Final Report Summary - KINCON (Molecular bases of kinetochore-microtubule attachment and their implications for cell cycle control.)
Kinetochores are multi-subunit assemblies that promote the attachment of chromosomes to spindle microtubules during mitosis. Kinetochores also host a feedback control mechanism known as the spindle assembly checkpoint (SAC), which imparts on chromosome segregation the necessary accuracy. The ultimate goal of KINCON was the biochemical reconstitution of large segments of the kinetochore, with a strong focus on the microtubule-binding machinery, as well as the reconstitution of the biochemical reactions that subtend to SAC function. The two main expected outcomes of this work of reconstitution were identified in 1) the structural characterization of the kinetochore and SAC proteins, and in 2) the characterization of the systems-level properties of SAC function.
In the first area, our studies focused on the Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, a 10-subunit kinetochore complex that acts as the main microtubule receptor and as an integrative device for SAC signaling. A highlight of KINCON was the reconstitution of the KMN network, linked with the dissection of crucial interaction interfaces and with the characterization of its structural features (Petrovic et al. 2010; Screpanti et al. 2011; Petrovic et al. 2014). This detailed dissection of the KMN network has enabled experiments on this crucial kinetochore complex at an unprecedented level of accuracy.
Also in area 1, biochemical reconstitution of SAC components within KINCON led to the initial determination of the crystal structure of Zwilch (Çivril et al. 2010), a subunit of the Rod-Zwilch-ZW10 (RZZ) complex that plays a crucial role in SAC signaling and silencing. The subsequent achievement was the reconstitution of a recombinant version of the entire 800-kDa RZZ complex and the characterization of its structural organization by cryo-electron microscopy (in preparation). Structure determination of the Bub1:Bub3:phosphoKnl1 complex (Primorac et al. 2013) allowed us to gain very valuable information on a crucial aspect of SAC signaling by the Mps1 kinase, which preluded to a number of new studies on the mechanism of Bub1 and BubR1 recruitment to kinetochores.
In Area 2, KINCON aimed to characterize how two SAC kinases, Mps1 and Aurora B, become recruited to SAC-signaling kinetochores (Santaguida et al. 2010; Santaguida et al. 2011; Maffini, Faesen & Musacchio, in preparation). The demonstration that Aurora B is a bona fide SAC kinase (Santaguida et al. 2011) put an end to a decade of controversies, finally allowing a redressing of the function of this kinase in the SAC mechanism.
Also in Area 2, we reconstituted in vitro crucial aspects of kinetochore recruitment dynamics of spindle assembly checkpoint proteins (Maffini, Faesen & Musacchio, in preparation; Overlack, Primorac & Musacchio, submitted), focusing in particular on Bub1, BubR1, and Mps1. These studies shed light on the molecular basis of kinetochore recruitment of these SAC proteins, allowing the dissection of the specific functions of these proteins with unprecedented accuracy.
Finally, our identification and characterization of the small molecule agent Reversine as a very specific Mps1 inhibitors and a SAC antagonist has provided the entire SAC field with an invaluable tool for the acute inhibition of SAC signaling, rendering Reversine an extremely popular tool.
Overall, KINCON has achieved its goals. It has provided the community with invaluable tools and information that will have very significant influence on kinetochore and SAC studies in the next decade.
In the first area, our studies focused on the Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, a 10-subunit kinetochore complex that acts as the main microtubule receptor and as an integrative device for SAC signaling. A highlight of KINCON was the reconstitution of the KMN network, linked with the dissection of crucial interaction interfaces and with the characterization of its structural features (Petrovic et al. 2010; Screpanti et al. 2011; Petrovic et al. 2014). This detailed dissection of the KMN network has enabled experiments on this crucial kinetochore complex at an unprecedented level of accuracy.
Also in area 1, biochemical reconstitution of SAC components within KINCON led to the initial determination of the crystal structure of Zwilch (Çivril et al. 2010), a subunit of the Rod-Zwilch-ZW10 (RZZ) complex that plays a crucial role in SAC signaling and silencing. The subsequent achievement was the reconstitution of a recombinant version of the entire 800-kDa RZZ complex and the characterization of its structural organization by cryo-electron microscopy (in preparation). Structure determination of the Bub1:Bub3:phosphoKnl1 complex (Primorac et al. 2013) allowed us to gain very valuable information on a crucial aspect of SAC signaling by the Mps1 kinase, which preluded to a number of new studies on the mechanism of Bub1 and BubR1 recruitment to kinetochores.
In Area 2, KINCON aimed to characterize how two SAC kinases, Mps1 and Aurora B, become recruited to SAC-signaling kinetochores (Santaguida et al. 2010; Santaguida et al. 2011; Maffini, Faesen & Musacchio, in preparation). The demonstration that Aurora B is a bona fide SAC kinase (Santaguida et al. 2011) put an end to a decade of controversies, finally allowing a redressing of the function of this kinase in the SAC mechanism.
Also in Area 2, we reconstituted in vitro crucial aspects of kinetochore recruitment dynamics of spindle assembly checkpoint proteins (Maffini, Faesen & Musacchio, in preparation; Overlack, Primorac & Musacchio, submitted), focusing in particular on Bub1, BubR1, and Mps1. These studies shed light on the molecular basis of kinetochore recruitment of these SAC proteins, allowing the dissection of the specific functions of these proteins with unprecedented accuracy.
Finally, our identification and characterization of the small molecule agent Reversine as a very specific Mps1 inhibitors and a SAC antagonist has provided the entire SAC field with an invaluable tool for the acute inhibition of SAC signaling, rendering Reversine an extremely popular tool.
Overall, KINCON has achieved its goals. It has provided the community with invaluable tools and information that will have very significant influence on kinetochore and SAC studies in the next decade.