An In Vitro Reconstitution Investigation into the Kinetic Mechanisms Underlying Kinesin-based Vesicle Transport
Open Access
- Author:
- Jiang, Rui
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 08, 2021
- Committee Members:
- Donna Korzick, Program Head/Chair
Paul Cremer, Outside Unit Member
William Hancock, Chair & Dissertation Advisor
Peter Butler, Major Field Member
Christopher Yengo, Outside Field Member - Keywords:
- kinesin
microtubule
kinetics
in vitro reconstitution
lipid membrane
total internal reflection fluorescence microscopy - Abstract:
- Bidirectional intracellular transport is driven by kinesin and dynein motors that move along microtubules towards the cell periphery and the nucleus, respectively. Intracellular cargos travel for significantly longer distances than individual motors because they are carried by teams of motors that work together such that when one or more motors detach, others remain to sustain transport. The distance that a cargo can travel is ultimately determined by the kinetic race between attachment of unengaged motors to the microtubule and detachment of engaged motors from the microtubule. Motor detachment rates are routinely measured by single-molecule motility assays, by dividing the velocity by the run length. In contrast, motor attachment rates are more challenging to measure and their impacts on multi-motor transport are less understood. Because most intracellular cargos are membrane-bound, motors are coupled to their cargo through a fluid lipid bilayer in which they can freely diffuse. Membranes can serve as platforms for motor reorganization through phase separation of lipid domains or by assembly of multimotor complexes by multivalent scaffold proteins. It is not understood how diffusion of motors in the plane of the membrane bilayer modulates motor binding kinetics to the microtubule. Furthermore, it also remains obscure whether differences in motor organization have a direct impact on the speed and persistence of cargo motility. A thorough understanding of how motor binding kinetics are impacted by membrane diffusion and motor reorganization has been hampered by the lack of suitable experimental systems. This dissertation introduces two novel reconstitution systems for answering these questions. Because the signals from different motors attached to a single vesicle overlap under the microscope, it is unfeasible to directly measure motor binding events using motor-functionalized vesicles. This challenge is overcome by performing this measurement using an inverted geometry where kinesin motors are connected to a 2D supported lipid bilayer and the rate of binding to the microtubule is monitored by a local increase in fluorescence from motor accumulation along a newly landing microtubule. Our understanding of the roles that motor organization plays in regulating vesicle transport is hindered by our limited knowledge of the cellular machinery that controls motor organization in membranes. This difficulty is overcome by designing a DNA scaffold that can induce motor cluster formation and thereby precisely control the number of clustered motors on the vesicle surface. Beyond the introduction of new reconstitution systems, this dissertation provides four major contributions in advancing our understanding of long-range vesicle transport. First, a direct measurement of the microtubule binding kinetics of membrane-bound kinein-1 is provided and its relation to membrane diffusivity is explored. Second, transport of liposomes of comparable size to intracellular cargos by multiple kinesin-1 motors is reconstituted and quantified, filling the previous gap in reconstituting transport of physiologically relevant cargos by defined numbers of motors. Third, a comparison between vesicle travel distances and total motor copy numbers is provided, directly supporting predictions previously made based on motor binding kinetics. Finally, a causational relationship between motor clustering and changes in vesicle motility behaviors is investigated and established, providing new insights into regulation of intracellular transport. Overall, this dissertation sheds light on the roles that lipid membrane and motor organization play in motor binding kinetics and long-range vesicle transport, and the two new reconstitution systems open up future avenues for studying bidirectional vesicle transport.