Minus-End-Directed Motor Ncd Exhibits Processive Movement that Is Enhanced by Microtubule Bundling In Vitro

SummaryDrosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis [1–7]. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle [8–12]. Although a tailless Ncd has been shown to be nonprocessive [13–16], the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along “single” microtubules, whereas it exhibited processive movement along “bundled” microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization

Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 forms homotetramers and that Klp9 is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon a nuclear localisation signal and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 alone is impaired in spindle elongation during anaphase B, and furthermore, cut7-122klp9Δ double mutants exhibit additive defects. We propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent that ensures structural integrity of spindle microtubules together with Ase1.This work was supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI Scientific Research (A) 16H02503 to T.T., a Challenging Exploratory Research grant 16K14672 to T.T., Scientific Research (C) 16K07694 to M.Y., Scientific Research (C) 15KT0155 to K.F. and Grantin-Aid for Scientific Research on Innovative Areas 18H05420 to K.F.), the Naito Foundation (T.T.) and the Uehara Memorial Foundation (T.T.)

Dynamic control of microbial movement by photoswitchable ATP antagonists

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site

A Single Protofilament Is Sufficient to Support Unidirectional Walking of Dynein and Kinesin

<div><p>Cytoplasmic dynein and kinesin are two-headed microtubule motor proteins that move in opposite directions on microtubules. It is known that kinesin steps by a ‘hand-over-hand’ mechanism, but it is unclear by which mechanism dynein steps. Because dynein has a completely different structure from that of kinesin and its head is massive, it is suspected that dynein uses multiple protofilaments of microtubules for walking. One way to test this is to ask whether dynein can step along a single protofilament. Here, we examined dynein and kinesin motility on zinc-induced tubulin sheets (zinc-sheets) which have only one protofilament available as a track for motor proteins. Single molecules of both dynein and kinesin moved at similar velocities on zinc-sheets compared to microtubules, clearly demonstrating that dynein and kinesin can walk on a single protofilament and multiple rows of parallel protofilaments are not essential for their motility. Considering the size and the motile properties of dynein, we suggest that dynein may step by an inchworm mechanism rather than a hand-over-hand mechanism.</p> </div

Single molecule motility of dynein and kinesin on tubulin polymers.

<p>(<b>A</b>) Tubulin polymers (left panels) and kymographs of GFP-GST-Dyn1_331kDa and RK430-GFP movements (right panels). (<b>B</b>) Mean-square displacement (MSD) plots of GFP-GST-Dyn1_331kDa (left) and RK430-GFP (right) movements on MTs, MTs (GA) and zinc-sheets (GA) with fitted quadratic curves (solid line). Each plot represents the mean ± SEM. (<b>C</b>) Velocities of GFP-GST-Dyn1_331kDa (left) and RK430-GFP (right) movements on MTs, MTs (GA) and zinc-sheets (GA) determined from MSD plots. Bars represent the mean ± SEM.</p

MT and zinc-sheet gliding movements on dynein- and kinesin-coated glass surfaces.

<p>(<b>A</b>) Movement trajectories of tubulin polymers plotted by positions marked every 3 s from the starting points (open circles). MT movements on dynein (upper left) and RK430-Avi (upper right). Zinc-sheet movements on dynein (lower left) and RK430-Avi (lower right). (<b>B</b>) Velocities of the gliding movement. Bars represent the mean ± SEM, and (N) is the number of measured gliding tubulin polymers.</p

Structure of MTs, zinc-sheets and zinc-macrotubes.

<p>(<b>A</b>) The protofilament arrangements are modified from Wolf et al. (1993). Arrows represent the polarity of a protofilament, and color discriminates the outer side (green) from the inner side (blue) of a MT. Dashed lines indicate the cut plane of the cross-cut views in B. (<b>B</b>) The cross-cut views of a MT (left), zinc-sheet and zinc-macrotube (right). Red stars denote a helix 12 that is closely related to dynein and kinesin binding sites. (<b>C</b>) General views, close-up views and diffraction patterns of MTs (left), zinc-sheets (middle) and zinc-macrotubes (right). (<b>D</b> and <b>E</b>) The width and length distribution of zinc-sheets measured in EM images. The mean ± SD and number of counted zinc-sheets (N) are shown.</p