Author: Salha Alshumrani
Alshumrani, Salha, 2015 The role of dynamin, actin/myosin II and clathrin in controlling catecholamine release from mouse adrenal chromaffin cells, Flinders University, College of Medicine and Public Health
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Exocytosis is a vital cellular process that regulates hormone or neurotransmitter release. This release occurs mainly via intracellular vesicles that fuse with the cell membrane. This fusion creates a pore that enables the vesicles to release their contents into the extra-cellular space. The fusion pore is a channel created between the vesicle and the cell membrane and it can undergo a secondary expansion in response to multiple factors. The mechanism of controlling the fusion pore expansion (FPE) is poorly understood; however, the dynamin protein has been recently discovered to be a key player in FPE. Dynamin is a GTP-ase that plays a well-known role in endocytosis and which can self-assemble and oligomerize into helices or rings. Through this GTP-ase activity, dynamin directly acts on the fission reaction at the neck of clathrin-coated pits to generate a free endocytic vesicle. Dynamin function in vivo depends on its ability for efficient GTP hydrolysis and its oligomerization, and this GTP-ase activity may be responsible for the regulation of fusion pore expansion. However, there is not enough research that shows dynamin as a significant part in the exocytosis process. Furthermore, there are other proteins that may have a critical role in assisting dynamin function in FPE. One of these important binding proteins are the actin/myosin II cytoskeleton; however, the interaction mechanism between dynamin and actin to control FPE is unclear. Additionally, clathrin, a coat-protein that recruits dynamin to retrieve vesicles during endocytosis, is also thought to play a role in exocytosis; however, there are no current studies to show its participation in the FPE. In this project we had access to novel small molecules that stimulate dynamin through their GTPase activity. Dynamin’s interaction with the actin/myosin II motor proteins and the involvement of clathrin in FPE was explored by using different chemical effectors. Mouse chromaffin cells were exposed to these chemical effectors, and the kinetics of vesicle release was measured using carbon fiber amperometry before and after treatment with these modulators. This study found that quantal transmitter release increased significantly upon dynamin activation, and less during dynamin inhibition. This may indicate that dynamin alteration directly regulates the amount of catecholamine released per fusion event by either making the fusion pore open wider or for longer periods of time. Dynamin’s ability to control FPE is assisted by actin/myosin II cytoskeleton, possibly by providing enough force on the vesicle to accelerate and evacuate vesicle content. For the first time, even though it is an endocytosis protein, clathrin has been found to play a role in exocytosis, either by controlling the recycling process of the vesicles or by interacting with actin/myosin II proteins.
Keywords: Exocytosis, Dynamin, Actin, Myosin II, Clathrin, fusion pore, fusion pore expansion, Catecholamine, Chromaffin Cells, amperometry, dynamin activation, Ryngo, dynamin inhibition, Dynole
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2015
School: College of Medicine and Public Health
Supervisor: Damien Keating