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Trafficking and Activity Dependent Function of Vesicular Transporters

Colgan, Lesley Anne (2009) Trafficking and Activity Dependent Function of Vesicular Transporters. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Vesicular neurotransmitter transporters (VNTs) are a small family of proteins responsible for packaging neurotransmitter into secretory vesicles. Their presence and function are required for regulated secretion from neuronal and neuroendocrine cells. During both the biogenesis and the activity-dependent recycling of secretory vesicles, VNTs undergo trafficking that can determine the quality, quantity, and location of packaged neurotransmitter. Thus understanding the signals and mechanisms of VNT trafficking is essential to understanding the regulation of neurotransmission. Here, the synaptic vesicle specific trafficking of Vesicular Acetylcholine Transporter (VAChT) is investigated. A dileucine containing targeting motif, with dual properties for internalization and synaptic vesicle targeting, is identified in the C-terminus of VAChT. Chimeras between this motif and an unrelated plasma membrane protein localize to synaptic-vesicle-like vesicles in a neuroendocrine cell line. The specificity and generalization of this motif is assessed. Next, sorting nexin 5 (SNX5), implicated in the regulation of membrane traffic, is identified as a novel regulator of VAChT targeting to synaptic vesicles. Disruption of SNX5 function leads to a decrease in VAChT-directed synaptic vesicle targeting and a concomitant increase in targeting to large dense core vesicles. This shift between secretory granules suggests an important mechanism of VNT regulation with the potential to shape properties of neurotransmission. In order to understand the physiologic importance of VNT regulation, vesicular transport and its influence on activity-dependent release must be assessed in living neurons. However, this has not been possible. Therefore, a live cell assay was established to measure vesicular transport and its contributions to release in brain slice. Using a pH sensitive, fluorescent serotonin analog visualized by two-photon microscopy, activity dependent somatic release and vesicular monoamine transporter (VMAT) activity were measured in the dorsal raphe nucleus. Interestingly, while a portion of monoamine packaged at rest was held in reserve, monoamine packaged during stimulation was released efficiently. The work presented in this thesis provides a greater understanding of VNT trafficking and activity-dependent function. Furthermore, it provides the foundation for the comprehensive study of the active role of VNTs in shaping the properties of neurotransmission.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Colgan, Lesley Annelac31@pitt.eduLAC31
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAmara, Susanamaras@pitt.eduAMARAS
Committee MemberMichael, Adrianamichael@pitt.eduAMICHAEL
Committee MemberLevitan, Edwinlevitan@pitt.eduLEVITAN
Committee MemberTorres, Gonzalogtorres@pitt.eduGTORRES
Committee MemberHorn, Johnjph@pitt.eduJPH
Committee MemberMartin,
Committee MemberLiu, Yongjianyjliu@pitt.eduYJLIU
Date: 11 March 2009
Date Type: Completion
Defense Date: 23 February 2009
Approval Date: 11 March 2009
Submission Date: 26 February 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Neurobiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: 57-dihydroxytryptamine; dHT; multiphoton microscopy; secretory granules; synaptic vesicle cycle; neurotransmitter release; imaging; neurotransmitter dynamics; somatodendritic release; fluoxetine; PC12
Other ID:, etd-02262009-143623
Date Deposited: 10 Nov 2011 19:31
Last Modified: 15 Nov 2016 13:36


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