Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal archit

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Title: Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal architecture. P.I. – Richard J. McKenney Research Summary Intracellular transport is essential for cellular homeostasis in eukaryotes. This process is carried out by molecular motors that convert the chemical energy from ATP hydrolysis into motion along the actin and microtubule cytoskeletal networks. Decades of research has uncovered structural and molecular details that explain how many of these motors move along their filament tracks. In the cellular milieu, most of these motors act in concert with complex regulatory machinery that links them to their respective cargos, modulates their motile properties, and dictates spatiotemporal activity. How individual motor output is controlled by this machinery is currently not clear and difficult to dissect in the complex environment of the cell. In addition, many cargos are moved by the concerted action of simultaneously bound, opposite polarity motors, in a process called bidirectional transport. How individual motors are recruited to cargo, activated, and integrated with other classes of motors presents a large open challenge to the field. Importantly, defects in this process lead to a wide variety of human diseases and these questions are thus directly related to human health. Microtubule network organization, dynamics, and motor activity along microtubules are all impinged upon by non-enzymatic microtubule-associated proteins (MAPs) that dynamically bind to microtubules. The specific functions, molecular properties, and dynamics of MAPs remains underexplored. The tau family of MAPs are critically important for human health, as tau is well- characterized to form insoluble inclusions/aggregates in a host of human neurodegenerative diseases such as Alzheimer’s disease and Pick’s disease. Despite their identification over four decades ago, the specific molecular functions and molecular properties of tau family MAPs remains unclear. This application seeks to develop novel assays and tools to study the complexity of microtubule motor regulation, bidirectional transport of cargos, and cytoskeletal functions driven by motors and MAPs. Our approach to combine biochemistry and single-molecule analysis towards in vitro reconstitutions that test molecular function, and translate our findings into in vivo systems that test hypotheses generated by these reconstitutions, will open up fruitful long-term avenues of research. We propose to: 1) Reconstitute and study the recruitment, regulation, and motility of cytoplasmic dynein and kinesin motors bound to native cellular cargo scaffolding molecules, and 2) Reconstitute and study evolutionary functions, dynamics, and pathological behaviors of tau family MAPs. These broad goals build and expand upon our expertise and previous work in dissecting the regulatory mechanisms of the cytoplasmic dynein motor, and aim to provide powerful new tools useful towards dissecting complex motor function. Our work will illuminate basic molecular and cell biological principles that drive cellular homeostasis and provide insight into the pathological mechanisms that arise from molecular motor malfunction.