Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

No abstract

Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis. 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. Although a tailless Ncd has been shown to be nonprocessive, 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.

The atomic force microscope (AFM), which was invented by Binnig et al. in 1986, can image at nanometer resolution individual biological macromolecules on a substrate in solution. This unique capability awoke an expectation of imaging processes occurring in biological macromolecules at work. However, this expectation was not met, because the imaging rate with available AFMs was too low to capture biological processes. This expectation has at last been realized by the high-speed AFM developed by our research group at Kanazawa University. In this article, after a brief review of the development of our apparatus, its recent advancement and imaging data obtained with motor proteins are presented.

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected the regulatory effects of LIS1 and NDEL1 on dynein motility using full-length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1, and the mechanisms of cytoplasmic dynein regulation.Cytoplasmic dynein is a multi-subunit protein complex that moves along microtubules. It is involved in various cellular and subcellular activities, such as cell migration, vesicle transport, and organelle positioning (1, 2). Dynein interacts with several accessory proteins to accomplish this wide range of activities throughout the cell cycle (3). The best-characterized of these accessory proteins is dynactin, a multiprotein complex that is almost universally associated with dynein-dependent functions (4). Platelet-activating factor acetylhydrolase, isoform 1b, subunit 1 (PAFAH1B1; commonly known as lissencephaly 1, LIS1) 3 and its binding partner nuclear distribution gene E homolog (A. nidulans)-like 1 (NDEL1) also contribute to many dynein functions involving nuclear and spindle positioning and centrosomal movement (3).LIS1 was first identified as a protein associated with the smooth brain disease, lissencephaly (5, 6). Homozygous loss of Lis1 or Ndel1 is lethal in mice (7, 8). The C terminus of LIS1 binds to cytoplasmic dynein (9), whereas the N terminus contains a LisH homodimerization domain (10). Between these domains is a coiled-coil region that imparts flexibility to the LIS1 dimer (11), suggesting that LIS1 can alter its conformation between an "open state," in which the coiled-coil regions form a random helix, and a "closed state," in which the coiled-coil regions form a superhelix (12). NDEL1 contains an N-terminal coiled-coil domain that interacts with LIS1, and an unstructured C terminus that directly binds to dynein (13,14). LIS1 and NDEL1 are thought to associate with cytoplas...