During the development of atherosclerosis, contractile vascular smooth muscle cells (VSMCs) change to cells capable of migrating and proliferating to mediate repair, where the responses may be adaptive or mal-adaptive in effect. Cyclic adenosine monophosphate (cAMP)-elevating agents have been shown to inhibit migration of VSMC. cAMP activity within the cell is known to be ubiquitous and dynamic, requiring control through signal termination mechanisms for cellular homeostasis. Phosphodiesterase (PDE) enzymes are central to this critical regulatory process catalyzing the hydrolysis of cAMP. A great deal of insight into the role of PDEs in defining compartmentalization of cAMP signaling has arisen predominately from recent studies on the cAMP-specific PDE4 family. Compartmentalization of PDE4 is mediated by their unique N-terminal domains, which have been proposed to provide the “postcodes/zipcodes” for cellular localization. PDE4D isoforms vary widely, yet their conservation over evolutionary time suggests important non-redundant roles in distinct cellular processes. To study the potential role of individual PDE4D isoforms we seek to utilize the unique N-terminal targeting domains that are proposed to be responsible for their protein-protein interactions and site-directed localization. Herein, we report on the expression, targeting and localization of five “long” PDE4D isoforms and the impact on cell morphology of certain amino-terminal domains of individual PDE4D constructs expressing green fluorescent protein (NT-PDE4D/GFP) in human aortic smooth muscle cells (HASMCs). Through the development of engineered NT-PDE4D/GFP expression plasmids, we were able to study the cell biological impacts associated with the overexpression of individual PDE4D amino-terminal variants in HASMCs. We show that NT-PDE4D5/GFP and NT-PDE4D7/GFP expressing cells exhibited an elongated cell morphology, where this effect was much more marked in NT-PDE4D7/GFP expressing cells, exhibiting multiple leading edge structures and highly elongated “tails”. We identify a potential role for PDE4D7 targeting in the regulation of cell polarity and migration. Our results suggest the novel idea that PDE4D7, rather than the four other long PDE4D isoforms (PDE4D3, PDE4D5, PDE4D8, or PDE4D9), represents the dominant PDE4D variant involved in controlling cAMP-mediated effects on cell tail retraction dynamics. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2014-03-13 13:00:31.684 / Video I: Time-lapse video of GFP-expressing cell migration in HASMC. GFP expressing cells did not differ in cell migration or morphology compared to non-injected control cells. HASMCs were microinjected with GFP construct. Representative images of micoinjected GFP cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X) / Video II: Time-lapse video of NT-PDE4D7/GFP-expressing cell migration in HASMC. NT-PDE4D7/GFP expressing cells exhibit elongated tail and decrease in cell migration compared to non-injected control cells. HASMCs were microinjected with NT-PDE4D7/GFP construct. Particle tracking of NT-PDE4D7 cells showed cleaving and full detachment of elongated tail. Representative images of micoinjected NT-PDE4D7 cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X)
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/8658 |
| Date | 14 March 2014 |
| Creators | Truong, Tammy |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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