Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC)‬‬‬‬‬‬‬

Abstract

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…KIFC5A has an additional ATP-independent microtubule-binding domain (Zhang and Sperry, 2004) and Ncd has a positively charged sequence in its tail domain that tethers it to the C-terminal E-hook of tubulin . Mouse KIFC1 has tail domain sequences that target it to membrane-bound organelles (Zhang and Sperry, 2004); the tail domain of Pkl1 direct it to γ-tubulin (Olmsted et al, 2013(Olmsted et al, , 2014. Additional studies in which chimeric kinesin-14 proteins of human HSET, Drosophila Ncd and fission yeast Pkl1 were generated also support this paradigm and confirm that tail domain sequences orchestrate function (Simeonov et al, 2009).…”

“…An exception to this is Pkl1, for which the tail domain was shown to be able to regulate nucleation without the motor domain in vitro and in vivo (Fig. 1B), and to do so in yeast and human cells (Olmsted et al, 2013(Olmsted et al, , 2014. Microtubule-dependent D. melanogaster Ncd motors have been shown to localize to parallel microtubules where they exert their movement into the opposite direction, thus creating a 'tug-of-war' effect.…”

KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis

“…Interestingly, S. pombe kinesin-14 Pkl1 promoted severing of γ-tubulin-anchored microtubules from the centrosome, which led the authors to suggest that microtubules released from the spindle poles contributed to central spindle organization. 6 We found that the longer and wider central bridges in DN-KIFC3 cells contained more microtubules that extended beyond the border of the central bridge than those in WT-KIFC3-expressing cells (Fig. 6A).…”

“…22 Pkl1 and HSET, by contrast, appeared to bind via microtubule-nucleating factors (γ-tubulin and augmin) that were associated with microtubule minus-ends. 3,6 To determine whether KIFC3 likewise tracked with moving microtubule ends, we examined mCherry-KIFC3 localization relative to growing microtubules in MDCK cells during the recovery from microtubule depolymerization by nocodazole. In these cells, microtubules are nucleated at the centrosome but released and captured elsewhere to give rise to the characteristic non-centrosomal microtubule organization of polarized epithelial cells.…”

“…1,2 Furthermore, all mitotic kinesin-14 motors characterized so far, including Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, Xenopus laevis XCTK2, and mammalian KIFC1/HSET possess active nuclear import signals that restrict their function to the stages of mitosis where the nuclear envelope is absent, i.e., from pro-metaphase to early telophase. [3][4][5][6][7] To date, no minus-end-directed motors have been implicated in the dramatic remodeling of the central spindle that precedes abscission when the daughter nuclei have reformed and mitotic kinesin-14 proteins are once again sequestered from the cytoplasm.…”