Collective interactions between motor proteins play an essential role in the transport of intracellular cargo by providing a means to optimize transport for the heterogeneous environment of the cytoplasm. As a result, collective biomotor transport is essential for the maintenance of healthy cellular function. However, significant challenges remain to fundamentally understand how motor proteins function collectively. These challenges largely stem from experimental difficulties in connecting structural information that describes the supramolecular properties of interacting groups of motor proteins to collective modes of intracellular transport. This research will bridge these gaps by establishing techniques that enable collective motor transport to be investigated from both a structural and dynamical standpoint. This goal will be accomplished by developing biosynthetic technologies that can be used to construct experimental model systems of interacting motor proteins. Based on self-assembled DNA nanostructures and engineered artificial proteins, these technologies will be harnessed to build molecular platforms that enable the position, number, and type of motors contained in an assembly, as well as the elasticity of motor interconnects to be encoded at the molecular level. The supramolecular architecture of the assemblies will be characterized using novel single-molecule imaging methods based on total-internal reflection microscopy (TIRFM). In addition, collective motor dynamics will be investigated using optical trapping instrumentation. This apparatus will allow the intricate stepping mechanics of interacting motor proteins to be investigated with single-molecule resolution and in real time. Together, both techniques will facilitate development of detailed structure-activity relationships that define how critical transport properties (velocities, load dependence, step size, dwell times, etc...) depend on the molecular architecture of assemblies. Because the mechano-chemistry of biomotors is known to be strongly dependent on strain, and hence on the mechanical coupling between motors, establishing these relationships will provide insight into how sharing force between motors influences their mechano-chemistry. Furthermore, by developing a platform technology to construct and probe a variety of architecturally rich systems of motor proteins, this research will allow mechanisms of collective transport that are typically masked by the complexities of cellular environments to be precisely determined.
The philosophical framework of this research is also harnessed in an education plan that strives to enhance the scientific literacy of students and educators by empowering them with the knowledge base and skills necessary to confront frontier challenges in the biosciences and bioengineering. These goals will be accomplished by using this research as a foundation for creating research and education opportunities for both graduate and undergraduate students. Furthermore, this work incorporates outreach activities designed to expose high school students to college-level research and education. These activities will also serve to recruit under-represented minority groups to bioscience research and will be used to disseminate new instructional tools to high school educators.
This project developed new experimental and computational methods to dissect how cytoskeletal motor proteins function collectively during intracellular transport processes in eukaryotic cells. The coordination of these motors is critical to various cellular functions. Most subcellular commodities (ribonucleoprotein particles, organelles, vesicles…) are transported by teams of motor proteins that are composed of multiple copies of the same motors and even mixtures of different motors that move in opposite directions, with different velocities, or along different types of filament tracks. Consequently, numerous intracellular transport processes and regulatory mechanisms depend fundamentally on how collections of similar and dissimilar motors either cooperate or compete antagonistically with one another. Understanding these behaviors is important to elucidating various disease pathologies stemming from motor and other transport-related protein mutations since their impact on cargo transport will ultimately depend on the extent to which they perturb the composite dynamics of motor systems that also contain groups of wild-type motors. Finally, systems of interacting motors constitute ideal test beds for the development experimental tools for examining multiple protein functions since they offer unique opportunities to probe collective protein dynamics at the single-molecule level by monitoring protein motions, while being able to tune their enzymatic properties simultaneously through the application of controlled applied loads. Existing studies of multiple motor dynamics have been largely confounded by the fact that the number and spatial arrangement of motors on intracellular cargos, as well the elasticity of motor-cargo linkages, are all unknown in most experimental circumstances. This project has confronted these problems directly by developing methods to synthesize structurally-defined motor complexes that are organized on genetically-engineered protein polymers and DNA/protein-based polymer templates. Such control provides unique abilities to characterize how each of these parameters influences a motor complex’s motile and force-generating properties and has allowed our group to uncover several features of multiple motor dynamics that have important mechanistic implications. Our comparisons of single and multiple kinesin behaviors in precision particle tracking assays demonstrated that groups of processive and efficient microtubule motors such as kinesin tend to exhibit negative cooperative behaviors and are unable to transport cargo over the distances predicted by prior theoretical studies. While providing new windows into the richness and complexity of this problem, our optical trapping studies of multiple kinesin force production uncovered similarly weak collective responses in the presence of applied loads. Although multiple kinesin complexes are capable of producing large forces and are able to move with high velocities, their average velocities and detachment forces were found to be remarkably similar to those of single kinesin motors. Our approach also provided key insights into this behavior by allowing vectorial force-distributions within a complex to be modeled from precision measurements of the non-linear elastic properties of single-motor molecules. Moreover, the optical trap’s exceptionally-high spatial and temporal resolution facilitated the first experimental analyses of how rapidly motor complexes transition between different filament-bound configurations due to the binding, detachment and stepping of individual motors within a complex. Together, these analyses uncovered that multiple kinesin complexes face fundamental mechanical and kinetic constraints that limit their ability to share their applied load and function productively as a team. We have also established that these constraints depend on the that efficiency at which motors can transport cargos under applied loads and that this property is a key determinant of whether they can cooperate productively as a team. Overall, we believe this behavior constitutes an important principle that dictates the extent to which variation in the number of a particular class of motor will contribute to mechanisms that regulate cargo motion. In line with these predictions, we have recently shown that multiple myosin V motors exhibit more sensitive collective responses since these motors are less efficient and weaker than kinesin. Finally, the experimental challenges associated with investigating these problems project has led to the establishment of an array of new synthetic techniques to create organized protein complexes, imaging probe technologies and procedures facilitating highly-multiplexed imaging of proteins, and analytical /computational tools for dissecting multiple protein functions. Thus, while opening new opportunities to resolve the role of motor cooperation in development and diseases, our tools and approach can be applied to various other biological problems requiring analyses of dynamic multiple protein behaviors.
