A Novel Synthetic Biology Method to Study the Cooperative Behavior of Kinesin Motors in Cells.

Abstract

Collective motor dynamics drives important cellular processes ranging from muscle contraction to spindle organization to vesicle trafficking (Chapter 1). Although the biomechanical and biochemical properties of individual motors have been widely studied, how motors coordinate their motility when attached to the same cargo is largely unknown. In this dissertation, I present a synthetic biology technique (Chapter 2) to generate multi-motor assemblies whose biological properties can be examined in vitro and in living cells. To do this, we assembled a “toolbox” of protein components consisting of scaffolds and linkers. We characterized scaffold proteins of different lengths that allow for specific separation distances between the components. We then characterized four different linker systems that enable constitutive or regulated attachment of individual motors to scaffolds. We then showed, through FRET and subcellular localization experiments, that this toolbox could be used to generate defined assemblies in living cells. Next, I present a characterization of fluorescent tags for use in single-molecule experiments (Chapter 3), and show that certain tags lead to aberrant kinesin-1 run lengths due to oligomerization. This study will provide a valuable reference for the field in choosing proper fluorescent tags for single-molecule experiments. I then present a series of experiments where we use this system to investigate the behavior of two motors attached to a scaffold (Chapter 4). We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor-cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. Overall, the approach presented in this dissertation is applicable to other biological questions such as the generation of complex signaling networks as well as the assembly of artificial biological systems for engineering applications (Chapter 5).