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Abstract

The Saccharomyces cerevisiae protein Slk19 (Synthetic Lethal with Kar3) has multiple functions during the cell cycle and is required for an error-free chromosome segregation in mitosis (Pfiz et al., 2002). Slk19 is a non-essential protein with 821 amino acid (aa) residues and seven predicted coiled coil (cc) domains (referred to as cc1-7). Slk19 localizes to attached kinetochores throughout the cell cycle and localizes to the spindle midzone in anaphase (Zeng et al., 1999). Additionally, Slk19 localizes at unattached kinetochores (uaKTs) where it efficiently and interdependently sequesters together with the microtubule (MT) rescue factor Stu1 (Funk et al., 2014), (Ortiz et al., 2009), (Kolenda et al., 2018). The sequestering of Slk19 and Stu1 was shown to facilitate KT capturing due to a reorganization of the MT network (Kolenda et al., 2018).

Here I identified two Slk19 domains that are essential for the Slk19-Stu1 sequestering process: the C-terminus (aa 709–821) and the cc1 domain (aa 300–410). I showed that both domains contribute to Stu1 interaction, while the C-terminus is required for Slk19 tetramerization and for its localization at the kinetochore (KT). Moreover, I determined Spc105 as the KT protein that mediates the basal Slk19 binding at uaKTs.

Slk19 was also shown to be required for metaphase spindle stability (Zeng et al., 1999),(Ye et al., 2005),(T. Zhang et al., 2006),(Richmond et al., 2013). However, whether Slk19 can bind to the metaphase spindle per se and what exact function it has at the spindle was so far unknown. The data obtained in this study suggest that Slk19 does not possess an intrinsic MT binding activity but can efficiently bind to MTs via prebound Stu1 or Ase1 in vitro. Also in vivo, Slk19 localization to the metaphase spindle depends on Ase1 and Stu1. Interestingly, I found that Slk19 localizes to the center of the metaphase spindle rather than along the complete spindle, indicative for a localization specifically at the interpolar MT (ipMT) overlaps. I showed that Slk19 can strongly enhance Ase1 and moderately enhance Stu1 amounts at the metaphase spindle in vivo and at MTs in vitro.

It was shown in previous studies that Ase1 as well as Stu1 can both bind and crosslink MTs by themselves in vitro (Schuyler et al., 2003), (Funk et al., 2014). Here I found that Slk19 can enhance this MT crosslinking ability, when incubated together with Stu1 or Ase1 in vitro. In contrast, Slk19 alone could not crosslink MTs. Absence of Slk19 from metaphase spindles results in defective ipMT overlaps in vivo, as assessed by reduced tubulin levels at the metaphase spindle center, increased appearance of long unaligned nuclear MTs and by altered Ase1 and Stu1 localizations at the ipMT overlaps. Taken together, the data suggest that Slk19 contributes to increased ipMT crosslinking and spindle stabilization by enhancing the binding of Stu1 and of the MT-crosslinking protein Ase1 at the metaphase spindle overlaps. Thus, tetrameric Slk19 might promote protein network formation specifically stabilizing ipMT overlaps of metaphase spindles.

Furthermore, I found that Slk19 amounts at the metaphase spindle center are increased in situations with high tension and decreased in situations with low or no tension at the KTs and the spindle. Thus, the results suggest a tension-regulated mechanism controlling Slk19 spindle localization according to demand.

Moreover, Slk19 is a component of the Cdc14 Early Anaphase Release (FEAR) pathway and thus is involved in the formation of the anaphase midzone and anaphase spindle stabilization (Sullivan et al., 2001), (Stegmeier et al., 2002),(Khmelinskii et al., 2007). At the meta-to-anaphase transition, binding of Stu1 at the anaphase spindle switches from a binding via the microtubule-binding domain (MBD) to a (probably indirect) binding via the domain 4 (D4) (Funk et al., 2014). So far, it was not known how this altered binding mode is achieved and whether the FEAR function of Slk19 might regulate the D4-mediated Stu1 binding in anaphase. Here I found that Slk19 promotes the midzone localization of Stu1 by two distinct mechanisms: The FEAR function of Slk19 as well as the physical presence of Slk19 at the anaphase spindle are both required for the D4-mediated Stu1 binding at the anaphase midzone.

Taken together, this study reveals new valuable insights into the Slk19 functions for KT capturing, spindle stabilization and possible regulation mechanisms for the Slk19 spindle localization.