1. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
Localization of Proteins in Presynaptic
Terminals of Caenorhabditis elegans
Kaylee Racs1, Han Wang2 and Derek Sieburth2
1Eagle Rock High School, Los Angeles, USA
2Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, USA
Abstract
Chemical synapses play an important role in mediating the communication between
neurons and muscles, and are therefore critical for regulating motor movement. Many
proteins are located in the presynaptic terminals of neurons to regulate the release of
neurotransmitters and thus impact the locomotion of the organism in which the neuron
is located. Here a project was designed and carried out in order to determine whether
or not five proteins important for the release of neurotransmitters in C. elegans are in
fact present at presynaptic terminals in the C. elegans nervous system. To determine if
this was the case, transgenic C. elegans which expressed each of these proteins tagged
with GFP were generated and subsequently analysed to determine the distribution of
the tagged proteins in neurons using fluorescence microscopy. Our results are as
follows: the localization of four of the proteins was examined and all four appear to be
at synapses as well as in the axons of motor neurons. One of them may exist at synapses
in higher concentrations than in between synapses, though it is present throughout the
entire axon. These results represent an advance in understanding how these proteins
may regulate neurotransmitter release.
Keywords: C. elegans, proteins, synapse.
Introduction
Through a large scale RNAi screen, the Sieburth lab has identified over 180 proteins
that are involved in the regulation of synaptic transmission in C. elegans [1]. Wild-type
C. elegans move in a smooth, sinusoidal shape, whereas locomotion mutants move
hesitantly and jerkily, though they have normal muscles and normal wiring and number
of neurons [2]. Among these180 proteins, 132 had not been previously implicated in
the process of synaptic transmission, which led to questions regarding the function of
these proteins [1]. The lab’s preliminary data implies that it is highly probable that five
of these 132 proteins regulate presynaptic function in neurons in C. elegans, but not the
postsynaptic function of muscles in C. elegans. Here we are testing the hypothesis that
these five proteins are localized to the presynaptic regions of the neurons where they are
likely to function. In order to visualize the subcellular localization of these five proteins

2. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
[magu-2, Y4C6A.1, C27B7.7, Frm-8a, Frm-8b], molecular cloning techniques were
used to create five plasmids in which each of the proteins in question was fused with
green-fluorescent-protein (GFP) and put under the control of the unc-129 promoter.
The unc-129 promoter can direct the expression of these fusion proteins in a small
subset of motor neurons in C. elegans that reside in the ventral side of the animal and
project their axons to the dorsal side to form en passant synapses on muscles. After the
molecular cloning process, transgenic worms were created by microinjection to allow
for examination of the localization of these proteins in vivo. We expect that proteins
that are concentrated at synapses should adopt a punctate pattern of fluorescence along
the length of axons. Proteins that are localized to synapses as well as axons should
adopt a diffuse pattern of fluorescence. For those candidates showing punctuated
patterns of GFP expression, further confirmation of their presynaptic location was
carried out by comparing their location to that of proteins that are known to be localized
to the presynaptic regions of neurons in C. elegans.
The study of C. elegans synaptic transmission is important and beneficial to the studies
of other animals because many of these synaptic proteins are highly conserved
evolutionary relics, and an understanding of the basic mechanism of synaptic
transmission in an organism as simple as C. elegans provides the essentials for studying
the communication between neurons in more complex organisms [3].
Materials and Methods
C. elegans strains. Strains were kept at a temperature of twenty degrees Celsius and
healthy adults animals were transferred to new agarose plates every three days, or every
generation. The strains used were of the N2 genome and plates of both hermaphrodites
and male animals were maintained.
Molecular cloning. The molecular cloning procedure began with the polymerase chain
reaction (PCR) of the messenger RNA corresponding to each of the five proteins. PCR
allows DNA to be amplified to high enough levels such that it can be used in ligation.
The DNA fragments generated from PCR were then extracted using a Qiagen gel
extraction kit [4] and were digested using the enzymes Nhe1-HF and Age1-HF. This
DNA was then ligated into the expression vector that had been cut with the same
enzymes to form a plasmid. This plasmid contained the unc-129 promoter, which allows
for the expression of the inserted genes in the correct motor neurons (the DA/DB class).
The vector also contained the gene for GFP, the green fluorescent protein that was
attached to the C terminus of each of the five proteins of interest. Once the ligation was
complete, the ligation products were transformed into bacteria, and the bacteria were
allowed to form colonies on agar plates. To identify the colonies that contained the
correct plasmid DNA, four to twelve colonies were grown in culture (inoculated), and
the plasmid DNA was purified from the bacteria using a Qiagen Miniprep kit. [5] The
correct plasmids were identified by diagnostic digestion of the plasmids from these
colonies with Nhe I-HF and Age I-HF.
Creating transgenic worms. A microinjection mixture was created and injected into
the gonads of healthy adult N2 hermaphrodites. The mixture consisted of the plasmids
created from the protein, an injection marker called KP708, pbs, and distilled water.
Once it was determined that the hermaphrodites passed along the injection marker (two

3. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
generations, six days), pictures were taken of the injected transgenic C. elegans in order
to see the distribution of the fusion proteins.
Fluorescence imaging of five candidate proteins. The five proteins in question were
tagged with a fusion protein known as green-fluorescent-protein (GFP) during the
molecular cloning process so that they would be easily identifiable in the injected
animals by fluorescence microscopy Additionally, proteins that are known to be
concentrated in the presynaptic sites of neurons in C. elegans were tagged with another
fusion protein known as mCherry and injected into the GFP-expressing transgenic
worms to allow for comparison of the location of the known presynaptic proteins and
the five proteins in question.
Results
Plasmids were successfully made from four of the five proteins [magu-2, Y4C6A.1,
Frm-8a, Frm-8b] using various primers and enzymes (Table 1). A plasmid was not
successfully obtained from the fifth protein [C27B7.7].
Table 1.Construction of plasmids from magu-2, Y4C6A.1, Frm-8a, Frm-8b.
Plasmid Primers Enzymes
pDS396 (magu-2)
pDS397 (Y4C6A.1)
pKR1 (Frm-8a)
pKR2 (Frm-8b)
pKR3 (C27B7.7)
ODS902, ODS904
ODS903, ODS904
ODS901, ODS902
ODS903, ODS902
ODS904, ODS905
Age1-HF, Kpn1-HF
Nhe1-HF, Age1-HF
Nhe1-HF, Age1-HF
Nhe1-HF, Age1-HF
Nhe1-HF, Age1-HF
The plasmids made from the magu-2, Y4C6A.1, Frm-8a, and Frm-8b proteins were
injected into healthy adult N2 hermaphrodites, which were then allowed to in order to
allow for observation of the stability of the co-injection marker (which was detected as
red fluorescence in the head). The first injection, ISKR1, used the plasmid derived from
the magu-2 protein (pDS396) (Table 2). About thirty animals were initially injected
(the parent generation), but only five of their progeny (the F1 generation) expressed the
trait. Of the F1 generation’s progeny, only one expressed the trait. A second injection
mixture was made using the plasmid derived from the magu-2 protein and was injected
(ISKR1 Line 2). About thirty animals were injected and seventeen of their progeny
expressed the trait. Of the F1 generation’s progeny, the trait was expressed in twelve of
the F2 animals.
Expression of the trait refers only to the existence of the green GFP marker within the
animal. Once the animals from the F2 generation that expressed the trait were imaged,
it was revealed that about half of them showed diffusion of the GFP tagged protein
along the axons in their neurons (Figure 1), and about half showed relatively punctuated
expression of the GFP tagged protein along their axons (Figure 2).

4. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
Table 2. Injection solutions and injection results.
Injection Solution Plasmids # of Animals Injected # of Stable Lines
Produced
ISKR1 pDS396(1.1),
pDS396(1.6)
30 3
ISKR2 pDS397(2.2),
pDS397(2.3)
30 1
ISKR3 pKR1(1.1), pKR1(1.7) 30 0
ISKR4 pKR2(2.1), pKR2(2.2),
pKR2(2.4)
30 1
Figure 1. Diffusion of GFP tagged protein on dorsal side of C. elegans ISKR1 worm 1.
Figure 2. Punctate expression of GFP tagged protein on dorsal side of C. elegans ISKR1 worm 3.

5. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
The second injection, ISKR2, used the plasmid made from the protein Y4C6A.1
(pDS397) (Table 2). Only two worms from the F2 generation expressed the trait, and
once imaged both it was revealed that both of the animals showed a diffusion of the
GFP tagged protein and were not punctuated at all (Figure 3).
The third injection, ISKR3, used the plasmid made from the Frm-8a protein (pKR1)
(Table 2). No transgenic worms were produced from the initial injection of this
mixture, leading us to believe that something must have gone wrong during the
molecular cloning process. The fourth injection, ISKR4, used the plasmid made from
the Frm-8b protein (pKR2) (Table 2). Once again, there was not a large amount of
transgenic F2 animals, but about half of those that did express the GFP tagged protein
showed diffusion of the protein along the dorsal side (Figure 4) while the other half
showed punctuated expression of the protein (Figure 5).
Figure 4. Diffusion of GFP tagged protein along the dorsal side of C. elegans ISKR4 worm 1.
Figure 3. Diffusion of GFP tagged protein in dorsal side of C. elegans ISKR2 worm 2.

6. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
Discussion
Of the four proteins examined, three adopted a “diffuse” pattern of localization and one
adopted a more “punctate” pattern (in about half of the animals that were examined).
We interpret this to mean that all the proteins are at synapses, and that the one of the
four proteins may be more concentrated at synapses than the other three. This does not
mean that the proteins in question were not located in the presynaptic regions of
neurons in those worms, but that they were not more concentrated in those areas than in
other regions of the neuron.
Knowing the subcellular location of each of the five proteins under observation will be
beneficial to future researchers in that it will allow them to determine the exact function
of each of the proteins in C. elegans. For example, if it turns out that they are localized
in the presynaptic regions of neurons, it will provide evidence to future researchers that
the proteins disrupt the transmission of neurotransmitters that regulate normal muscle
movement.
The relatively small amount of C. elegans that expressed the trait in the F2 generation is
also troubling. Typically, the F2 generation yields at least thirty worms that express the
trait, which increases the possibility of finding animals that the majority of the animals
will show the desired result. We are unsure why there were so few animals that
expressed the stable co-injection marker, because there is no concrete way to prove
what went wrong.
The inability to derive a plasmid from the C27B7.7 protein could be attributed to one of
two possibilities. Due to an initial absence of C27B7.7 DNA during the gel extraction
procedure, it is possible that there is something faulty about the DNA itself. Another
possibility is that the enzymes used to cut and ligate the C27B7.7 DNA once it was
Figure 5. Punctuated expression of GFP tagged protein along the dorsal side of C. elegans ISKR4 worm 4.

7. Science High School Advanced Research Program 10 June - 2 August 2013, Los Angeles,USA
obtained were not functioning correctly, though we do not have any ideas as to why this
could have happened since they are stored in a -25 degrees Celsius freezer and are not
exposed to temperatures high enough to denature them.
Additional experiments must be carried out in order to determine whether this initial
project yielded accurate results and to quantify its results. Once the locations of the five
proteins under observation are determined and quantified, further investigations will be
done to determine the function of any of the proteins that seem to be most heavily
concentrated in the presynaptic regions of the C. elegans nervous system. Additionally,
experiments will need to be developed and performed to discover the location and
function of the other 127 proteins thought to cause uncoordinated movement in C.
elegans.
Acknowledgements
I would like to thank my mentor, Han Wang, and my PI, Derek Sieburth, for their
guidance throughout this project and for teaching me everything I needed to know in
order to be able to put this paper together, as well as for allowing me to join their lab. I
would also like to thank Dr. Joe Cocozza and Diana Sabogal for coordinating the
SHSARP program and granting me this opportunity.
We would like to thank the C. elegans Genetic Stock Center for providing the strains
we worked with.
We would like to thank Windsong Trust and the National Science Foundation for their
generous financial support of SHSARP.
References
[1] Derek Sieburth et al., “Systematic Analysis of Genes Required for Synapse
Structure and Function”, Nature, Vol. 436, No.28, pp. 510-517, July 2005.
[2] D. H. Hall and Z. F. Altun, The C. elegans Atlas, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 2005.
[3] William B. Wood, The Nematode Caenorhabditis Elegans, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1988.
[4] "QIAquick Gel Extraction Kit Protocol." Qiagen, n.d. Web. 23 July 2013.
<https://www.mcdb.ucla.edu/Research/Banerjee/protocols/gelextraction--
Qiagen.pdf>.
[5] "QIAprep Spin Handbook." pp. 25-26. Qiagen, n.d. Web. 24 July 2013.
<http://public.wsu.edu/~kahn_sci/Flow/E2-QIAprep_Miniprep_Handbook.pdf>.