Characterizing and Influencing Intracellular Transport

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2023

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Abstract

For over 200 years, cell function and behavior has been the subject of significant interest. Although microscopic, mammalian cells are fantastically complicated and need to overcome tremendous environmental inertia to maintain homeostasis, such as facilitating ion gradients and intracellular transport, the movement of cargo through a viscous, crowded cytosol. This latter point is especially important because many diseases, including Alzheimer’s disease, are associated with aberrant transport. Thus, determining how a cell responds to environmental stimuli is critical to building an understanding of fundamental biophysics and working toward future curative measures for transport-related disease.This dissertation begins with a high-level, epidemiological perspective on the co-occurrence of pulmonary diseases in the United States with pneumoconiosis to contextualize microscopic cell responses to damage with macroscopic outcomes. Pneumoconiosis is caused by inhaled dusts and nanomaterials, which can become embedded in and inflame lung tissues. We found that pneumoconiosis is associated with increased rates of chronic obstructive pulmonary disease (COPD), lung cancer, and pneumonia at time of death. When we combined pneumoconiosis diagnoses with the known covariate of smoking history, smokers with pneumoconiosis had the highest rate of COPD in our data, indicating a potential synergistic effect of lung damage. Interestingly, we found that smoking history and pneumoconiosis were more associated with lung cancer and pneumonia, respectively. Through presented case studies in non-mining/construction occupations, we note that specific pneumoconioses can occur on local scales, demonstrating that even if nanomaterials are too varied to appear in an aggregate population, the risks of increased disease rate as a result of microscopic lung injury are still present. This is essential for future regulation and policy decisions as nanomaterial production continually increases. To further explore microscopic cell responses to controlled stimuli, we used particle tracking microscopy to follow trafficked organelles and evaluate how the cell uses intracellular transport and reacts to disruptions. The classic approach to analyzing particle tracking data is the mean squared displacement (MSD). Despite its ubiquity, recent work has shown that the MSD is a flawed method because it is unstable with respect to noise, curve fitting choices, and observation window. Here, we present a novel tracking framework that uses a Bayesian changepoint segmentation strategy and then infers population motility from segment velocities. This method avoids the use of MSDs and is efficient and stable in response to trajectories of different quality and length. We demonstrate this software on tracked lysosomes in epithelial cells. We found that there is a clear difference in the frequency of motion for lysosomes depending on where in the cell they were located, with lysosomes in the perinuclear region moving less often than those in the periphery. This is an extremely important finding because it robustly distinguishes these two regions over thousands of lysosome observations and much of the current particle tracking literature ignores region as a factor, potentially exposing any results to selection bias. Separately, we found that the size of lysosomes, which was controlled with sucrose-induced osmotic swelling, had no effect on transport frequency; however, the speed of large lysosomes was slower than with small lysosomes. Next, we generalize our tracking system to include all vesicles, rather than only lysosomes. With these conditions, we present an exciting new result: disruption of the endoplasmic reticulum with palmitate, a fatty acid found at elevated levels in patients with diabetes and obesity, significantly decreases vesicle motility. This effect was independent of any reduction in ATP levels or cell viability and appears to be associated with the distortion of the ER we observed under these conditions. This result points to areas of future research in the biophysical complications associated with these diseases and further underscores recent work detailing the extensive interactions between the ER and endocytic vesicles. Paired with this analysis, we also observed that macromolecular crowding has no effect on directed transport through the reduction of ribosome concentration, indicating that directed intracellular transport is quite efficient despite significant obstacles in the cytosol. Looking further at the cytoskeleton, we show that disruption of actin filaments and microtubules both decrease vesicle motility as expected. However, we found that disruption of intermediate filament organization with withaferin A significantly decreases vesicle motility in a dose-dependent fashion. Unlike microtubules and actin filaments, there are no molecular motors associated with intermediate filaments, so this result may be tied to cytoskeletal interactions and merits further exploration. In summary, this dissertation details analyses that explore and characterize disruptions to cellular homeostasis. We first provide an updated perspective of dust inhalation diseases in the United States to provide context for cell damage on a macroscopic level and advocate for intentional regulation of nanomaterials as production and exposure risk increase. We also demonstrate an effective Bayesian particle tracking analysis alternative to the mean squared displacement, which overcomes the latter’s limitations. We then use this new tool to learn significant new information about intracellular transport, particularly that there are regional distinctions in vesicle behavior and that ER disruption with palmitate causes a dramatic decrease in transport without affecting viability. Overall, we are most excited for the potential for this analysis to be used across a variety of problems and disciplines and look forward to its implementation.

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Rayens, Nathan (2023). Characterizing and Influencing Intracellular Transport. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/27717.

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Dukes student scholarship is made available to the public using a Creative Commons Attribution / Non-commercial / No derivative (CC-BY-NC-ND) license.