Structural and Mechanical Elements Important for Kinesin Force Generation in Cells
Budaitis, Breane
2020
Abstract
Eukaryotic cells contain a cytoskeleton that forms the structural framework for fundamental cellular processes including cell division, cell motility, intracellular trafficking, and cilia function. The functional output of the microtubule cytoskeleton depends on a family of molecular motor proteins called kinesins. Genetic analysis has indicated that the human genome encodes for over 45 kinesin motor proteins belonging to 19 different families. All kinesins share a highly-conserved catalytic motor domain that converts the chemical energy of ATP hydrolysis into the mechanical outputs of force generation and directed “stepping” along microtubule tracks. How individual kinesins have adapted this basic enzymatic output for their specific functional roles in cells is an outstanding question in the field. Previous work has largely focused on characterizing kinesin motility (e.g. velocity, run length) under single-motor, unloaded conditions using in vitro assays. However, in the crowded cellular environment, kinesins work in teams to transport membrane-bound cargos along a complex network of microtubules. Therefore, although we have a better understanding of the functional diversity of the kinesin motor domain under single molecule conditions, how divergent motility properties lead to emergent mechanisms of transport in cells is not clear. To bridge this gap, I adapted artificial cargo transport assays in cells to correlate how changes in a motor’s single molecule properties impact their ability to collectively drive transport under physiological conditions. Recent theoretical, biophysical, and computational studies predict that a motor’s behavior under force is a critical parameter that dictates transport in cells. Generation of force by kinesin motors involves ATP-induced docking of the neck linker (NL) along the motor’s core; however, the contributions of the proposed substeps of NL docking are unclear. Furthermore, whether sequence changes that modulate NL docking is a strategy to tune the functional output of a motor in cells has not been addressed. In this dissertation, I first present work that uses a combination of techniques (computational, biophysical, and cellular) to address how NL docking impacts the functional output of kinesin-1 motors. Surprisingly, I find that motors with weakened NL docking are faster and more processive but at a cost to their force production. Furthermore, motors with weakened NL docking are crippled in their ability to drive transport of high-load cargo in cells, providing the first evidence that a power-stroke mechanism of force generation is critical for multi-motor driven transport under physiological conditions (Chapter 2). I further extend these results to other kinesin family members (Chapter 3). Although biophysical studies have led to a better understanding of how family-specific sequence divergence within the kinesin superfamily tunes motility, how single residue changes implicated in disease impacts the functional output of a motor can be difficult to predict. We find that a majority of disease-associated mutations cluster in elements of the motor domain important for microtubule binding, force generation, and nucleotide binding/hydrolysis. We characterized the effect of two mutations predicted to impair NL docking in the kinesin-3 motor KIF1A under different load regimes to provide insight into how these mutations manifest in disease (Chapter 4). Collectively this work confirms the proposed role of the NL as a mechanical element important for force generation by kinesin motors and drives our understanding of how kinesins adapted this feature for their functional roles in the cell.Subjects
kinesin motor protein cytoskeleton
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.