Akane Furuta

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

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.

Autoinhibition and cooperative activation mechanisms of cytoplasmic dynein

Cytoplasmic dynein is a two-headed microtubule-based motor responsible for diverse intracellular movements, including minus-end-directed transport of organelles. The motility of cargo transporters is regulated according to the presence or absence of cargo; however, it remains unclear how cytoplasmic dynein achieves such regulation. Here, using a recombinant and native dynein complex in vitro, we show that lone, single dynein molecules are in an autoinhibited state, in which the two motor heads are stacked together. In this state, dynein moves diffusively along a microtubule with only a small bias towards the minus end of the microtubule. When the two heads were physically separated by a rigid rod, the movement of dynein molecules became directed and processive. Furthermore, assembly of multiple dynein molecules on a single cargo enabled them to move unidirectionally and generate force cooperatively. We thus propose a mechanism of autonomous on–off switching of cargo transport, in which single dynein molecules in the cell are autoinhibited through intramolecular head–head stacking and become active when they assemble as a team on a cargo.

Creating biomolecular motors based on dynein and actin-binding proteins

Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors-combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins-robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

Re-engineering of protein motors to understand mechanisms biasing random motion and generating collective dynamics

A considerable amount of insight into the mechanisms of protein-based biomolecular motors has been accumulated over decades of research. However, our knowledge about the design principles of these motors is still limited. Even less is known about the design of multi-motor systems that perform various functions within the cell. Here we focus on constructive (or synthetic) approaches to biomolecular motors that could make a breakthrough in our understanding. Recent achievements include studies at different hierarchical levels of complexity: re-engineering of individual motors, construction of multi-motor systems, and generation of large-scale complex behaviour. We then propose a strategy where the collective behaviour can be repeatedly tested upon modifying individual motors, which may provide important clues about how biomolecular motors and their systems are designed.