Alushin, G., Nogales, E. and Westermann, S. (2013) Molecular requirements for the formation of a kinetochore-microtubule interface by Dam1 and Ndc80 complexes.  J Cell Biol1,21-30.

Musinipally V, Howes S, Alushin GM, Nogales E. (2013) The Microtubule Binding Properties of CENP-E's C-Terminus and CENP-F  J Mol Biol.,425(22)4427-41

Stuart C. Howes, Gregory M. Alushin, Toshinobu Shida, Maxence V. Nachury and Eva Nogales (2014) Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure  MBoC25(2)257-66