Anchoring geometry is a significant factor in determining the direction of kinesin-14 motility on microtubules.

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.

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5-Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance.

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron 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.

Nucleotide- and Mal3-dependent changes in fission yeast microtubules suggest a structural plasticity view of dynamics.

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.

Alp7/TACC-Alp14/TOG generates long-lived, fast-growing MTs by an unconventional mechanism.

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.

Ectopic A-lattice seams destabilize microtubules.

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold.

S. pombe kinesins-8 promote both nucleation and catastrophe of microtubules.

The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S.

Regulation of microtubule dynamics by kinesins.

The simple mechanistic and functional division of the kinesin family into either active translocators or non-motile microtubule depolymerases was initially appropriate but is now proving increasingly unhelpful, given evidence that several translocase kinesins can affect microtubule dynamics, whilst non-translocase kinesins can promote microtubule assembly and depolymerisation. Such multi-role kinesins act either directly on microtubule dynamics, by interaction with microtubules and tubulin, or indirectly, through the transport of other factors along the lattice to the microtubule tip. Here I review recent progress on the mechanisms and roles of these translocase kinesins.

Purification of tubulin from the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies.

Mal3 masks catastrophe events in Schizosaccharomyces pombe microtubules by inhibiting shrinkage and promoting rescue.

Schizosaccharomyces pombe Mal3 is a member of the EB family of proteins, which are proposed to be core elements in a tip-tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences microtubule dynamics is unclear. We tested the effects of full-length recombinant Mal3 on dynamic microtubules assembled in vitro from purified S.

The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism.

The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules.

Mal3, the Schizosaccharomyces pombe homolog of EB1, changes the microtubule lattice.

In vitro studies of pure tubulin have suggested that tubulin heterodimers in cells assemble into B-lattice microtubules, where the 8-nm dimers in adjacent protofilaments are staggered by 0.9 nm. This arrangement requires the tube to close by forming a seam with an A-lattice, in which the protofilaments are staggered by 4.

An ATP gate controls tubulin binding by the tethered head of kinesin-1.

Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing tubulin-activated release of adenosine diphosphate (ADP).

Distinct roles of doublecortin modulating the microtubule cytoskeleton.

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules.

Kinesin heads fused to hinge-free myosin tails drive efficient motility.

The rat kinesin motor domain was fused at residues 433, 411, 376 or 367, respectively, to the C-terminal 1185, 1187, 1197 or 1185 residues of the brush border myosin tail. In motility assays, K433myt and K411myt, which preserve the head-proximal kinesin hinge, and K367myt, which deletes it, drove rapid microtubule sliding ( approximately 0.6 microms(-1)) that was optimal when the head-pairs were spaced apart by adding 1:1 headless myosin tails.