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GENETICALLY-ENCODED PROBES FOR VISUALIZING PROTEIN
TRAFFICKING DYNAMICS IN LIVING NEURONS
by
Rudy J Mora
A Dissertation Presented to the
FACULTY TO THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
May 2012
Copyright 2012 Rudy J Mora
Object Description
| Title | Genetically-encoded probes for visualizing protein trafficking dynamics in living neurons |
| Author | Mora, Rudy J. |
| Author email | rudymora@usc.edu;littleboots1@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Neuroscience |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-03-27 |
| Date submitted | 2012-04-17 |
| Date approved | 2012-04-17 |
| Restricted until | 2012-04-17 |
| Date published | 2012-04-17 |
| Advisor (committee chair) | Arnold, Donald |
| Advisor (committee member) |
Butler, Samantha Liman, Emily Roberts, Richard |
| Abstract | Here I describe the development of genetically-encoded probes for visualizing the trafficking of neuronal proteins in living cells. The probes are derived from a diverse library of small fibronectin domains that resemble antibodies with the advantage of containing no disulfide bridges which facilitates proper expression and folding inside cells. Using the directed evolution technique mRNA display I selected fibronectins that specifically label the voltage-gated potassium channel Kv4.2 auxiliary subunit KChIP and Ca2+/Calmodulin-dependent Kinase II (CaMKII) α, proteins both involved in the regulation of long-term potentiation. As a fusion to GFP the CaMKIIα probe reveals the rapid translocation of kinase to post-synaptic densities upon excitatory stimulation. It also reveals a hitherto unreported localization pattern of large kinase clusters in the cell body. These cell body CaMKIIα clusters rapidly and reversibly disperse upon excitatory stimulation in a Ca2+-dependent manner and may represent a new mechanism by which neurons localize inactive CaMKIIα. These Fibronectin intrabodies generated by RNA display (FingRs) combine the advantages of recombinant DNA such as fluorescent fusion tags with the ability to visualize endogenous proteins. This technology is a leap forward in visualizing the trafficking dynamics of native proteins in living cells. |
| Keyword | probe; calcium; traffic; neuron; CaMKII |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Mora, Rudy J. |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume1/etd-MoraRudyJ-616.pdf |
Description
| Title | Page 1 |
| Full text | GENETICALLY-ENCODED PROBES FOR VISUALIZING PROTEIN TRAFFICKING DYNAMICS IN LIVING NEURONS by Rudy J Mora A Dissertation Presented to the FACULTY TO THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (NEUROSCIENCE) May 2012 Copyright 2012 Rudy J Mora |
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