Identifying Pathways that Regulate Flagellar Assembly

Keywords: Chlamydomonas reinhardtii, flagella assembly, kinase, length regulation

Introduction: Recent discoveries concerning cilia assembly suggest complex signaling
pathways play a prominent role in cilia length regulation and function.1 Far less is known about
the about the kinases that regulate these pathways. The proposed research will attempt to
uncover the signaling pathways responsible for growth and regulation by establishing which
kinases and mechanisms are responsible for the regulation of cilia length.
Background and Rationale: Cilia and flagella are found on almost every cell in the human
body and consist of microtubules that extend from the cell surface. Cilia are typically divided
into two types, primary and motile, which both sense extracellular signals. Primary cilia, found
on the majority of cells in the human body are immobile. Motile cilia, found on the majority of
epithelial cell surfaces, create wave-like patterns to propagate fluid. As flagella and motile cilia
have identical structures, the words cilia and flagella are used interchangeably.
The process of assembling cilia, ciliogenesis, is highly regulated as the centrioles that nucleate
cilia are also required for cell division. The mechanisms regulating ciliogenesis, including
initiation, assembly and resorption, are poorly understood. Learning more about these mechanisms
will facilitate the study and treatment of diseases involving ciliary dysfunction.
Flagella of the unicellular green alga Chlamydomonas reihardtii are essentially identical to
cilia of vertebrate cells and provide an excellent model
to study ciliogenesis. Chemical studies in
Chlamydomonas have demonstrated length-regulating
roles for G-protein coupled receptors2. Similarly,
flagella assemble in a length-dependent manner, with
rapid early assembly and very slow assembly as they
approach their steady-state length. The rate of
disassembly is length independent and the length at
which assembly and disassembly are in equilibrium is Figure 1. Preliminary kinase inhibitors with
known as the balance point3. To identify kinase increased rate of assembly during regeneration.
pathways that affect flagellar assembly, we performed
a small-molecule screen using a kinase inhibitor library. Preliminary data show inhibiting Protein
Kinase A and G causes an increased rate of flagellar assembly during regeneration over of 2 hours
as compared to wild type. Also, inhibiting Protein Kinase C causes a slower rate of assembly
during regeneration at 1 hour as compared to wild type (Figs. 1,2). We hypothesize these inhibitors
target regulators that control the switch from rapid to slow assembly rates. Reversing this switch
has the potential to rescue defects caused by slow or impaired assembly.
Aim 1: Validate Targets and Phenotypes with Novel
Inhibitors and Activators: To confirm phenotypes and
identify the kinases responsible for the observed
phenotypes, we will use different inhibitors of the same
targets. In contrast to the kinase inhibitors, the activation of
these kinases should show us the opposite effects,
confirming the targets and phenotypes previously identified
(Figs. 1,2). Following pH shock to induce flagellar loss and
regrowth, we will treat Chlamydomonas cells (CC125) with
inhibitors and activators of protein kinase A, G, C and Figure 2. Preliminary kinase inhibitors
tyrosine kinases (Table 1). Flagella will be imaged using with decreased rate of assembly during
regeneration.
automated phase contrast microscopy and flagellar length Table 1. Inhibitors and Activators of
measurements will be performed using ImageJ software. With proposed targets to be used in Aim 1
these experiments, we expect to confirm the data seen in
Figures 1 and 2 while helping us to further confirm these
kinases as regulators of the switch from rapid to slow
assembly.
Aim 2: Identify Regulatory Pathways for Flagellar
Assembly: As many of the kinases identified in the
preliminary screen have both cytoplasmic and nuclear
downstream targets, we will identify which subset of targets
are responsible for the flagellar assembly phenotype. To
discriminate between the targets, we will use inhibitors from
the preliminary screen simultaneously with cyclohexamide, a protein synthesis inhibitor that will
prevent gene expression in downstream transcription factors (Table 2). Next, we will perform an
epistasis experiment by inhibiting or activating a preliminary target as well as a potential
downstream targets to see if they are in the same pathway. The process of pH shock and flagellar
length measurement will be followed according to the steps described in Aim 1. These
experiments will narrow down the pathway components regulating the switch from rapid to
slower assembly rates.
Aim 3: Determine the Role of Identified Kinases in Trafficking of Flagellar Cargo: We will
use total internal reflection fluorescence (TIRF) microscopy to Table 2. Cytoplasmic and
determine the role of kinases in trafficking of flagellar cargo by Nuclear compounds to be used
assessing the preliminary targets’ effect on transportation of tagged for experiments in Aim 2.
proteins in regenerating flagella. We will treat the cells with the
kinase inhibitors during flagellar regeneration and use TIRF imaging
to compare the amount of cargo traveling from the base to the tip of
the regenerating flagella by quantifying fluorescence intensity of
tagged cargo.4,5 Results from this visualization and quantification of tagged cargo will identify the
mechanism with which identified pathways regulate flagellar assembly.
Intellectual Merit/Broader Impacts: My familiarity with the culture conditions and flagellar
phenotyping of the model organism Chlamydomonas reinhardtii will facilitate the proposed
experiments. I will gain the necessary skills to perform TIRF microscopy through future
mentoring from Dr. Avasthi. The initial microscopy work in the outlined project, allows
Rockhust University undergraduate students participating in research at the University of Kansas
Medical Center to be trained on microscopy. The findings from these experiments impact the
science community through the identification of fundamental principles of ciliary regulation.
Society is influenced by these findings as they will provide the foundation for the treatment of
diseases of ciliary dysfunction. Results will be shared in relevant conferences, preprints and
publications. Also, the proposed experiments and results, will be shared with undergraduate
students at Rockhurst University with the intention of using relevant basic science research to
engage future students. This will capture their attention and spark their interest for research
opportunities at the University of Kansas Medical Center. Support from the NSF through the
Graduate Fellowship Research Program will promote my success as a future scientist by
facilitating research during my graduate career, but will also benefit society by providing a more
approachable path to science careers for women.
[1] Nachury,Maxence V.(2014) Philosophical Transactions of the Royal Society B: Biological Sciences 369.1650
[2] Avasthi, Prachee et al. (2012). ACS Chemical Biology 7.5 [3] Marshall, Wallace F. et al. 2005.Molecular Biology
of the Cell 16.1 [4] Engel, Benjamin D. (2009) Method Cell Biol.93 [5] Avasthi, Prachee et al. (2014).Curr Biol. 24.
NSF Graduate Research Fellowship Program Brittany Jack
Personal Statement:
Life is challenging and plans can change in a split second. Hurricane Katrina became one of
those challenges during my first week of classes at Louisiana State University. This natural
disaster made an impact on my family and how I decided to pursue the next several years of my
life. Experiencing this was not an ideal way to begin my college career but provided strength that
I was unaware I possessed. I made the decision to finish the semester and take some time to help
my family rebuild after the storm. In order to keep busy, I began waiting tables and have
continued through my entire undergraduate career. I have witnessed both sides of success and
without a doubt know what I want to do for the rest of my life. I am committed to a future career
in scientific research and academia. Waiting tables was more than just a way to pay bills, I
believe it was fundamental in teaching teamwork, training skills, work ethic, as well as
leadership due to my role as head server at multiple restaurants. The leadership skills and
discipline I have learned while waiting tables have aided in success during my undergraduate
career and will continue in the future.
The motivation to pursue a career in the STEM field began when I was a young child
witnessing my Nonna suffer with rheumatoid arthritis. She was always taking medication for her
symptoms that appeared to give her relief but always seemed to result in another symptom or
aliment. Observing this continuous cycle left me wondering how the human body metabolizes
pharmaceuticals and how side-effects are determined to be safe. My idea of a career in the
STEM field has progressed into a more well-defined plan throughout my undergraduate career
and personal experiences. Now, my goal as a woman in the STEM field is to pursue a PhD. in
Cell Biology followed by post-doctoral research position and an academic position where I can
continue to do cutting edge scientific research and provide opportunities to women entering this
field.
Relevant Background (Intellectual Merit):
When I returned to college in 2011, I knew a career in STEM was in my future. The pivotal
moment in my undergrad career that unequivocally confirmed I wanted to pursue a career in
scientific research was after I made acetaminophen in Organic Chemistry lab. Following this
pivotal moment, I was determined to find a way to obtain a Biochemistry degree as a stepping
stone to my future career in STEM.
Attending a small university has offered the opportunity of being a teaching assistant for the
Organic Chemistry I and II lab since my sophomore year. During this time, I performed all
experiments with the lab professor before the students did to ensure the ease and reliability of the
experiments. In addition, I prepared all solutions necessary for completing experiments and was
responsible for the overall cleanliness of the lab.
In addition to teaching assistant, I have been a private tutor in Chemistry, Math, and Biology
since the end of my sophomore year. My role as a tutor allowed me to both solidify my
knowledge and help others. The leadership and effective teaching strategies I have acquired from
tutoring will aid in my success in academia in the future.
Building on my experience as a tutor, I began to work on a chemistry education research
project at Rockhurst University with Dr. Paula Morehouse. Primarily, the project involved
developing a concept-based pre-lab curriculum for Organic Chemistry. A secondary part of the
project is the assessment of the developed curriculum. I found myself mesmerized by the
research process as a whole and not with any specific topic. Over the course of four semesters, I
developed conceptual demonstrations for students to be completed before lab by identifying
concepts that students tend to have trouble understanding. These topics were identified through
personal experience with these topics and through literature reviews of chemistry education. In
addition, we have developed two surveys to be given to the students for evaluation and
satisfaction of mini-demos. The work from this project was presented at the ACS Midwestern
Regional conference as an invited oral presentation in the fall of 2015, where I was the only
undergraduate invited to give an oral presentation in the educational division of the conference.
Also, I presented a poster at the Festival of Student Achievement at Rockhurst University Spring
2016 and the Undergraduate Women in Physical Sciences Conference at the University of
Nebraska at Lincoln in Nebraska during the Fall of 2016. My work on this project will ultimately
lead to a first author publication likely to be published in the American Chemical Society Journal
of Chemistry Education.
The summer of 2015 I was accepted as an American Society of Pharmaceutical and
Therapeutics (ASPET) summer scholar which provided my first opportunity to work in a
scientific research laboratory where I was able to develop and perform scientific research. I
worked with Dr. Wen-Xing Ding at the University of Kansas Medical Center on the role of
parkin in autophagy and lipid droplet accumulation in alcoholic liver disease. Specifically, I
studied parkin and p53 expression in HEK293 cells and lipid droplet accumulation in alcohol fed
mice. I learned many skills such as, western blot, cell culture, protein isolation, BSA analysis,
microscopy, and quantification of lipid droplets using a method I developed. Lab meetings and
journal club became second nature after this experience. My time in Dr. Ding’s lab provided me
an invaluable experience toward my future as a scientist in academia.
Due to excitement following my experience in the laboratory at the University of Kansas
Medical Center, I participated in another project at Rockhurst with Dr. Mary Haskins which
began early in my junior year. Dr. Haskins and I developed a project to identify the prevalence of
parasitic eggs in dog parks in the metropolitan Kansas City area. This project involved my
development of a protocol for separating parasite eggs from dog feces and soil. After the
protocol was established we collected samples from several dog parks in the Kansas City metro
area and analyzed the samples. I presented a poster with my results at the Festival of Student
Achievement at Rockhurst University and the Missouri Academy of Science Annual Conference
both in the Spring 2016.
The summer of 2016 I returned to the University of Kansas Medical Center as a K-INBRE
summer scholar and worked in the lab of Dr. Prachee Avasthi on identifying pathways that
regulate flagellar assembly in an algal model system. The preliminary data obtained from my
independent work on this project is the basis of this graduate research proposal. The opportunity
to write and work through the grant writing process early in my career has given me an
advantage I am fortunate to have experienced. My results will be presented as a poster at the
2016 American Society of Cell Biology Annual Conference in San Francisco, California and will
ultimately lead to a first author publication in a relevant journal. This year, I plan to apply for the
Interdisciplinary Graduate Program in Biomedical Science at the University of Kansas Medical
Center and hope to rejoin Dr. Avasthi’s laboratory as a graduate student.
I received a number of honors and awards throughout my undergraduate career. I received
the Phi Theta Kappa scholarship and Dr. Don Gibbs Memorial science division scholarship from
Rockhurst University. I was also accepted into Phi Theta Kappa, Phi Lambda Upsilon, and
Sigma Pi Sigma honor societies. Due to demonstrated academic excellence, I was included on
the Dean’s List and President’s List. Through summer opportunities at the University of Kansas
Medical Center, I was selected as an ASPET summer scholar and a funded K-INBRE student
scholar. Finally, I served as the Vice-President of the American Chemical Society Rockhurst
Student Chapter. The opportunities provided to me through my continued hard work were vital
to becoming familiar with scientific research as well as my desire to pursue an academic career.
Broader Impacts:
In order to achieve my goal of providing opportunities to undergraduate students as well as
breaking barriers for women in the STEM field I have established a partnership between
Rockhurst University and the University of Kansas Medical Center. This partnership provides
the opportunity for cutting edge scientific research to undergraduates. The goal of this
partnership is to provide undergraduate students with an invaluable experience that includes
research opportunities, journal clubs, lab meetings, and grant writing at a facility performing
cutting edge scientific research which is necessary to be a competitive applicant at the time of
graduation.
I will return to Rockhurst as a guest speaker to introduce students to this program in their
“freshman in science” course, where I will present the results from the proposed experiments
outlined in the research plan. Students that were unable to take advantage of the research
opportunity early in their undergraduate career will also be approached later. Students will be
evaluated on the following criteria: reliability, level of interest and academic standing. Rockhurst
University has strong leadership of women in STEM including, Dr. Nancy Donaldson (Professor
and Department Chair of Physics), Dr. Laura Salem (Associate Professor and Department Chair
of Biology) and Dr. Liza Felzein, (Associate Professor of Biology, Division Chair of Natural,
Applied and Quantitative Sciences). These women played a critical role in my development as a
student and with my interest in a career in STEM. They will continue to engage undergraduate
women by offering an outlook on the importance of STEM as a career and research opportunities
available through Rockhurst University and the University of Kansas Medical Center. Through
continued interaction with partners at Rockhurst, I will be involved with the selection process of
interested students, who will be placed with a mentor at the University of Kansas Medical Center
where the student will be given a project to work on with their mentor for the duration of their
time as a student at Rockhurst University, in order to facilitate the completion of scientific
projects and acquiring the necessary research skills to be competitive at the time of graduation.
Working hand in hand with professors at both Rockhurst University and the University of
Kansas Medical Center provides an opportunity to help undergraduate students work early in
their career on undergraduate research that is crucial to being a competitive applicant for STEM
careers in the future. My goal is to create a more equal playing field for women by providing
opportunities and participating as a role-model that can demonstrate scientific research as a
career option.
Future Goals:
I hope to obtain an academic position in a university in the field of basic science research for
a two simple reasons: I enjoy both research and teaching. My goal to pursue a career in research
stems from internal motivation to continue learning and be at the forefront of scientific
knowledge. Teaching other people has become a part of my life over the years and I delight in
witnessing others make connections of complex material and hope to continue this in the future.
Obtaining a doctorate degree in Cell Biology is a stepping stone to my future career as an
academic researcher. Funding from the NSF GRFP will provide a solid foundation for my
lifelong commitment to research and education, with a particular emphasis to promote research
opportunities for women in STEM. Throughout my life, I have had a strong desire to help people
and I look forward to providing opportunities for students while pursuing a career that I love.