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Title:A tensed pathway to vesicle clustering
Author(s):Fan, Anthony
Director of Research:Saif, Taher
Doctoral Committee Chair(s):Saif, Taher
Doctoral Committee Member(s):Gillette, Rhanor; Popescu, Gabriel; Juarez, Gabriel
Department / Program:Mechanical Sci & Engineering
Discipline:Mechanical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Vesicle clustering
Mechanical tension
Lab on a chip
Abstract:Synaptic vesicles play a central role in the functionality and adaptability of the nervous system. They carry inside them molecules called neurotransmitters which allow information to be transmitted from one neuron to another cell. This transmission process can only happen if the vesicles are tightly clustered at the cell junctions, known as synapses, such that a supply of neurotransmitters can be maintained. The mechanism of vesicle clustering had always been considered as mainly biochemistry driven. About a decade ago, newer evidence surprisingly demonstrated that mechanical tension can influence vesicle clustering as well. However, what allowed tension to influence clustering was not understood, and will be explored in this dissertation. The first part of this work explores whether neurons maintain internal tension, and identifies the origin of the tension generators. The existence of internal tension generators allows neurons to regulate their tension state independent of their surrounding tissues, providing a potential pathway for neuronal tension to regulate clustering. Towards this goal, we inhibited multiple proteins in in vivo drosophila motor neurons and subsequently studied their contractility in both the axial and circumferential directions. Contractility was hampered in both directions when either F-actin or myosin motors were inhibited, revealing that there exist internal tension generators that consist of acto-myosin machinery. Our results also showed that this acto-myosin driven contractility is coupled in the axial and circumferential direction, pointing to a misaligned network architecture of the tension generators. The second part describes 2 new enabling methods. The first method is a microfluidic setup that allows partial perfusion of an insuspendable tissue sample. Preexisting partial treatment methodologies can only be applied to suspendable samples. By extending this capability to insuspendable samples, I would be able to perform partial treatment on in vivo drosophila neurons to study neuronal tension. The second method was developed to support the assembly process of the microfluidic setup, which relies on natural adhesion between a soft polymeric material and a stiff substrate. This method uses the delamination induced by a trapped bead at the soft-stiff interface to quantify the adhesion energy at the interface. Both methodologies were verified by experimental results. The final part of this work attempts to explain the relationship between tension and vesicle clustering. By disrupting the tension generators (myosin motors) identified in the first part, I observed the declustering of vesicles after the disassembly of F-actin. I further used the microfluidic device described in the second part to demonstrate that a partial inhibition led to the same result. The microfluidic experiments isolated the treatment region from the synapse such that myosin motors disruption would only hamper neuronal tension but nothing else. It also showed that tension was generated in series along the entire length of the neuron; any failure along the length would lead to a total tension loss. I further accounted for the dynamics of vesicle clustering and declustering by photobleaching the fluorescence proteins fused to the vesicles and subsequently observing the recovery due to other vesicles migrating into the bleached area. Based on all of these experimental observations and results, it appears that F-actin and myosin motors form an in-series network along the neuron to generate tension. This tension is responsible for sustaining the F-actin network at the synapse. The synaptic F-actin is then able to serve as a scaffold for vesicles, such that vesicles can stay clustered. This pathway allows tension to influence, and potentially regulate, vesicle clustering.
Issue Date:2018-09-28
Rights Information:Copyright 2018 Anthony Fan
Date Available in IDEALS:2019-02-07
Date Deposited:2018-12

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