Regulated by the interplay of multiple CLASP domains, the localization of the TOGL2 domain to microtubules drives spindle formation in Saccharomyces cerevisiae

The kinetochore (KT) is a complex structure that enables attachment of chromosomes to spindle microtubules (MTs). Several MT associated proteins (MAPs) contribute to the KT-MT interface and regulate the dynamics of kinetochore microtubules (kMTs). In addition, these MAPs localize to interpolar MTs and regulate spindle stability. One of these proteins is the S. cerevisiae CLASP (cytoplasmic linker associated protein) Stu1, an essential protein that has several functions during mitosis and therefore localizes differently in the course of each cell division. The aim of this work was to investigate which domains of Stu1 are important for its cell cycle specific localization, how this contributes to a coordinated action of Stu1 and how localization and function of Stu1 are regulated. Structural predictions of Stu1 suggest the organization in six domains. The following observations were made with the focus on three of them: the TOGL2 domain, the minimal MT-binding loop (ML) and the C-terminal loop (CL). The TOGL2 domain solely achieves binding of αβ-tubulin and drives spindle formation Co-immunoprecipitations identified the TOGL2 domain to be sufficient to bind free αβ-tubulin. This feature of the TOGL2 domain is essential and solely responsible for the important role of Stu1 in driving spindle formation. Thus, the TOGL2 domain ensures the function of Stu1 as a MT polymerase or rescue factor. Domain swapping experiments demonstrated that the function of the TOGL2 domain of Stu1 is very specific and cannot be easily taken over by another TOG domain. MT binding via the ML domain is required for efficient metaphase spindle formation, but is dispensable for midzone localization Besides the TOGL2 domain, efficient spindle formation in metaphase additionally depends on the binding of Stu1 to the MT lattice via the ML domain. Thereby, the CL domain specifies Stu1 localization to the region of the MT overlap. Midzone localization of Stu1 in anaphase, however, is independent of the ML domain and therefore must be ensured in a manner that is not based on MT binding. An interplay of the CL domain with the ML domain specifies Stu1’s sequestration at unattached KTs The CL domain was found to specify Stu1 for the sequestration at unattached KTs, most likely by inhibiting the MT binding affinity of the ML domain. Thus, the CL domain indirectly prevents spindle formation in the presence of unattached KTs. Efficient KT capture relies on unperturbed MT dynamics ensured by the Stu1 TOGL2 activity Capturing experiments of the CL deletion mutant revealed that Stu1 localization to unattached KTs is not a prerequisite for efficient capturing. However, the ML domain and especially the TOGL2 activity are mandatory in this respect. The contribution of Stu1 to unperturbed MT dynamics seems to be more important for the capturing pro-cess than KT localization. This may involve the polymerization of capturing kMTs but also, as analyzed, the temporal regulation of KT-generated MTs. The CL domain makes kMT length dependent on the tension on the KT-MT inter-face The localization of Stu1 to attached KTs is a prerequisite for the polymerization of kMTs. In this respect, the CL domain was found to inhibit Stu1’s ability to stabilize kMTs and to make the kMT length dependent on tension on the KT-MT interface. The CL domain prevents precocious spindle formation to ensure biorientation The data showed that the CL domain facilitates bipolar attachment by ensuring unperturbed dynamics of interpolar MTs. Therefore, the CL domain seems to fine-tune the MT polymerizing activity of Stu1 by regulating the MT affinity of the ML domain. Stu1 phosphorylation within the CL domain contributes to Stu1 regulation Finally, this work revealed that phosphorylation of Stu1 contributes to the regulation of Stu1. SILAC analyses identified 15 phosphorylation sites that mainly reside within the ML and the CL domain of Stu1 and are putative target sites of various serine/ threonine kinases like Cdk1, polo-like kinase, Ipl1 and Mps1. Furthermore, these analyses demonstrated that Stu1 gets phosphorylated and dephosphorylated throughout the cell cycle suggesting a regulatory role for kinases. Consistent with that, in vitro kinase as-says identified Stu1 N- and C-terminus as targets of Ipl1 and Mps1 kinases. Analyses of phosphomutants eventually suggested that phosphorylation of the CL domain con-tributes to the regulatory impact of the CL domain on the MT affinity of the ML domain. Taken together, the present study supports the theory that Stu1, similar to other CLASP proteins acts as a local modulator for MT dynamics and stability. While the TOGL2 domain accomplishes the essential function of tubulin incorporation in MT plus-ends, the other domains are required to regulate the localization of Stu1 and (probably therefore) control the MT polymerizing activity.