An unconventional interaction between Dis1/TOG and Mal3/EB1 in fission yeast promotes the fidelity of chromosome segregation

Abstract

Dynamic microtubule plus-ends interact with various intracellular target regions such as the cell cortex and the kinetochore. Two conserved families of microtubule plus-end-tracking proteins, the XMAP215, ch-TOG or CKAP5 family and the end-binding 1 (EB1, also known as MAPRE1) family, play pivotal roles in regulating microtubule dynamics. Here, we study the functional interplay between fission yeast Dis1, a member of the XMAP215/TOG family, and Mal3, an EB1 protein. Using an in vitro microscopy assay, we find that purified Dis1 autonomously tracks growing microtubule ends and is a bona fide microtubule polymerase. Mal3 recruits additional Dis1 to microtubule ends, explaining the synergistic enhancement of microtubule dynamicity by these proteins. A non-canonical binding motif in Dis1 mediates the interaction with Mal3. X-ray crystallography shows that this new motif interacts in an unconventional configuration with the conserved hydrophobic cavity formed within the Mal3 C-terminal region that typically interacts with the canonical SXIP motif. Selectively perturbing the Mal3–Dis1 interaction in living cells demonstrates that it is important for accurate chromosome segregation. Whereas, in some metazoans, the interaction between EB1 and the XMAP215/TOG family members requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module.

Purification and characterisation of the fission yeast Ndc80 complex

“…Our previous results indicated that Ndc80 directly or indirectly interacts with the MAP Dis1/chTOG [23] that tracks the plus end of dynamic MTs and possesses a MT polymerase activity as other members of this family [32]. Intriguingly, Dis1 forms a stable complex with the autonomous MT plus-end tracking protein Mal3/EB1, thereby regulating mitotic progression [32].…”

“…Intriguingly, Dis1 forms a stable complex with the autonomous MT plus-end tracking protein Mal3/EB1, thereby regulating mitotic progression [32]. In addition, it was reported that budding yeast and human orthologues of Dis1, Stu2 and chTOG are capable of directly interacting with the Ndc80 complex [43].…”

“…The purification of a number of fission yeast MAPs required for kinetochore capture has been achieved and the in vitro characterisation of these proteins has been reported [32], [35], [36], [37]. By contrast, the Ndc80 complex from fission yeast has not been purified yet.…”

Mapping the kinetochore MAP functions required for stabilizing microtubule attachments to chromosomes during metaphase

“…The XMAP215 ortholog in Xenopus and yeast, XMAP215/Dis1/ Stu2 interacts with the MT plus-end tracking protein EB1 (Kronja, Kruljac-Letunic, Caudron-Herger, Bieling, & Karsenti, 2009;Matsuo et al, 2016;Wolyniak et al, 2006) to regulate MT dynamics at the kMT interface (Figures 1e1,e2,g1,g2 and 2b,c). An in vitro study using the purified Ndc80 complex from fission yeast showed that the complex interacted with EB1 to track the growing MT plus-ends, but no detectable binding was observed between the complex and Dis1 (Matsuo, Maurer, Surrey, & Toda, 2017).…”

“…TIRF microscopy has shown that the budding yeast Stu2 binds preferentially to MT plus‐ends in vitro and that the fission yeast Dis1 weakly associates with the MT lattice and accumulates specifically at the growing MT plus‐ends (Figures e2 and b,c). In contrast, human chTOG accumulates at both the growing and shrinking MT plus‐ends, and it is thought that MT polymerase activity promotes MT assembly at both growing and shrinking (also called rescue) MT tips (Figures d2 and a (Matsuo et al, ; Podolski et al, ; Roostalu, Cade, & Surrey, ; van Breugel, Drechsel, & Hyman, ). Budding yeast Stu2 and Xenopus XMAP215 have also been reported as MT‐destabilizing factors at the plus‐ends, possibly by inducing catastrophe (Shirasu‐Hiza, Coughlin, & Mitchison, ; van Breugel et al, ).…”