In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surfaceimmobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. molecular motors | lipid bilayers | transport efficiency | motor-cargo coupling | streptavidin-binding peptide I ntracellular transport of membrane-bound vesicles and organelles is a process fundamental to many cellular functions including morphogenesis, signaling, and growth (1-4). Active cargo transport inside eukaryotic cells is mediated by ensembles of motor proteins, such as kinesins and dynein, walking on microtubule tracks (5), and myosins walking on actin filaments (6). Gaining mechanistic insight into the functioning of these motors inside the complex environment of cells is challenging. Several studies have thus used in vitro approaches to investigate transport mediated by groups of same or different motors attached to cargos such as silica beads (7), quantum dots (8), glass coverslips (9, 10), or DNA scaffolds (11,12). Although these approaches provide us with knowledge about the collective dynamics of multimotor transport, a key anomaly in these in vitro systems is the use of rather nonphysiological rigid cargo. Vesicular cargo transport by molecular motors requires their attachment to a fluid lipid bilayer either directly or via different adaptor molecules. The anchoring of motors in a diffusive lipid environment induces loose intermotor coupling along with the motors diffusing within the lipid bilayer, thereby increasing the flexibility of the system....

Kinesin-1 plays a major role in anterograde transport of intracellular cargo along microtubules. Currently, there is an ongoing debate of whether α-tubulin K40 acetylation directly enhances the velocity of kinesin-1 and its affinity to the microtubule track. We compared motor motility on microtubules reconstituted from acetylated and deacetylated tubulin. For both, single- and multi-motor in vitro motility assays, we demonstrate that tubulin acetylation alone does not affect kinesin-1 velocity and run length.

Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer's. However, little is known about how motor proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼ 0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼ 70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor's specific intracellular tasks and environments.

Cytoplasmic dynein is a microtubule-based molecular motor that participates in a multitude of cell activities, from cell division to organelle transport. Unlike kinesin and myosin, where different tasks are performed by highly specialized members of these superfamilies, a single form of the dynein heavy chain is utilized for different functions. This versatility demands an extensive regulation of motor function. Using an improved application of an optical trap, we were now able to demonstrate that cytoplasmic dynein can generate a discrete power stroke as well as a processive walk in either direction; i.e., towards the plus-or towards the minus-end of a microtubule. Thus, dynein's motor functions can be described by four basic modes of motion: processive and nonprocessive movement, and movement in the forward and reverse directions. Importantly, these four modes of movement can be controlled by two switches. One switch, based on phosphate, determines the directionality of movement. The second switch, depending on magnesium, converts cytoplasmic dynein from a nonprocessive to a processive motor. The two switches can be triggered separately or jointly by changing concentrations of phosphate and magnesium in the local environment. The control of four modes of movement by two switches has major implications for our understanding of the cellular functions and regulation of cytoplasmic dynein. Based on recent studies of dynein's structure we are able to draw new conclusions on cytoplasmic dynein's stepping mechanism. molecular motors | motor mechanics | single molecule I n eukaryotic cells almost all organelle transport is performed by the three families of cytoskeleton-based molecular motors, myosin, kinesin, and dynein (1-3). While myosin and kinesin have evolved into large families with multiple members, each of which specialized for different tasks, a single dynein heavy chain performs all of the dynein related activities in the cytoplasm of eukaryotic cells (1).One key function of cytoplasmic dynein is to power cargo transport over long distances. Here, motor processivity is required if long distance transport is to be achieved by a single motor molecule. To facilitate cargo delivery processivity must be switched off, when the target area is reached. Regulation becomes even more important when multiple motors of the same or even different type act together. For example, a model situation for the cooperation of different motors occurs during axonal transport driven by kinesin-1 and cytoplasmic dynein. During long distance travel, frequent changes in direction are observed. It is generally thought that the reversals of direction result from counteracting motor activity (4, 5). Though, a simple tug-of-war model in which only the winner determines the direction of movement appears rather inefficient to deliver cargo to specific locations. Moreover, it has been demonstrated in vitro and in vivo that dynein's activity dominates over kinesin (6, 7). Therefore, being able to modulate dynein's processivity and directional...

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.single ATP turnover assay | ATP dwell times | dwell time distribution | TIRF microscopy F orces and movements, generated when myosins interact with actin filaments, are driven by the coupling of conformational changes in the myosin head domain to particular steps of the ATP hydrolysis cycle (1-3). Essential steps of the ATP hydrolysis cycle for skeletal muscle myosin and acto-myosin were characterized in solution studies (1,4,5). With the concept of Hill (6), it became possible to relate solution studies to mechanical, biochemical, and structural studies on muscle fibers (7, 8) to form a general concept for the coupling of ATP hydrolysis to the generation of mechanical work (6, 9).In studies on large ensembles of myosin molecules such as solution and fiber studies, however, crucial reaction steps could be masked by the ensemble averaging and thus may complicate relating solution kinetics to structural changes and the generation of forces and movements. In addition, ensemble studies can be complicated by mixed populations of myosin molecules-for example, by the presence of different myosin isoforms or different posttranslational modifications. Here we explored the feasibility to study ATPase kinetics from ...

Microtubules and actin filaments function coordinately in many cellular processes(1-3). Although much of this coordination is mediated by proteins that statically bridge the two cytoskeletal networks(4-6), kinesin-14 motors with an actin binding calponin homology domain (KCHs) have been discovered as putatively dynamic crosslinkers in plants(7,8). OsKCH1, a KCH from rice, interacts with both microtubules and actin filaments in vivo and in vitro(9). However, it has remained unclear whether this interaction is dynamic or if actin binding reduces or even abolishes the motor's motility on microtubules(10,11). Here, we directly show in vitro that OsKCH1 is a non-processive, minus-end-directed motor that transports actin filaments along microtubules. Interestingly, we observe two distinct transport velocities dependent on the relative orientation of the actin filaments with respect to the microtubules. In addition, torsional compliance measurements on individual molecules reveal low flexibility in OsKCH1. We suggest that the orientation-dependent transport velocities emerge from OsKCH1's low torsional compliance combined with an inherently oriented binding to the actin filament. Together, our results imply a central role of OsKCH1 in the polar orientation of actin filaments along microtubules, and thus a contribution to the organization of the cytoskeletal architecture.

Isopolar arrays of aligned cytoskeletal filaments are components in a number of designs of hybrid nanodevices incorporating biomolecular motors. For example, a combination of filament arrays and motor arrays can form an actuator or a molecular engine resembling an artificial muscle. Here, isopolar arrays of microtubules are fabricated by flow alignment, and their quality is characterized by their degree of alignment. We find, in agreement with our analytical models, that the degree of alignment is ultimately limited by thermal forces, while the kinetics of the alignment process are influenced by the flow strength, the microtubule stiffness, the gliding velocity, and the tip length. Strong flows remove microtubules from the surface and reduce the filament density, suggesting that there is an optimal flow strength for the fabrication of ordered arrays.

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