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

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, XMAP215/TOG and EB1, play pivotal roles in regulating microtubule dynamics. Here we study the functional interplay between fission yeast Dis1/XMAP215 and Mal3/EB1. 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. Xray crystallography shows that this novel 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 EB1-XMAP215/TOG interaction requires an additional binding partner, fission yeast relies on a direct interaction, indicating evolutionary plasticity of this critical interaction module. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le INTRODUCTION Microtubules (MTs) are structurally polar and highly dynamic tubulin polymers that undergo spontaneous transitions from growing to shrinking phases called dynamic instability (Mitchison and Kirschner, 1984). Such dynamic properties of MTs play an essential role in many cellular processes including intracellular transport, cell polarity and cell division (Desai and Mitchison, 1997). MT dynamics are regulated by a cohort of evolutionarily conserved microtubule-associated proteins (MAPs) (Akhmanova and Steinmetz, 2008). A subclass of MAPs, microtubules plus-end tracking proteins (+TIPs) have unique properties, as they can specifically interact with the dynamic ends of MTs, thereby playing a decisive role in determining the characteristics of MT plus ends in cells (Buey et al., 2012; Duellberg et al., 2013). In particular, EB1 and XMAP215 are important +TIPs because they can both accumulate autonomously at MT ends independent of other MAPs, however by different mechanisms (Akhmanova and Hoogenraad, 2005). EB1 family proteins (EBs) bind in a nucleotide-dependent manner to an extended region at the growing MT end, resulting in a comet-like appearance at MT plus ends in cells (Bieling et al., 2008; Bieling et al., 2007; Maurer et al., 2011; Maurer et al., 2014; Maurer et al., 2012; Mohan et al., 2013; Zanic et al., 2009; Zhang et al., 2015). EBs can have direct and indirect effects on the MT dynamics (Galjart, 2010); several in vitro experiments suggest that purified EBs promote the MT growth rate and simultaneously increase the catastrophe frequency (Bieling et al., 2007; Li et al., 2012; Mohan et al., 2013; Vitre et al., 2008; Zanic et al., 2013). In vivo EBs recruit several other MAPs to MT plus ends through direct proteinprotein interactions. EBs consist of four functional regions; the N-terminal calponinhomology (CH) domain required for MT binding (Hayashi and Ikura, 2003), the medial coiled-coil region involved in homo-dimerisation (De Groot et al., 2010) followed by the EB homology (EBH) domain and finally the C-terminal EEY/F motif (Duellberg et al., 2013). The EBH domain specifically binds to an SXIP motif found in a variety of +TIPs (Buey et al., 2012; Duellberg et al., 2014; Honnappa et al., 2009), whereas the EEY/F motif at the Cterminus of EBs binds to some CAP-Gly domains found in some MAPs (Duellberg et al., 2013; Honnappa et al., 2006; Weisbrich et al., 2007). MT plus end recruitment of other +TIPs by EBs is responsible for the indirect effects EBs can have on MT behaviour and hence on a variety of MT dependent cellular processes. Mal3, the sole EB1 homologue in fission yeast Schizosaccharomyces pombe, is nonessential for cell division, yet mal3 deletion mutants display a variety of defects derived Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le from abnormal MT architectures and dynamics. These include cell polarity defects during interphase (Beinhauer et al., 1997; Browning et al., 2003; Busch and Brunner, 2004; Busch et al., 2004) and chromosome segregation errors during mitosis (Asakawa et al., 2006; Asakawa and Toda, 2006; Asakawa et al., 2005; Beinhauer et al., 1997; Mana-Capelli et al., 2012). Mal3 has been shown to interact with the SXIP and CAP-Gly domain containing MAP Tip1, the fission yeast CLIP-170 orthologue and the Tea2 kinesin, thereby playing a critical role in regulation of interphase MT organisation and cell polarisation (Bieling et al., 2007; Browning et al., 2003; Busch et al., 2004). By contrast, our understanding of how Mal3 regulates mitotic progression remains poorly understood despite several earlier studies (Asakawa et al., 2006; Kerres et al., 2004). Work performed in vitro indicated that Mal3 alone has some impact on MT dynamics (Bieling et al., 2007; des Georges et al., 2008; Katsuki et al., 2009), however is likely that Mal3 cooperates with other +TIPs during mitosis through direct interactions as in interphase. The XMAP215/TOG family comprises another class of +TIPs that play pivotal roles in many MT-mediated processes (Al-Bassam and Chang, 2011; Kinoshita et al., 2002; Ohkura et al., 2001). Members of this protein family contain N-terminal TOG domains that bind soluble tubulin and a separate microtubule binding site (Al-Bassam et al., 2006; Widlund et al., 2011) which in their combination allow them to act as microtubule polymerases accelerating microtubule growth (Al-Bassam et al., 2012; Ayaz et al., 2014; Ayaz et al., 2012; Brouhard et al., 2008; Li et al., 2012; Podolski et al., 2014; Reber et al., 2013; Takeshita et al., 2013). Consequently, these XMAP215/TOG proteins localise to the very MT end in contrast to EBs that bind to an extended region (Maurer et al., 2014). In the absence of tubulin, XMAP215/TOG has been shown to catalyse MT depolymerisation (Brouhard et al., 2008; Roostalu et al., 2015; Shirasu-Hiza et al., 2003). Fission yeast contains two XMAP215/TOG orthologues, Alp14/Mtc1 and Dis1 (Garcia et al., 2001; Nakaseko et al., 2001; Ohkura et al., 2001). These two proteins share essential functions; each single deletion mutant is viable, while double deletions are inviable (Aoki et al., 2006; Garcia et al., 2002; Hsu and Toda, 2011; Kakui et al., 2013; Nabeshima et al., 1995; Nakaseko et al., 2001). Whereas Alp14 has been shown to be a microtubule polymerase like other family members (Al-Bassam et al., 2012; Hussmann et al., 2016), a biochemical characterisation of the catalytic properties of Dis1 at MT ends has not been performed yet. In this study we explore the biochemical and physiological interplay between Dis1 and Mal3. Dis1 is a microtubule polymerase that, different from other XMAP215/TOG Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le family members also directly binds to Mal3. In combination, the two proteins promote MT dynamicity synergistically. Intriguingly, Dis1 does not have a canonical EB1 binding motif. Instead, crystallographic analysis has unveiled a non-canonical binding mode of a novel motif in Dis1 to the conserved hydrophobic cavity in the EBH domain that is also found in Mal3; these structural data demonstrate similarities and differences in the interaction between EB1 and the SXIP motif versus the unconventional EB binding motif in Dis1. Genetic studies demonstrate that the interaction between Dis1 and Mal3 is of physiological significance.