Characterization and cooperative dynamics of kinesin motor proteins in intracellular transport

Intracellular transport is typically carried out by teams of motor proteins rather than by isolated molecules, yet the rules governing multi-motor coordination remain poorly understood because motor number, spacing, and composition are difficult to control experimentally. In this dissertation, I developed both the molecular engineering and nanostructural framework needed to address this problem. I designed a modular plasmid toolkit for controlled kinesin expression and purification, and I created a novel DNA origami scaffold that allows motors to be positioned with defined stoichiometry and geometry on a rigid cargo-like platform. Together, these advances provided a programmable experimental system for dissecting how kinesin-1 ensembles and mixed kinesin-1/kinesin-14 assemblies behave under well-defined structural conditions.Using this platform, I found that motor valency strongly enhances attachment persistence and transport robustness, but that the magnitude and interpretation of this effect depend on nucleotide state and ensemble architecture. In weak-binding ADP conditions, increasing kinesin-1 copy number strongly stabilized microtubule association, revealing that additional motors provide a disproportionate rescue benefit in vulnerable binding regimes. In ATP, processivity increased with motor number while velocity changed comparatively little, but the motor-number dependence was more geometry- and preprocessing-sensitive, motivating statistical and modeling analysis. Direct mean-field modeling captured the ADP results reasonably well but proved fragile for ATP, whereas a microstate continuous-time Markov chain model with a global attachment scale regularized this instability and supported an ensemble-rescue interpretation. Finally, introducing antagonistic kinesin-14 reduced kinesin-1-driven transport and produced tunable mixed-motor behavior, showing that this engineered platform can reveal how motor copy number, geometry, and opposition collectively regulate cargo transport. These findings have broader significance for understanding transport defects in neurodegenerative and other trafficking-related diseases, where failures in motor coordination and cargo delivery can have major cellular consequences. More broadly, the ability to engineer and quantitatively tune multi-motor assemblies may inform future bioengineering efforts in nanoscale transport design, including programmable cargo handling and motor-based drug delivery systems.

Grantor dc:publisher

Temple University. Libraries

Year dc:date.issued

2026

Author dc:creator

• Alizadeh, Vahid

Advisor dc:contributor.advisor

• Tuzel, Erkan

Committee members dc:contributor.committeemember

• Gligorijevic, Bojana
• Spence, Andrew J.
• Petrie, Ryan

• Bioengineering
• Biophysics
• Molecular biology
• Cooperative motility
• DNA origami
• Kinesin
• Plasmid cloning
• Single molecule assay

Statement dc:rights

• IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

eng

Repository record dc:identifier.uri

https://scholarshare.temple.edu/handle/20.500.12613/12244

OAI identifier oai:identifier

oai:scholarshare.temple.edu:20.500.12613/12244