Controlling organization and forces in active matter through optically defined boundaries.

Living systems are capable of locomotion, reconfiguration and replication. To perform these tasks, cells spatiotemporally coordinate the interactions of force-generating, 'active' molecules that create and manipulate non-equilibrium structures and force fields of up to millimetre length scales1-3. Experimental active-matter systems of biological or synthetic molecules are capable of spontaneously organizing into structures4,5 and generating global flows6-9. However, these experimental systems lack the spatiotemporal control found in cells, limiting their utility for studying non-equilibrium phenomena and bioinspired engineering. Here we uncover non-equilibrium phenomena and principles of boundary-mediated control by optically modulating structures and fluid flow in an engineered system of active biomolecules. Our system consists of purified microtubules and light-activatable motor proteins that crosslink and organize the microtubules into distinct structures upon illumination. We develop basic operations-defined as sets of light patterns-to create, move and merge the microtubule structures. By combining these operations, we create microtubule networks that span several hundred micrometres in length and contract at speeds up to an order of magnitude higher than the speed of an individual motor protein. We manipulate these contractile networks to generate and sculpt persistent fluid flows. The principles of boundary-mediated control that we uncover may be used to study emergent cellular structures and forces and to develop programmable active-matter devices.

Optogenetic control of organelle transport and positioning.

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems.

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail.

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.

Photo-control of kinesin-microtubule motility using caged peptides derived from the kinesin C-terminus domain.

To design a nanoscale biodevice that can be controlled by an external stimulus, we have introduced photochemical switching peptides derived from the kinesin C-terminus domain into the kinesin-microtubule in vitro motility system.

Electrical docking of microtubules for kinesin-driven motility in nanostructures.

We demonstrate localized electrical control of the docking of microtubules onto engineered kinesin-coated structures. After applying a voltage to a gold electrode, we observe an enhanced transport of microtubules from solution toward the surface and a subsequent increase of the amount of moving microtubule shuttles. Switching off the voltage leads to a partial detachment of microtubules from the surface. The surface coverage of microtubules, during both the docking and undocking events, follows an exponential time dependence. We provide a simple kinetic model, incorporating the equilibrium between free and surface-bound microtubules, that explains these data.

Microscale transport and sorting by kinesin molecular motors.

As biomolecular detection systems shrink in size, there is an increasing demand for systems that transport and position materials at micron- and nanoscale dimensions. Our goal is to combine cellular transport machinery-kinesin molecular motors and microtubules-with integrated optoelectronics into a hybrid biological/engineered microdevice that will bind, transport, and detect specific proteins, DNA/RNA molecules, viruses, or cells. For microscale transport, 1.5 microm deep channels were created with SU-8 photoresist on glass, kinesin motors adsorbed to the bottom of the channels, and the channel walls used to bend and redirect microtubules moving over the immobilized motors. Novel channel geometries were investigated as a means to redirect and sort microtubules moving in these channels. We show that DC and AC electric fields are sufficient to transport microtubules in solution, establishing an approach for redirecting microtubules moving in channels. Finally, we inverted the geometry to demonstrate that kinesins can transport gold nanowires along surface immobilized microtubules, providing a model for nanoscale directed assembly.

Chromophore-assisted light inactivation and self-organization of microtubules and motors.

Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.