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MECHANISMS OF ACTIVE DIFFUSION OF VIRAL AND CELLULAR PARTICLES IN THE CYTOPLASM OF MAMMALIAN CELLS

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title
MECHANISMS OF ACTIVE DIFFUSION OF VIRAL AND CELLULAR PARTICLES IN THE CYTOPLASM OF MAMMALIAN CELLS
author
Moran, Steven Javier
abstract
Most of the focus on intracellular transport has been on directed motion, yet motion that is random in direction is the more predominant type of motion in the cytoplasm. Small particles move by Brownian diffusion while large particles undergo ATP-dependent random motions to circumvent the limitations imposed on them due to their size. Those random motions are mediated by the cytoskeleton, an extensive network of proteins that also acts a barrier to diffusion for large particles. To understand the molecular mechanisms behind ATP-dependent random motions, the ribonucleoprotein (RNP) core of vesicular stomatitis virus (VSV) served as a molecular probe. VSV is a prototype RNA virus commonly used to understand processes relevant to basic and translational science. Fluorescently labelled VSV RNPs were used by our group to track particles in living cells. And a variational Bayesian analysis method was used to analyze the data. It was determined that VSV RNPs infrequently undergo directed motion, and that their random motion can be classified as either confined motion or hopping-like motion. This research project further investigated the random motions of VSV RNPs with live-cell fluorescence imaging, variational Bayesian analysis, and treatment with two cellular inhibitors: latrunculin A to depolymerize actin filaments and blebbistatin to inhibit non-muscle myosin II motor proteins. It was determined that actin filament traps mediate the active diffusion of VSV RNPs through non-muscle myosin II ATPase activity. The random motion of other large particles with different physical and biological properties was then investigated to determine whether the mechanisms behind their random motion were similar to mechanisms for VSV RNPs. Those other large particles were VSV inclusion bodies and cellular early endosomes. It was shown through our previous experimental approach and treatment with latrunculin A and the microtubule depolymerizing inhibitor nocodazole that actin filaments mediate the movement of traps occupied by particles larger than VSV RNPs. Furthermore, while microtubules mediate the directed motion of early endosomes, they only play a minor role in trap composition along with actin filaments. The results of this research project provide experimental support for the molecular mechanisms behind the random motions of large viral and cellular particles.
subject
active diffusion
early endosome
inclusion body
ribonucleoprotein
variational Bayesian analysis
vesicular stomatitis virus
contributor
Lyles, Douglas S. (advisor)
McDonald-Esstman, Sarah (committee member)
Hollis, Thomas (committee member)
Ornelles, David A. (committee member)
(Palmer) Allred, Nicholette (committee member)
date
2023-07-25T17:48:42Z (accessioned)
2023 (issued)
degree
Biochemistry and Molecular Biology (discipline)
embargo
2024-06-06 (terms)
2024-06-06 (liftdate)
identifier
http://hdl.handle.net/10339/102261 (uri)
language
en (iso)
publisher
Wake Forest University
type
Dissertation

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