Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

David M. Eckmann

Abstract

Energetic and structural maintenance are both critical to cellular homeostasis, and clinical disease is often characterized by alterations in both of these realms. While the manifestation of pathology in each of these fields has been extensively studied, little research has been done to characterize basic, direct interactions between mitochondrial function and cell mechanics. The experiments described in this dissertation endeavored to address that gap, first by investigating the cytoskeletal and mechanical effects of mitochondrial dysfunction and then by considering the mitochondrial consequences of cytoarchitectural breakdown.

Mechanical integrity of the cell following mitochondrial dysfunction was investigated through multiple experimental platforms, including the quartz crystal microbalance with dissipation (QCM-D). Early work thus focused on improving the suitability of QCM-D for cell experimentation by developing a method of covalently conjugating fibronectin to QCM-D sensors. We then subjected cells to mitochondrial toxins in order to address whether and how mitochondrial dysfunction affects cell mechanics and the cytoskeleton. Cells showed characteristic rounding after long-term exposure to rotenone, an inhibitor of complex I of the mitochondrial respiratory chain. Since mitochondrial dysfunction can also be caused by genetic defects in the mitochondrial DNA (mtDNA), we also studied the cytoskeletal and mechanical variations in cells heteroplasmic for the m.3243A>G mutation. We found a conserved, nonmonotonic relationship between m.3243A>G heteroplasmy and cell mechanics, originating in expression of actin-related genes and persisting at the levels of protein production, cytoskeletal structure, and single cell stiffness.

The second half of the dissertation considered how cytoarchitectural breakdown influences mitochondria. We first developed a novel tool for tracking individual mitochondria throughout entire cells, and then used this method to demonstrate that microtubule and microfilament depolymerization affect mitochondrial motility in opposing ways. Another set of experiments found that cytoskeletal breakdown significantly decreased mitochondrial respiration, which sometimes only occurred when mitochondria were pre-stressed by increased demands of calcium maintenance.

Together, these studies highlight direct, bidirectional interactions between mitochondrial function and cell mechanics. These findings will inform future mechanistic studies focused on a comprehensive understanding of human disease at the cellular level, which will hopefully contributing to advancing development of therapeutics.

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