The Biogenic Pathways of Malignant and Non-Malignant Microvesicles

Taylor, Jack Dylan

The Biogenic Pathways of Malignant and Non-Malignant Microvesicles

Taylor, Jack Dylan

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Publication Type:: Thesis
Issue Date:: 2019

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Field	Value	Language
dc.contributor.author	Taylor, Jack Dylan
dc.date.accessioned	2020-08-21T02:06:38Z
dc.date.available	2020-08-21T02:06:38Z
dc.date.issued	2019
dc.identifier.uri	http://hdl.handle.net/10453/142275
dc.description	University of Technology Sydney. Graduate School of Health.	en_AU
dc.description.abstract	Plasma membrane-derived extracellular vesicles (EV) are released for most cells types and are important mediators of intercellular communication. In the context of cancer, EVs mediate the intercellular transfer of deleterious traits including multidrug resistance (MDR), enhanced metastatic capacity, evasion of immune surveillance, and altered tissue biomechanics. The biogenesis of plasma membrane EVs is characterised by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. However, the precise biogenic mediators and differences between malignant and non-malignant plasma membrane-derived EV biogenesis remain undefined. Uncovering discrete pathways of biogenesis in malignant and non-malignant cells holds potential in the search for new strategies to circumvent the acquisition and transfer of plasma membrane EV-meditated deleterious traits in cancer. In this thesis, the molecular pathways that regulate plasma membrane EV biogenesis in malignant and non-malignant cells are investigated. This study evidences that discrete EV biogenic pathways exist in malignant and non-malignant cells. At rest, biogenesis of cancer EVs is regulated by a calcium-calpain dependent pathway whereas non-malignant biogenesis is driven by alternative pathways. Comparing the surface topography of malignant and non-malignant cells using atomic force microscopy demonstrates malignant cells have intrinsically higher vesiculation at rest and this is shown to be driven by high basal calcium mobilisation. Interrogating the calcium signalling pathways that regulate biogenesis with pharmacological modulators of calcium revealed increases in free cytosolic Ca²⁺ via endoplasmic reticulum (ER) Ca²⁺ store depletion with thapsigargin increases EV biogenesis in both malignant and non-malignant cells. Evidencing a role for the ER in plasma membrane EV biogenesis for the first time. Furthermore, the store-operated calcium entry (SOCE) plays an essential role in the maintenance of plasma membrane EV biogenesis after store depletion. The calcium-calpain pathway is also a known regulator of αII-spectrin structural dynamics and this work evidences that it plays a role in increased vesiculation in malignant cells. High resolution confocal microscopy in combination with a custom designed, automated image analysis plugin, provided an unbiased platform to show that the subcellular localisation of spectrin is distinctly different in malignant and non-malignant cells at rest. Non-malignant cells display prominent αII-spectrin borders and are low vesiculators. It is the redistribution of αII-spectrin from the membrane to the cytoplasm that drives plasma membrane-derived EV biogenesis in malignant cells. These findings provide new insight into the biogenic pathways regulating EV biogenesis in both malignant and non-malignant cells and identifies strategies for the selective modulation of plasma membrane EV biogenesis in malignancy.	en_AU
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/142275/2/02whole.pdf
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	The Biogenic Pathways of Malignant and Non-Malignant Microvesicles	en_AU
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Plasma membrane-derived extracellular vesicles (EV) are released for most cells types and are important mediators of intercellular communication. In the context of cancer, EVs mediate the intercellular transfer of deleterious traits including multidrug resistance (MDR), enhanced metastatic capacity, evasion of immune surveillance, and altered tissue biomechanics. The biogenesis of plasma membrane EVs is characterised by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. However, the precise biogenic mediators and differences between malignant and non-malignant plasma membrane-derived EV biogenesis remain undefined. Uncovering discrete pathways of biogenesis in malignant and non-malignant cells holds potential in the search for new strategies to circumvent the acquisition and transfer of plasma membrane EV-meditated deleterious traits in cancer. In this thesis, the molecular pathways that regulate plasma membrane EV biogenesis in malignant and non-malignant cells are investigated. This study evidences that discrete EV biogenic pathways exist in malignant and non-malignant cells. At rest, biogenesis of cancer EVs is regulated by a calcium-calpain dependent pathway whereas non-malignant biogenesis is driven by alternative pathways. Comparing the surface topography of malignant and non-malignant cells using atomic force microscopy demonstrates malignant cells have intrinsically higher vesiculation at rest and this is shown to be driven by high basal calcium mobilisation. Interrogating the calcium signalling pathways that regulate biogenesis with pharmacological modulators of calcium revealed increases in free cytosolic Ca²⁺ via endoplasmic reticulum (ER) Ca²⁺ store depletion with thapsigargin increases EV biogenesis in both malignant and non-malignant cells. Evidencing a role for the ER in plasma membrane EV biogenesis for the first time. Furthermore, the store-operated calcium entry (SOCE) plays an essential role in the maintenance of plasma membrane EV biogenesis after store depletion. The calcium-calpain pathway is also a known regulator of αII-spectrin structural dynamics and this work evidences that it plays a role in increased vesiculation in malignant cells. High resolution confocal microscopy in combination with a custom designed, automated image analysis plugin, provided an unbiased platform to show that the subcellular localisation of spectrin is distinctly different in malignant and non-malignant cells at rest. Non-malignant cells display prominent αII-spectrin borders and are low vesiculators. It is the redistribution of αII-spectrin from the membrane to the cytoplasm that drives plasma membrane-derived EV biogenesis in malignant cells. These findings provide new insight into the biogenic pathways regulating EV biogenesis in both malignant and non-malignant cells and identifies strategies for the selective modulation of plasma membrane EV biogenesis in malignancy.

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