Download Exploration of PAR2-like Proteins present in Culex Mosquitoes

Exploration of PAR2-like Proteins present in Culex Mosquitoes using the CRISPR/Cas9 System.
Scientific Merit:
A PAR2-like protein is hypothesized to be responsible for activation of sperm motility. PARs are GPCRs that are
activated through the cleavage of a specific protease (Cottrell et al 2003). In this pathway, the N-terminus is cleaved
at a conserved amino acid sequence, which exposes a new N-terminus that forms a tethered ligand that binds to a
receptor on the second loop of the seven transmembrane domain resulting in receptor activation (Yau et al 2013).
PAR2 is cleaved by trypsin between the arginine and serine residues of an RSLIGKV (human) or an RSLIGRL
(mouse) sequence (Cottrell et al 2003) and receptor activation stimulates MAPK signaling via a β-arrestindependent mechanism (Cottrell et al 2003; Ramachandran et al 2009). Although PAR2 has been implicated in a
number of physiological processes in mammals, it has only recently been discovered in invertebrates where it plays
a behavioral role in the activation of insect sperm motility. In Culex quinquefasciatus and Aquarius remigis, a
trypsin-like molecule was discovered to initiate sperm motility in vitro and Culex accessory glands contain a trypsinlike protease (Miyata et al 2012; Thaler et al 2014). In A. remigis, trypsin activates sperm, which induces the
MAPK-pathway by presumably phosphorylating axonemal proteins to stimulate motility. Further evidence of a
PAR2-like protein being cleaved by trypsin to stimulate activation is the presence of a PAR2-like protein on the A.
remigis sperm flagellum detected by an anti-PAR2 antibody directed at the N-terminus of the mouse PAR2. This
particular method of sperm motility activation is, to date, unique to insects.
This project will allow me to examine the activation of a PAR2 pathway in a biologically tractable system. Insect
sperm have a unique advantage over many other cell types, mainly, the activation of an easily detectable and
quantifiable behavior, activation of flagellar motility. The focus of this project will be on the activation of the
MAPK pathway via PAR2 cleavage in Culex spp. sperm and to demonstrate that PAR2-like proteins are involved in
sperm activation. The only insect in which a PAR2-like protein has been detected is Aquarius remigis where it is
hypothesized to be involved in sperm motility activation. Culex spp. sperm are a good system to study the
involvement of PAR2-like proteins because it is a tractable system with a sequenced genome, which will be helpful
for utilizing insect genetic technologies such as CRISPR/cas9 to study pathways involved in sperm activation. The
sperm have a unique motility pattern, specifically the sperm cycle through three separate and distinctive waveforms
(Thaler et al 2014). The three wave forms consist of a low amplitude, long wavelength form (Wave A), a double
waveform with two superimposed waveforms over the length of the flagellum (Wave B) and a helical waveform that
propels the sperm at high velocity (Wave C; Thaler et al 2014). These waveforms can be monitored after knocking
out genes using CRISPR/cas9 to determine if these genes are involved in motility activation or regulation.
Goals:
The major question being addressed in this proposal is: are PAR2-like proteins that are present on the sperm of
Culex involved in sperm motility activation? My hypothesis is that a PAR2-like protein is involved in sperm
motility activation in Culex because in another insect, A. remigis, sperm motility is activated by trypsin leading to
the initiation of a MAPK pathway and A. remigis sperm flagellum has PAR2 present as evidenced by
immunofluorescence studies (Miyata et al 2012). In addition, our laboratory has recently shown that, Culex sperm
can be activated by both a male reproductive tract accessory gland extract and purified trypsin (Thaler et al 2014)
and that a trypsin-like molecule is present in the accessory glands. If successful, results from this project will
demonstrate a role for PAR2-like proteins in initiating or regulating sperm motility in insects. Potential PAR2-like
proteins will be verified to be transcribed in the testis via qPCR and packaged into the sperm via mass spectrometry.
I will then determine if these proteins are involved in motility activation and regulation by injecting Culex
mosquitoes with CRISPR/Cas9 constructs designed specifically against genes that encode PAR2-like proteins that
are transcribed in the testis and packaged into the sperm. In order to do this, I will require training in needle pulling,
vector construction, microinjection, and insert confirmation, which can be provided by the host lab.
Significance to the Applicant and Host Laboratory
Collaboration between the two labs will allow for the development of protocols in Culex mosquitoes that will be
useful for exploring the significance of PAR2-like proteins in insect sperm. Developing the tools for Culex
transgenics will allow me to not only investigate the problem I am currently working on but to also utilize the
techniques developed through this project for potential projects in the future. As I move forward in my career, I will
not only be able to further utilize the skills learned through this collaboration but also will be able to train/assist
colleagues with learning these techniques. I will also gain a skill set that will allow me to develop transgenics in
other insect species. I am currently finishing up a PhD program and will be able to train members in the lab I am
currently in on the techniques I learn. I will also be able to take these techniques with me when I move on to a
different lab. Collaboration between the Cardullo lab and the IBBR's Insect Transformation Facility (ITF) will allow
for the facility we use to broaden the repertoire of transgenic services that they are able to provide. The ITF is a
great resource for developing new modification technologies in new research organisms while also providing
training in established insect genetic modification models. All the resources and expert personnel necessary to
transform insect species are available at the ITF facilities. Some of these resources include strain production,
microinjection and characterization. Namely, the facilities will be able to develop transgenic methods for Culex
mosquitoes that will allow them to assist in a wider variety of projects as the tools for producing transgenic Culex
mosquitoes will have been developed.
Activity Plan and Timetable
I will perform experiments to determine if the PAR2-like proteins (and the cleavage of the canonical RSLIGRL
sequence) are involved in sperm motility activation after the PAR2-like sequences that are expressed in the testes
and packaged in the sperm have been identified (this work is in progress). It is important to experimentally validate
that trypsin cleavage of the PAR2-like protein is involved in motility activation. In order to do this, the CRISPRcas9 system will be used. Primers for gRNA will be designed using a gRNA design tool available online and E. coli
will be used to produce gRNA. MaxiPrep will be used to isolate the plasmid DNA. For cas9 to bind, the genomic
target sequence must have a protospacer adjacent motif (PAM) immediately following the target sequence. Guide
RNAs (gRNA) then form a complex, which localizes the cas9 to the target sequence resulting in cas9 cutting
resulting in a double strand break (DSB) of the DNA. The DSB is repaired leading to either gene disruption via Non
Homologous End Joining (NHEJ) or gene editing via homology directed repair (HDR). I will use both types of gene
disruption to study the function of potential PAR2-like proteins that I identify. I will specifically target the
RSLIGRL sequence for either deletion or replacement. The constructs (gRNA, cas9 and donor plasmid necessary to
replace RSLIGRL sequence) will be introduced into Culex mosquitos by injection into the embryo. I will then
design primer pairs flanking the gRNA cut sites and primers specific for the sequence used to replace the HDR.
Thirty-two individual embryos will be transferred to a PCR tube and disrupted by trituration in buffer containing
tritonX100 and proteinase K using a P200 pipettor. Samples are then incubated at 37C for 40 minutes and then 95C
for 5 minutes. This sample will be used for PCR to determine if the DNA has inserted correctly. Complementation
studies will be performed in order to ensure that phenotypes assessed are from mutation of the candidate par2 gene.
Once we have PAR2-like mutants, I will determine if the mutations affect sperm motility. To do this, I will treat
mutant and non-mutant mosquito sperm with trypsin or accessory gland fluids. If mutant sperm is not activated by
trypsin that indicates that the PAR2-like protein that has been mutated is indeed involved in activating sperm
motility.
Goal
June July August September
Activity 1 Injection demonstration and practice
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Activity 2 Microinjection prep
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A
Injection mix preparation
B
Coverslip preparation
C
Injection Station use
D
Embryo collection
E
Embryo alignment
F
Needle filling and needle filler creation
Activity 3 Needle design and fabrication
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Activity 4 Post-injection care and rearing
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Justification and Relation to Goals of the IGTRCN
Hands-on laboratory based training in CRISPR/Cas9 will be important for answering the questions my proposal will
seek to answer. The peer-to-peer training program will foster collaboration between the two labs in developing and
implementing CRISPR/Cas9 technologies in Culex mosquitoes. Once these technologies have been fully developed
in mosquitoes, practical and theoretical knowledge on how to implement CRISPR/Cas9 in Culex mosquitoes can be
shared with other research groups via symposia, workshops, conferences and publications. The resources developed
will be fully available to other research groups and other researchers will be able to access the information that
comes from this project in order to design experiments utilizing CRISPR/Cas9 technology in Culex mosquitoes.
Data Dissemination Plan
The techniques learned and developed at The University of Maryland Insect Transformation Facility (UM-ITF) can
be shared via publications and presentations at scientific meetings. Also, I will be able to teach these techniques to
members of the Cardullo lab and will be able to utilize the techniques learned when I leave the Cardullo lab.
Specifically, while in the Cardullo lab, an undergraduate student will also be recruited from University of California
– Riverside to assist with this project and that student will be able to learn genetic manipulation (CRISPR). I am also
involved in outreach through the entomology outreach program where I will be able to discuss CRISPR and her
project with the general public including K-12 students and undergraduate students.