Language：English   Publishing type：Research paper (scientific journal)   Publisher：Scientific Reports  

Kinesin-14 microtubule-based motors have an N-terminal tail attaching the catalytic core to its load and usually move towards microtubule minus ends, whilst most other kinesins have a C-terminal tail and move towards plus ends. Loss of conserved sequences external to the motor domain causes kinesin-14 to switch to plus-end motility, showing that an N-terminal attachment is compatible with plus-end motility. However, there has been no systematic study on the role of attachment position in minus-end motility. We therefore examined the motility of monomeric kinesin-14s differing only in their attachment point. We find that a C-terminal attachment point causes kinesin-14s to become plus-end-directed, with microtubule corkscrewing rotation direction and pitch in motility assays similar to that of kinesin-1, suggesting that both C-kinesin kinesins-14 and N-kinesin kinesin-1 share a highly conserved catalytic core function with an intrinsic plus-end bias. Thus, an N-terminal attachment is one of the requirements for minus-end motility in kinesin-14.

DOI： 10.1038/s41598-022-19589-4

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Language：English   Publishing type：Research paper (scientific journal)  

Kinesin-14 microtubule-based motors have an N-terminal tail attaching the catalytic core to its load and usually move towards microtubule minus ends, whilst most other kinesins have a C-terminal tail and move towards plus ends. Loss of conserved sequences external to the motor domain causes kinesin-14 to switch to plus-end motility, showing that an N-terminal attachment is compatible with plus-end motility. However, there has been no systematic study on the role of attachment position in minus-end motility. We therefore examined the motility of monomeric kinesin-14s differing only in their attachment point. We find that a C-terminal attachment point causes kinesin-14s to become plus-end-directed, with microtubule corkscrewing rotation direction and pitch in motility assays similar to that of kinesin-1, suggesting that both C-kinesin kinesins-14 and N-kinesin kinesin-1 share a highly conserved catalytic core …

DOI： https://doi.org/10.1038/s41598-022-19589-4

Other Link： https://rdcu.be/dIvDu

Language：English   Publishing type：Research paper (scientific journal)  

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryoelectron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motormicrotubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

DOI： 10.1016/j.jmb.2019.01.011

Other Link： https://www.sciencedirect.com/science/article/pii/S0022283619300105?via%3Dihub

Repository Public URL： https://hdl.handle.net/2324/7218282

Language：English   Publishing type：Research paper (scientific journal)  

Using cryo-electron microscopy, we characterize the architecture of microtubules assembled from Schizosaccharomyces pombe tubulin, in the presence and absence of their regulatory partner Mal3. Cryo-electron tomography reveals that microtubules assembled from S. pombe tubulin have predominantly B-lattice interprotofilament contacts, with protofilaments skewed around the microtubule axis. Copolymerization with Mal3 favors 13 protofilament microtubules with reduced protofilament skew, indicating that Mal3 adjusts interprotofilament interfaces. A 4.6-Å resolution structure of microtubule-bound Mal3 shows that Mal3 makes a distinctive footprint on the S. pombe microtubule lattice and that unlike mammalian microtubules, S. pombe microtubules do not show the longitudinal lattice compaction associated with EB protein binding and GTP hydrolysis. Our results firmly support a structural plasticity view of microtubule dynamics in which microtubule lattice conformation is sensitive to a variety of effectors and differently so for different tubulins.

DOI： 10.1038/s41467-017-02241-5

Other Link： https://www.nature.com/articles/s41467-017-02241-5

Repository Public URL： https://hdl.handle.net/2324/4842493

Language：English   Publishing type：Research paper (scientific journal)  

Alp14 is a TOG-family microtubule polymerase from S. pombe that tracks plus ends and accelerates their growth. To interrogate its mechanism, we reconstituted dynamically unstable single isoform S. pombe microtubules with full length Alp14/TOG and Alp7, the TACC-family binding partner of Alp14. We find that Alp14 can drive microtubule plus end growth at GTP-tubulin concentrations at least 10fold below the usual critical concentration, at the expense of increased catastrophe. This reveals Alp14 to be a highly unusual enzyme that biases the equilibrium for the reaction that it catalyses. Alp7/TACC enhances the effectiveness of Alp14, by increasing its occupancy. Consistent with this, we show in live cells that Alp7 deletion produces very similar MT dynamics defects to Alp14 deletion. The ability of Alp7/14 to accelerate and bias GTP-tubulin exchange at microtubule plus ends allows it to generate long-lived, fast-growing microtubules at very low cellular free tubulin concentrations.

DOI： 10.1038/srep20653

Other Link： https://www.nature.com/articles/srep20653

Repository Public URL： https://hdl.handle.net/2324/4842492

Responsible for pages："Microtubules in Non-conventional yeasts" pp 237 -296 in “Non-conventional yeasts: from basic research to application” ed A. Sibirny, Springer   Language：English   Book type：Scholarly book

Microtubules polymerise from tubulin proteins and play a significant role in the growth and proliferation of eukaryotic cells. In yeasts, most studies on microtubules and tubulins have utilised the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe model systems. However, more recently interest in the microtubules of other non-conventional yeast and fungal species has increased, both for investigation of biological processes such as fungal evolution and for applications such as developing antifungal drugs. We review the microtubule cytoskeleton and its role in yeast and fungal cellular processes in vivo and the tubulin proteins found in yeast cells and their study in vitro, together with the recent advances in cryoEM leading to detailed molecular structures of yeast microtubules. We examine what is known about the microtubule cytoskeleton in non-conventional yeasts and highlight the significant differences, as well as many conserved aspects, in their microtubule biology compared to the two model yeasts. Finally, we discuss the potential role of microtubules as drug targets for treatment of yeast and fungal infections.

DOI： 10.1007/978-3-030-21110-3_8

Repository Public URL： https://hdl.handle.net/2324/7218283

Event date： 2017.9

Language：English  

Venue：Kumamoto University   Country：Japan  

Event date： 2017.6

Language：English  

Venue：Tskuba International Congress Center, Tskuba   Country：Japan  

Purified tubulins will self-assemble to form dynamic microtubules (MTs), however MT dynamics are normally modulated by MT associated proteins in vivo. We have used darkfield microscopy to measure the dynamics of MTs assembled from purified S. pombe yeast tubulin in vitro and found MT growth rates were reduced compared to those we previously observed in live S. pombe cells. However, Alp14 a TOG-family MT plus end tracking protein can accelerate S. pombe MT assembly both in vivo and in vitro. We also found that the Alp14 binding protein Alp7/TACC was essential for this function in vivo. TIRF microscopy in vitro showed that Alp7 increases the occupancy of Alp14 on MT ends. Together Alp7 and Alp14 promote MT formation at the low concentrations of tubulin found in vivo.

Other Link： http://www.aeplan.co.jp/bsj2016/

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Other Link： http://dx.doi.org/10.1016/j.semcdb.2011.09.021