development of multiplexable biosensors to quantify the spatiotemporal dynamics of rho gtpases ...

252 Pages·2012·11.34 MB·English

DEVELOPMENT OF MULTIPLEXABLE BIOSENSORS TO QUANTIFY THE
SPATIOTEMPORAL DYNAMICS OF RHO GTPASES AND PROTEIN KINASES
IN THE SAME LIVING CELL

Chia-Wen Hsu

A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill
in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the
Division of Chemical Biology and Medicinal Chemistry at the Eshelman School of
Pharmacy

Chapel Hill
2012

Approved by:
Klaus M. Hahn, Ph.D.
David S. Lawrence, Ph.D.
Alexander Tropsha, Ph.D.
Brian Kuhlman, Ph.D.
Qisheng Zhang, Ph.D.
© 2012
Chia-Wen Hsu
ALL RIGHTS RESERVED
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ABSTRACT
CHIA-WEN HSU: Development of Multiplexable Biosensors to Quantify the
Spatiotemporal Dynamics of Rho GTPases and Protein Kinases in the Same Living Cell
(Under the direction of Dr. Klaus M. Hahn)

Cell motility is a highly dynamic and heterogenous cellular process regulated by the
coordination of multiple Rho GTPases, Src family kinases, and the mitogen-activated
protein kinase (MAPK) cascades. However, it has been difficult to monitor more than
two protein activities in the same cell due to the overlapping spectra of current biosensors
and biological perturbations at high biosensor concentrations. Dye-based biosensors,
which rely on an affinity scaffold that binds only to the activated conformation of the
endogenous targets and an environment-sensing dye that changes its fluorescence
properties to report the specific binder-target interactions, possess great potential to
monitor multiple endogenous targets in the same cell. Here, I created novel environment-
sensing dyes and exploited novel affinity scaffolds to develop multiplexable dye-based
biosensors capable of quantifying the spatiotemporal dynamics of multiple Rho GTPases
and protein kinases in the same cell.
Src protein kinase is an upstream regulator of the Cdc42 GTPase. The coordination of
Cdc42 and Src at the leading edge has not been well characterized due to lack of
multiplexable biosensors to monitor Cdc42 and Src activities in the same living cell.
Therefore, I developed novel near infrared merocyanine dyes and a red ratiometric
merocyanine dye with an intrinsic ratiometric response that can be used to construct
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multiplexible biosensors. The relative timing and the subcellular localization of active
Cdc42 and Src during leading edge dynamics and during pinocytosis were revealed using
the new dyes.
Src also plays an important role in the MAPK-mediated cell motility. However, the
precise roles of MAPKKs and MAPKs at the leading edge remain poorly characterized
due to the lack of sensitive biosensors for each target. By taking advantage of the specific
interactions between MAPKKs and MAPKs, I developed the first substrate-based
biosensor designs to report the activity of endogenous MEK1/2 and MKK3/6. I also
developed a sensitive ERK1/2 biosensor based on artificial binders through
collaborations with the Plűckthun group.
This work will provide a foundation to study the crosstalk between Rho GTPases, Src
family kinases and the MAPK cascades via multiplexed live cell imaging.

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DEDICATION
To my parents Chun-Tseng Hsu and Yu-Yun Lin for their constant support and care
throughout out my life.

To my husband Wei Sun for his unlimited love throughout my graduate studies.
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ACKNOWLEDGEMENTS
First I would like to express my most sincere thanks to my advisor Prof. Klaus Hahn.
His great insight, patience, and critical thinking have nurtured me to be a better scientist.
I am happy to have so many exciting, challenging, and highly interdisciplinary projects
that forced me to exploit my creativity as much as I can and immerse myself in various
disciplines. As a foreign student, I am fortunate to have an advisor like Prof. Hahn who
is willing to take his time to improve my writing and presentation skills. More
importantly, because of his close mentorship, I have become a chemist who not only
knows how to make useful tools but also knows how to quantify cell biology to extract
new information.
I also truly appreciate the support and the valuable advice from my doctoral
committees: Prof. Alex Tropsha, Prof. Brian Kuhlman, Prof. David Lawrence, and Prof.
Qisheng Zhang. I thank Prof. Tropsha and his former student Yetain Chen for their
computation model of ERK that gave us a good starting point of biosensor designs. I am
grateful to have a joint journal club with Prof. Kuhlman and his group, where I learned a
lot about photosensory proteins and generated many ideas when designing MEK
biosensors. I truly appreciate the critical questions and the advice from Prof. Lawrence. I
still remember that he told me to be brave when I was about to give up the MEK
biosensor project. I thank Prof. Zhang for his kindness to let me work as his teaching
assistant during my fifth year.
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My graduate life could not be so colorful without my brilliant and hardworking
labmates: Chris MacNevin, Li Li, Dmitriy Gremyachinskiy, Yi Wu, Hui Wang, Jason Yi,
Marie Rougie, Jon Zawistowski, Scott Slattery, Chris Welch, Brian Mehl, Andrei
Karginov, Peihsuan Chu, Onur Dagliyan, Jianrong Wu, Akash Gulyani, Evan Trudeau,
Xin Zou, Tamara Davis, and Janet Doolittle. I benefited a lot from daily interactions with
these “in-house” experts with diverse backgrounds and critical thinking. I give my
special thanks to Dr. Chris MacNevin. I feel comfortable to work on chemistry-related
projects in a cell biology lab because I know Chris is always there and ready to answer
my chemistry problems throughout my graduate studies. I am very grateful to have
Dmitriy Gremyachinskiy to be my rotation mentor, who trained me all the essential
chemistry skills as well as lab management practice within the intensive six months of
our overlap. I truly thank Dr. Li Li for taking her time and for her patience to teach me
how to do microinjection, bead loading, and microscopy. I also enjoyed our extensive
collaborations and productive discussions. I thank Marie Rougie for teaching me the
techniques of cell culture, microscopy and imaging processing. I would like to thank Dr.
Yi Wu for guiding me into protein engineering and for his continued encouragement
when I worked on the caging project. I thank Dr. Hui Wang for teaching me molecular
cloning skills, and for providing me many advice and remarkable ideas during
troubleshooting. I thank Evan Trudeau for helping me prepare some of the ERK
biosensor proteins and clone all the p38 mutant constructs. I thank Dr. Jianrong Wu and
Dr. Scott Slattery for helping me understand the scripts in the MetaMorph and the Matlab
routines. I thank Onur Dagliyan for his expertise in molecular modeling. I thank Dr. Yi
Wu, Dr. Chris Welch, and Dr. Andrei Karginov for generation of several valuable stable
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cell lines that I found very useful for characterization and applications of my new dye-
based biosensors. I truly thank Dr. Jason Yi who frequently shares interesting science
stories and makes me more positive about science. I especially thank Dr. Chris
MacNevin, Dr. Jon Zawistowiski, Dr. Scott Slattery, and Dr. Li Li for reviewing this
dissertation.
Many of the great works I could not have accomplished without my collaborators. I
was very happy to work with Dr. Alexei Toutchkine on the ratiometric merocyanine
dyes, because it was his astounishing work on dyes that allured me to do my second
rotation in the Hahn laboratory. I really appreciate the collaboration work on the
optimization of red merocyanine dyes with Dr. Dmitriy Gremyachinskiy, Dr. Chris
MacNevin, and Dr. Li Li. This work set a solid foundation for the design of my near
infrared merocyanine dyes. Dmitriy also contributed to the intial design and the synthesis
of near infrared dyes. I also thank the collaboration efforts on the ERK biosensor project
with Dr. Lutz Kummer in the Plϋckthun group at the University of Zurich. They
discovered and characterized a specific artificial binder for phosphorylated ERK that our
group has been wanted for more than ten years. I strongly appreciated the support and the
fruitful discussions from Dr. Michelle Mendoza in the Blenis group at the Havard
University. Most of the valuable constructs and proteins for the MEK biosensor project
were generous gifts from them. I thank Dr. Elizabeth Hinde in the Gratton group at the
University of California at Irvine on the FLIM projects. She conducted all the in vitro
lifetime measurements. I also appreciate the joint efforts on FLIM with Dr. Li Li, Dr.
Brain Mehl, and Marie Rougie in the Hahn group. The addition of Brian to our lab has
enabled us to build a better FLIM scope and image analysis routines. Thus I was able to
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obtain the first FLIM image of dye-based biosensors with his help. I thank Dr. Tilo
Mathes in the Hegemann group for providing valuable reagents and for his support in the
caging project. I also thank Dr. Arlene Bridges and Dr. Wanda Bodnar for their
assistance in mass spectrometry.
Lastly, I would like to express my greatest thanks to my family and friends for their
endless love and support. Everytime I get together with them in Taiwan, I feel fully
recharged and get lots of inspirations in both science and life. I am very fortunate to have
warm-hearted parents—Chun-Tseng Hsu and Yu-Yun Lin who only wish me to be happy
and healthy, have a cheerful brother Shao-Kai Hsu who helps me take care of my parents
while I am studying abroad, and have Wei Sun to be my husband who shares all sorts of
ups and downs and works long hours together throughout my graduate studies. I
especially thank the continued encourgement from my previous undergraduate advisor,
Prof. Chao-Tsen Chen at the National Taiwan University. It was a great pleasure to join
her group since sophoremore. Most of my synthetic skills and my knowledge of dye
chemistry were established through the hardcore training in her group. I also especially
appreciate the kindness of Dr. Kuo-Hsiung Lee to give me the opportunity to join this
program and to conduct my first rotation in his laboratory. I thank Prof. Michiyuki
Mastuda at the Kyoto University, Prof. Peter Hegemann at the Humboldt University at
Berlin, and Prof. Chun-Hung Lin at the Academic Sinica for inviting me to present my
work at their institutes during my graduate studies. I also appreciate the critical questions
and valuable suggestions I got during my postdoctoral interviews in the laboratories of
Dr. Jin Zhang, Dr. Ryohei Yasuda, Dr. Loren Looger, Dr. Luke Lavis, Dr. Deborah
Morrison and Dr. Menghang Xia. I thank the knowledgeable teachers—Dr. Nigel Caplan,
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Dr. Gigi Taylor, and the active members in the writing group—Congying Wu, Hung-
ching Hsia, Xin Zou, Peihsuan Chu, Zaozao Chen and Wei Sun for their help in
improving my academic writing skills. I thank Chen-Yu Hsieh, Po-hung Wang, and Dr.
Feng Liu for their chemistry expertise and for constant encouragement throughout my
graduate studies. I especially thank Yin-Hsuan Lin, Peihsuan Chu, Jin Hong and Chih-Da
Wu for their friendship and their support throughout my time in Chapel Hill.

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