In vitro reconstitution and biochemical characterisation of the Dynein/Dynactin:RZZ/Spindly interaction

Kinetochores are large proteinaceous assemblies recruited to the centromere, a specialized region of the chromosome, where they form load-bearing interactions with the microtubules of the mitotic spindle, allowing for chromosome segregation into the two daughter cells. Together with this structural function, the kinetochores have a fundamental regulatory role, by recruiting the machinery responsible for the regulation of the Spindle Assembly Checkpoint (SAC), which ensures segregation does not start until all kinetochores are bioriented. SAC signaling is dependent on the corona, a fibrous structure that develops outside the unattached kinetochore, and in which the SAC machinery is embedded. Corona formation is dependent on the RZZ-Spindly complex, which also plays a critical role in its disruption upon microtubule attachment. Spindly, a member of the cargo adaptor protein family, directly recruits and activates Dynein/Dynactin, which strips the corona away from the kinetochore upon microtubule attachment, silencing the SAC and allowing the transition to anaphase.

My PhD work aimed at reconstituting in vitro the interaction between Dynein/Dynactin and RZZ-Spindly. Furthermore, I aimed at deciphering the role of Spindly in regulating the formation of this complex.

Using biochemical reconstitution, I revealed that Spindly is an autoinhibited Dynein cargo adaptor, and that its autoinhibition reflects an interaction between its N-terminal CC1 box conserved domain and the C-terminal CC3. In agreement with this, deletion of the N-terminal CC1 box, mutation or deletion of the CC3, as well as removal of a Spindly-specific hinge point released autoinhibition and allowed the formation of a complex with Dynein and Dynactin, and with their respective adaptor-binding subunits, the LIC2 and the pointed end.

I dissected the interaction between Spindly and the Dynactin pointed end, revealing that at least two independent sites on both binding partners are necessary for the interaction to take place, and I identified the location of these sites within the two binding partners.

Finally, I explored the role of the RZZ dimer in the regulation of Spindly interaction with Dynein/Dynactin, in the first in vitro study of the effect of cargo-binding on Dynein function. I discovered that the RZZ dimer inhibits the interaction of Spindly with the motor, through a pathway independent of Spindly autoinhibition.

In conclusion, this work represents a significant step forward in the understanding of the regulation of Dynein/Dynactin at the kinetochore, and of the mechanism of adaptor autoinhibition in general.


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