Jonathan W. Driver

Productive cooperation among processive motors depends inversely on their mechanochemical efficiency.

Subcellular cargos are often transported by teams of processive molecular motors, which raises questions regarding the role of motor cooperation in intracellular transport. Although our ability to characterize the transport behaviors of multiple-motor systems has improved substantially, many aspects of multiple-motor dynamics are poorly understood. This work describes a transition rate model that predicts the load-dependent transport behaviors of multiple-motor complexes from detailed measurements of a single motors elastic and mechanochemical properties. Transition rates are parameterized via analyses of single-motor stepping behaviors, load-rate-dependent motor-filament detachment kinetics, and strain-induced stiffening of motor-cargo linkages. The model reproduces key signatures found in optical trapping studies of structurally defined complexes composed of two kinesin motors, and predicts that multiple kinesins generally have difficulties in cooperating together. Although such behavior is influenced by the spatiotemporal dependence of the applied load, it appears to be directly linked to the efficiency of kinesins stepping mechanism, and other types of less efficient and weaker processive motors are predicted to cooperate more productively. Thus, the mechanochemical efficiencies of different motor types may determine how effectively they cooperate together, and hence how motor copy number contributes to the regulation of cargo motion.

Cooperative Responses of Multiple Kinesins to Variable and Constant Loads

Background: Multiple kinesin function is central to intracellular transport. Results: Unlike single-motor molecules, two kinesin velocities can depend on whether loads vary spatially or temporally. Conclusion: Kinesin cooperation is influenced appreciably by spatially dependent changes in load. Significance: Factors governing the force-time history and spatial dependence of loads must be examined to understand mechanisms regulating intracellular transport. Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fashion or antagonistically. Several lines of evidence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environmental factors, and the influence of these conditions has been largely overlooked. Here, we characterize the dynamics of structurally defined complexes containing multiple kinesins under the controlled loads of an optical force clamp. While demonstrating that there are generic kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple motor systems. We propose this dependence has implications for intracellular transport processes, especially for bidirectional transport.

Collective Dynamics of Elastically Coupled Myosin V Motors

Background: Collective myosin Va functions are important to various transport processes in eukaryotes. Results: Strain coupling between myosins affects multiple motors velocities and run lengths. Conclusion: The large step size and small stall force of myosin Va yields a dependence of multiple myosin behaviors on the structural and mechanical properties of cargos. Significance: The properties of myosin V motors lead to unique cooperative behaviors compared with other motor types. Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain.

How the Interplay between Mechanical and Nonmechanical Interactions Affects Multiple Kinesin Dynamics

Intracellular transport is supported by enzymes called motor proteins that are often coupled to the same cargo and function collectively. Recent experiments and theoretical advances have been able to explain certain behaviors of multiple motor systems by elucidating how unequal load sharing between coupled motors changes how they bind, step, and detach. However, nonmechanical interactions are typically overlooked despite several studies suggesting that microtubule-bound kinesins interact locally via short-range nonmechanical potentials. This work develops a new stochastic model to explore how these types of interactions influence multiple kinesin functions in addition to mechanical coupling. Nonmechanical interactions are assumed to affect kinesin mechanochemistry only when the motors are separated by less than three microtubule lattice sites, and it is shown that relatively weak interaction energies (~2 k(B)T) can have an appreciable influence over collective motor velocities and detachment rates. In agreement with optical trapping experiments on structurally defined kinesin complexes, the model predicts that these effects primarily occur when cargos are transported against loads exceeding single-kinesin stalling forces. Overall, these results highlight the interdependent nature of factors influencing collective motor functions, namely, that the way the bound configuration of a multiple motor system evolves under load determines how local nonmechanical interactions influence motor cooperation.

Construction and analyses of elastically coupled multiple-motor systems.

Precision analyses of the collective motor behaviors have become important to dissecting mechanisms underlying the trafficking of subcellular commodities in eukaryotic cells. Here, we describe a synthetic approach to create structurally defined multiple protein complexes containing two elastically coupled motor molecules. Motors are connected using a simple DNA-scaffolding molecule and DNA-conjugated, artificial protein polymers that function as tunable elastic linkers. The procedure to self-assemble these components produces complexes in high synthetic yield and allows individual multiple-motor systems to be interrogated at the single-complex level. Methods to evaluate cooperative motor responses in a static optical trap are also discussed. While enabling the average transport properties of single/noninteracting and coupled motors to be compared, these procedures can provide insight into the extent to which motors cooperate productively via load sharing as well as the roles loading-rate-dependent phenomena play in collective motor functions.