Microtubule associated proteins and motors required for ectopic microtubule array formation inS. cerevisiae

Abstract

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Further, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.

Stu2 has an essential kinetochore role independent of regulating microtubule dynamics

“…Previous work demonstrated that the TOG family of proteins, including budding yeast homolog Stu2, function as critical regulators of microtubules, including controlling microtubule polymerization, depolymerization, nucleation, and in regulating kinetochore-microtubule interactions (Ayaz et al, 2014;Brouhard et al, 2008;Chen et al, 1998;Geyer et al, 2018;Humphrey et al, 2018;King et al, 2020King et al, , 2021Kinoshita et al, 2002;Kosco et al, 2001;Miller et al, 2016;Podolski et al, 2014;Severin et al, 2001;Wang & Huffaker, 1997;Widlund et al, 2011). These described microtubule regulatory functions depend on two microtubule binding elements that are conserved within all TOG family members: TOG domain arrays that to bind to -tubulin dimers, and a basic linker domain important for binding the microtubule lattice.…”

“…One such family of MAPs is the TOG family of proteins, which conduct conserved functions across all eukaryotes (Al-Bassam & Chang, 2011;Kinoshita et al, 2002;Ohkura et al, 2001). This family of proteins is canonically known for their microtubule polymerase function; however, TOG proteins have additional roles in the cell, including nucleating and anchoring microtubules, regulating kinetochore-microtubule interactions, and regulating the dynamics and disassembly of microtubules (Chen et al, 1998;Geyer et al, 2018;Humphrey et al, 2018;King et al, 2020King et al, , 2021Kinoshita et al, 2002;Kosco et al, 2001;Miller et al, 2016;Podolski et al, 2014;Severin et al, 2001;Wang & Huffaker, 1997). They use conserved TOG domains to bind tubulin heterodimers at or near microtubule plus ends, where they stimulate large changes in the addition or loss of tubulin subunits to the microtubule polymer (Al-Bassam et al, 2006;Ayaz et al, 2014;Brouhard et al, 2008;Geyer et al, 2018;Nithianantham et al, 2018;Podolski et al, 2014;van Breugel et al, 2003;Widlund et al, 2011).…”

Cik1 and Vik1 Accessory Proteins Confer Distinct Functions to the Kinesin-14, Kar3

“…The functions of this complex are presently not known. Previous work showed that Kar3 absence increases the number and length of cytoplasmic MTs (Huyett et al, 1998;Saunders et al, 1997b), suggesting a role in assembly regulation, and provided evidence that Kar3Vik1 focuses MT minus ends on the cytoplasmic face of the spindle pole body (SPB), the budding yeast MT organizing center (King et al, 2021).…”