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Permanent link (DOI): https://doi.org/10.7939/R3942S

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Characterization of GBF1, Arfs and COPI at the ER-Golgi intermediate compartment and mitotic Golgi clusters Open Access

Descriptions

Other title
Subject/Keyword
GBF1
Exo1
BFA
immunofluorescence
mitosis
Golgi complex
live cell microscopy
COPI
brefeldin A
ADP ribosylation factor
Arf
ERGIC
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Chun, Justin
Supervisor and department
Melancon, Paul (Cell Biology)
Examining committee member and department
Hendzel, Michael (Oncology)
Rachubinski, Richard (Cell Biology)
Bergeron, John (Anatomy and Cell Biology)
Eitzen, Gary (Cell Biology)
Department
Department of Cell Biology
Specialization

Date accepted
2009-07-13T20:37:33Z
Graduation date
2009-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.
Language
English
DOI
doi:10.7939/R3942S
Rights
License granted by Justin Chun (jc@ualberta.ca) on 2009-07-13T07:10:10Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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Last modified: 2015:10:12 15:34:38-06:00
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File title: preface to pg 69 b.pdf
File title: Microsoft Word - First pages for defense July 9 2009
File author: Justin Chun
Page count: 188
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