Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Matthew C. Good

Abstract

A cell must tightly regulate the dynamics, stability, and positioning of microtubule networks, and mutation of microtubule associated proteins (MAPs) are associated with several human diseases. Improper regulation of microtubule stability has been linked to neurodegenerative disease, stem cell over proliferation, and improper keratinocyte morphogenesis leading to loss of skin barrier function4. While faulty expression of spindle positioning factors is linked to errors in cell division and improper cell-fate specification, it is often unclear whether the misregulation of microtubule networks are the origin or consequence of disease. Additionally, due to the complexities and redundant systems present in a living cell, it is challenging to dissect the contributions of the positioning and activities of MAPs to regulation of cytoskeletal architecture. To understand the dynamic nature of microtubule network reorganization, it is necessary to perturb its organization in real-time. Here, I implemented optochemical dimerization tools and a biochemically reconstituted system composed of a minimal set of parts to generate asymmetrically patterned synthetic boundaries and manipulate the spatial patterning of microtubules in cell-like compartments after encapsulation. By spatially micropatterning components within cell-like compartments, I remodeled microtubule networks in real-time. I anchored a bait protein to create a synthetic cortex within the cell-like compartment and used chemogenic or optical inputs to trigger cortical relocalization of a protein present in the compartment lumen. To achieve stable photopatterning of protein localization, I slowed lateral diffusion within the lipid monolayer. Further, by combining the optochemical tools with slowed lipid diffusion, I demonstrated stable protein micropatterning using focused light. To control microtubule network organization, I fused microtubule interacting proteins to the optochemical domains. This allowed induced repositioning of microtubule-interacting proteins and spatial reorganization of microtubule networks. Cortical patterning of polymerizing microtubules breaks network symmetry and the collective forces dramatically reshape the compartment boundary, reminiscent of the reorganization of the microtubule marginal band in platelets. This system offers a biomimetic platform for cell biology to characterize the contributions of biochemical components and physical boundary conditions to microtubule network organization. Additionally, active shape control has applications in protocell engineering and for the construction of synthetic cells.

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