1. General Introduction
Before even the first embryonic division has taken place in the C. elegans zygote,
a multitude of cytoplasmic proteins are already at work. A subset of these proteins share
a crucial task− to partition the cell's cytoplasmic contents into two cell-lineages: the soma
and the germline. While somatic cells will eventually differentiate to comprise most of
the mature worm, germline cells are special in that they were destined to become
reproductive cells from the time of the very first cell division, when the worm was still
just a zygote itself. The ability of a worm to reproduce and pass on its genes is therefore
unequivocally dependent on the success of the initial cytoplasmic division, which in turn
requires the proper formation of asymmetric protein gradients (reviewed in Lyczak et al.,
2002).
When a sperm cell pierces the egg, three events directly follow: the oocyte
continues to mature through meiosis I and II, a protective eggshell is produced, and the
anterior-posterior axis (A-P axis) is established by the sperm's pronucleus. As the
pronucleus enters the egg, a cytoplasmic flux causes the pronucleus-centrosome complex
to migrate toward one of the poles, which then becomes the posterior of the zygote.
Seemingly by default, the opposite end automatically becomes the anterior pole
(reviewed in Lyczak et al., 2002).
1
2. Figure 1. Establishment of the Anterior-Posterior Axis (reviewed in Lyczak et al., 2002)
The cytoplasmic flux responsible for specifying the A-P axis just after fertilization
also asymmetrically distributes many cytoplasmic regulator proteins. Widely conserved
cortical PAR proteins, are the first of these proteins to be localized to one of the two poles
within the actin-rich layer beneath the plasma membrane (reviewed in Lyczak et al.,
2002; Griffin et al., 2011). While PAR proteins all play a role in partitioning the contents
of the cytoplasm, it is worth noting that these proteins are not all related to each other and
can actually have drastically different mechanisms of action. As the PAR proteins
polarize, both the oocyte pronucleus and germline P granules (discussed later in more
2
3. detail) simultaneously migrate to the posterior. Finally, the spindle displaces posteriorly
and an asymmetric cleavage takes place, resulting in a larger anterior daughter cell
(denoted AB in Figure 1) and a smaller posterior daughter cell (denoted P1 in Figure 1),
(reviewed in Lyczak et. al., 2002).
During the initial phase of polarity formation, PAR-1 and PAR-2 localize to the
posterior of the single-cell embryo, while PAR-3, PAR-6, and PKC-3 migrate to the
anterior pole. Interestingly, all of the PAR proteins appear to be interdependent− if a
single PAR protein is mutated, none of the PAR proteins are able to properly localize to
their respective poles. Furthermore, mutations in PAR proteins often result in blastomeres
that are of undesirable size, have abnormal cleavage rates, and show altered protein
expression compared to wild-type cells (Schubert et al., 2000.)
Polarity mediator proteins, which are derived from maternally expressed mRNAs,
act downstream of the PARs to asymmetrically allocate other proteins throughout the cell
(reviewed in Lyczak et al., 2002; Schubert et al., 2000). Two of these mediator proteins,
PIE-1 and MEX-5 (whose name indicates that it is a maternally expressed gene), will be
the initial focus of this thesis.
MEX-5 and PIE-1 are both CCCH zinc finger proteins that bind RNA through
their CCCH finger domains. As the zygote prepares to divide, MEX-5 forms an anterior-
rich gradient, while PIE-1 forms an opposing gradient that is strongest at the posterior of
the cell (Figure 2). Once division occurs, the fate of the two daughter cells has already
been determined: the MEX-5-rich daughter cell is somatic, and the PIE-1-rich daughter
3
4. cell will go on dividing to create both somatic cells and the germline (reviewed in Lyczak
et al., 2002; Tenlen et al., 2008).
MEX-5
A! ! P
PIE-1
A! ! P
Figure 2. MEX-5 and PIE-1 Localization
The MEX-5 anterior-rich gradient is formed first and is dependent on PAR-1
kinase activity. Within 10 minutes of fertilization, the anterior-high/posterior-low MEX-5
gradient has already been established. Cytoplasmic PAR-1 (accumulated at the zygote's
posterior pole) directly phosphorylates the S485 and S404 residues of MEX-5. While
both S485 and S404 are already phosphorylated during oogenesis, in vivo studies have
shown that S404 is dephosphorylated far more rapidly than S485. Phosphorylation of
previously dephosphorylated S404 residues is therefore thought to play a critical role in
the detachment of MEX-5 molecules from relatively static RNA-containing complexes in
the posterior of the zygote, freeing them to travel toward the cell's anterior pole (Griffin
et al., 2011).
4
5. Migration of MEX-5 occurs at different rates, and a molecule's diffusion rate is
strongly correlated with its original location prior to gradient formation when MEX-5 is
distributed uniformly throughout the cell. MEX-5 sucrose gradients suggest that there is
more than one type of MEX-5 complex present in the C. elegans zygote. The number of
specific complexes MEX-5 forms is currently unknown, but there do seem to be two
main categories or classes of MEX-5 complexes: "fast" and "slow." Although both classes
are found throughout the cytoplasm, fast-diffusing MEX-5 (averaging 5.15 µm2/s) is
more prevalent in the posterior of the cell, while the slower variant of MEX-5 (averaging
0.086 µm2/s) exists in higher concentrations at the anterior pole (Griffin et al., 2011).
Recent studies suggest that slow-mobility MEX-5 is primarily due to RNA
binding through its CCCH fingers (as mentioned previously), with assistance from the N-
terminal domain. RNA binding mediates the association of MEX-5 with high-density
complexes containing 80S ribosomes, which in turn results in decreased mobility. When
the CCCH fingers are mutated, the diffusion of MEX-5 increases and the anterior-high/
posterior-low gradient weakens. Previously, it was thought that the accumulation of slow-
diffusing MEX-5 in the anterior could have been due to the asymmetric localization of a
subclass of mRNAs, but this is now considered unlikely because mutations that block the
phosphorylation of S404 result in the symmetric distribution of slow-moving, high-
density MEX-5 complexes throughout the cytoplasm. Furthermore, these mutants (in
which S404 phosphorylation is blocked) are enriched in the slow-diffusing complexes
compared to wild-type MEX-5. As for the formation of the heavy complexes themselves,
it is hypothesized that MEX-5 may interact with other MEX-5 molecules in addition to
5
6. RNA. The polyglutamine-rich N-terminus of MEX-5, which plays a role in the formation
of slow MEX-5, is a potential candidate for this interaction (Griffin et al., 2011).
While the development of fast- and slow-diffusing MEX-5 complexes are
independent of PAR-1 activity (in PAR-1 knockouts the distribution of fast to slow
MEX-5 complexes is 30:70), phosphorylation of the S404 residue by PAR-1 does appear
to favor the formation of fast-diffusing complexes (Griffin et al., 2011).
Okadaic acid-sensitive phosphatase (PP2A) is a heterotrimeric cytoplasmic
phosphatase that is a PAR-1 antagonist: its catalytic subunit, LET-92, dephosphorylates
the S404 residue of MEX-5, returning the protein to a slow-diffusing state. Experiments
conducted in the Seydoux lab with let-92(RNAi) demonstrated a predicted increase in
pS404 (Griffin et al., 2011).
Before the zygote polarizes, PP2A activity is greater than that of PAR-1, so that
MEX-5 exists primarily in slow-diffusing complexes. Once the cell begins to polarize,
however, PAR-1 activity suddenly exceeds PP2A activity (mechanism unknown),
resulting in high pS404 levels which cause the majority of MEX-5 molecules to shift to
fast-diffusing complexes. Since PAR-1 is distributed along a posterior-high/anterior-low
gradient, slow-diffusing unphosphorylated MEX-5 accumulates in the anterior of the
zygote, forming the anterior-high/posterior-low MEX-5 gradient (Griffin et al., 2011).
Mutations in PAR proteins result in the symmetric distribution of MEX-5
throughout the cytoplasm, and changes in the distribution of PAR-1 elicits rapid changes
in MEX-5 diffusion dynamics. In vivo observations further confirm that the posterior-
high/anterior-low cytoplasmic PAR-1 gradient is sufficient to both establish the anterior-
6
7. low/posterior high MEX-5 gradient as well as alter the rate of MEX-5 diffusion.
Interestingly, the PAR-1 gradient is always significantly larger than the opposing MEX-5
gradient it regulates. For example, a ~2.9-fold MEX-5 gradient is correlated with a 5.5-
fold PAR-1 gradient (Griffin et al., 2011). Although it is now generally accepted that the
PAR-1 gradient is responsible for the MEX-5 gradient, mutants lacking MEX-5 and
MEX-6 (discussed later) can impact PAR localization, suggesting that the interaction is
far more complex than previously thought (Tenlen et al., 2008).
PIE-1, a transcriptional repressor, is predominantly found in the nucleus of
germline blastomeres. Consequently, germline blastomeres are immune to somatic
differentiating factors. In addition to transcriptional repression, germline cells contain
cytoplasmic structures called P granules, which are absent in the somatic lineage. P
granules are characterized by unusual RNA molecules that are both capped and
polyadenylated, and associate with PIE-1 and MEX-1 in the early embryo. After
fertilization of the egg, P granules are localized to the posterior via the initial cytoplasmic
flux. While other cytoplasmic components appear to migrate back toward the anterior of
the zygote along the cortex, P granules associate with the posterior cortex and remain
localized at the posterior pole. This localization pattern of P granules and PIE-1 to the
posterior continues after the one-cell stage, as the early embryo continues to divide
(Guedes and Priess, 1997).
PIE-1 localization is regulated in two ways. Prior to cell division, the posterior
cytoplasm becomes enriched in PIE-1. Then after the cell divides, any residual PIE-1 in
the somatic daughter cell (originating from the anterior of the zygote) is degraded, a
7
8. process that is dependent on PIE-1's interaction with MEX-5 and MEX-6 through its first
CCCH zinc finger and a region including amino acids 174-335. (Reese et al., 2000).
MEX-6 has an amino acid sequence that is 70% identical and 85% similar to MEX-5, but
the two are only partially redundant. In the absence of MEX-6, PIE-1 is segregated and
degraded normally, and no embryo lethality is observed. When MEX-5 is knocked out,
58% of the mutants are defective for PIE-1 degradation in the anterior. But when both
MEX-5 and MEX-6 are knocked out, all double mutants are defective for both PIE-1
segregation and degradation. In addition, experiments have found ectopic expression of
MEX-5 and MEX-6 in the posterior cytoplasm to be sufficient to inhibit posterior
expression of PIE-1 and other CCCH finger germline proteins such as MEX-1 and
POS-1, implying that MEX-5 and MEX-6 are responsible for suppressing anterior
expression of germline proteins (Schubert et al., 2000). Whether MEX-5/MEX-6 interact
with PIE-1 directly, however, is not known.
Proper expression of MEX-1 is vital for both P granule segregation in the early
embryo and the development of germline blastomeres. In mex-1 mutants, P granules still
localize to the posterior of the zygote but fail to associate with the cortex. Therefore P
granules are mis-partitioned to somatic cells in subsequent divisions, and the
concentration of P granules found in the supposed germline lineage is progressively
reduced. MEX-1 also regulates the localization of PIE-1 to the posterior; mex-1 mutants
have lower levels of P granule-associated PIE-1. (Guedes and Priess, 1997). Whether
MEX-1 and PIE-1 interact directly is also unknown.
8
9. C. elegans share approximately 40% of their genome with humans (Worm
Classroom, 2012). By gaining a solid understanding of how proteins interact with one
another in the early C. elegans embryo, we are able to build a foundation of knowledge
that will ultimately enable us not only to better understand human diseases, but also
provide us with an educated starting point for developing treatments.
In this thesis, the yeast two-hybrid assay was first used to look for interactions
between full-length proteins PIE-1, MEX-5, MEX-6, and MEX-1. Next, MEX-5
fragments were assayed against full-length MEX-5 and full-length PIE-1 as a preliminary
step in mapping the interaction. In an attempt to replicate our results in vivo, HEK 293
tissue culture cells were transfected with the full-length proteins PIE-1, MEX-5, and
MEX-6. Though the HEK 293 cells are derived from human embryonic kidney
preparations, they are a popular choice due to the fact that they are relatively easy to
culture and transfect (TGR BioSciences). Protein-protein interactions within the
transfected HEK 293 cells were assayed via western blots.
9
10. Introduction to Yeast Two-Hybrid Experiments
The yeast two-hybrid assay is a popular first step for mapping protein-protein
interactions, and is the initial method used in this thesis. Proteins (or protein regions) of
interest are fused to either the DNA binding domain (DBD) or the activation domain
(AD) of a transcription factor, resulting in a DBD-X fusion and an AD-Y fusion. In order
for the transcription factor to activate the transcription of its target gene, the AD must be
brought into contact with the DBD, which sits on the upstream activating sequence.
Therefore, if protein Y interacts with protein X, the AD will consequently be brought into
close proximity of the DBD, creating a functional transcription factor (Fields and Song,
1989).
Figure 3. General Description of the Two-Hybrid System (Invitrogen, 2005)
10
11. In order to determine whether or not the interaction took place, the MAV203 yeast
strain is used because it contains three reporter genes that confer a specific phenotype to
the yeast when they are expressed: HIS3, lacZ, and URA3. These genes codes for
imidazoleglycerol-phosphate dehydratase, β-galactosidase, and orotidine 5-phosphate
decarboxylase, respectively. The HIS3 reporter gene has the advantage of allowing for the
most tuning sensitivity (by utilizing different His concentrations), while lacZ provides the
most quantitative results. URA3's read-out is also important, however, because it allows
for counter selection to perform a reverse-two-hybrid screen, which selects for mutants
that do not interact.
AD plasmids (pDEST22) and DBD plasmids (pDEST32) come with the
Invitrogen Proquest yeast-two-hybrid kit. Both destination vectors contain a ccdB death
gene, which enables selection for only those plasmids that have had this ccdB region
swapped out for the desired insert during an LR reaction. However, only pDEST22
contains the LEU2 gene, and only pDEST32 contains the TRP1 gene. Once the ccdB
region of pDEST32 has been replaced by the DNA encoding protein X and the ccdB
region of pDEST22 has been exchanged for the DNA encoding protein Y, the two
plasmids are cotransformed into MAV203 yeast, which is a -Leu, -Trp knockout strain.
Therefore, only those yeast cells that have taken up both pDEST32 and pDEST22
plasmids can grow on -Leu, -Trp selection plates. -Leu, -Trp, -Ura plates are then used to
assay for URA3 expression, and -Leu, -Trp, -His plates with various concentrations of 3-
AT (which competitively inhibits the HIS3 enzyme) are used to assay for HIS3
expression.
11
12. Materials and Methods for Yeast Two-Hybrid Experiments
Overview
Vectors of interest were co-transformed into the MAV203 yeast strain. 100µL of
the co-transformed yeast were plated on a -Leu, -Trp plate and allowed to grow up
overnight at 30° C. Next, four colonies from each transformation plate were patched onto
another -Leu, -Trp plate using sterile toothpicks and allowed to grow for 24-48 hours at
30° C. The patch plates were then replica-plated via sterile velvet onto a clean –Leu, -Trp
plate to clean up the patches. After these patches had grown up at 30° C for an additional
24-48 hours, they were then replica-plated onto the following plates in descending order:
-Leu, -Trp, -Ura
-Leu, -Trp, -His (10mM 3-AT)
-Leu, -Trp, -His (25mM 3-AT)
-Leu, -Trp, -His (50mM 3-AT)
-Leu, -Trp, -His (100mM 3-AT)
-Leu, -Trp
12
14. Truncations Used
Figure 5. MEX-5 Truncations (generated in the Seydoux lab)
Figure 6. PIE-1 Truncations (generated in the Seydoux lab)
14
15. BP Reaction
In a 1.5 mL microcentrifuge tube at room temperature, 3µL of PCR product were
added to 1µL of pDEST 22 or pDEST 32, and the total volume was brought to 8µL using
TE buffer at pH 8.0. 2µL of BP II reaction mix (Invitrogen) were then added to the 1.5µL
tube, and after briefly vortexing the reaction was incubated for one hour at 25°C. Finally,
1µL of Proteinase K solution (Invitrogen) was added to the reaction tube and allowed to
incubate at 37°C for ten minutes.
LR Reaction:
In a 1.5 mL microcentrifuge tube at room temperature, 1µg of the entry clone was
added to 1µL of pDEST 22 or pDEST 32, and the total volume was brought to 8µL using
TE buffer at pH 8.0. LR Clonase II enzyme mix (Invitrogen) was then removed from
-20°C and thawed on ice for approximately two minutes. After briefly vortexing this mix
twice (for about two seconds each time), 2µL were added to the 1.5 mL tube. This tube
was then vortexed two times for about two seconds each time. Following a one hour
incubation period at 25°C, 1µL of Proteinase K solution (Invitrogen) was added to the
reaction tube and allowed to incubate at 37°C for ten minutes.
15
16. Miniprep
At room temperature, pelleted bacterial cells were thoroughly resuspended in
250µL Buffer P1 (containing RNase A) and transferred to a 1.5 mL microcentrifuge tube.
250µL of Buffer P2 were then added, and the tube was gently inverted six times to mix
the contents. 350µL of Buffer N3 were next added, and once again the tube was gently
inverted six times. After centrifuging for ten minutes at 13,000 rpm in a table-top
microcentrifuge (this is the speed used for this entire protocol), the supernatant was
carefully removed from the tube and transferred to a QIAprep spin column using a
pipette. The spin column was then centrifuged for one minute, and the flow-through was
discarded. Next, the spin column was washed with 500µL Buffer PB, spun for one
minute, and the flow-through was discarded. A second wash was performed using 750µL
of Buffer PE, centrifuging for one minute, and discarding the flow-through. The spin
column was then centrifuged for an additional minute to remove any remaining buffer,
and then the column was placed into a clean 1.5 mL microcentrifuge tube. 50µL of Buffer
EB were then carefully added to the center of the column to elute the DNA, and after
being allowed to stand for one minute, this new tube (with the column) was centrifuged
for one minute (resulting in the DNA being captures by the 1.5 mL microcentrifuge tube).
(All buffers provided by QIAGEN miniprep kit.)
16
17. Yeast Transformation (adapted from Gietz and Schiestl, 2008)
A single colony of MAV203 yeast (grown on a YPAD plate) was inoculated in 5
mL of liquid YPAD and shaken at 200 rpm at 30°C until titer reached 2 x 107 cell mL-1
(which took about four hours). A 250 mL liquid culture of fresh YPAD was incubated
with the inoculum.
After the required titer was reached, the yeast cells were harvested by centrifuging
the inoculum for five minutes at 3,000g, and then washed by gently resuspending the
pellet in 25 mL of sterile water. The yeast were then centrifuged again at 3,000g for five
minutes at 20°C, and then the wash was repeated. After the second wash, cells were
resuspended in 1.0 mL of sterile water and transferred to a 1.5 mL microcentrifuge tube.
This tube was then centrifuged at 13,000g for 30 seconds, and the supernatant was
removed. 1.0 mL of sterile water was then used to resuspend the cells, and then 100 mL
aliquots were added to fresh microcentrifuge tubes for each transformation. These tubes
were then centrifuged at 13,000g for 30 seconds, and their supernatants were removed.
Next, the single-stranded carrier DNA (salmon sperm DNA) was prepared. 1.0
mL of salmon sperm DNA (Invitrogen) were boiled for five minutes in a heat block, and
then chilled immediately on ice until being used.
To each transformation tube containing a yeast cell pellet, the following were
added:
240µL PEG (50% w/v)
36µL LiAc 1.0 M
50µL salmon sperm DNA
1µL of each plasmid (so 2µL total per transformation tube)
32µL dH2O
17
18. The transformation tubes were then vortexed until all of the contents were
thoroughly mixed, and then incubated in a 42°C water bath for 40 minutes. Following
incubation, the tubes were centrifuged at 13,000g for 30 seconds and their supernatants
were removed. Pellets were resuspended in 1.0 mL of sterile water by first breaking the
clump with a micropipette tip and then vortexing.
The entire contents of each transformation tube were then plated onto -Leu,-Trp
plates and spread with a glass rod. After the transformation solution had seeped into the
agar at room temperature, the plates were incubated at 30°C for 3-4 days.
18
20. The yeast two-hybrid assay was validated by using the provided controls in the
Invitrogen kit, as well as negative controls containing experimental proteins of interest
that were fused to either the AD or DBD while the other domain was left empty. All of
the controls grew as expected, with the exception of RalGDS m2 showing slight growth
on the -LTU plate; since the readout was significantly less than the weak control of the kit
(RalGDS m1), however, this growth is not considered to be a failure of the control. In
addition to the controls, the first set of experimental combinations were assayed. Out of
the four experimental combinations, only MEX-5 fused to the AD and PIE-1 fused to the
DBD failed to produce strong growth on the 100mM 3-AT plate.
Figure 7. An Example of Patch Growth.
Plate A is the final –Leu, -Trp patch plate that was used to confirm equal growth
of each sample. Plate B is –Leu, -Trp, -Ura and shows limited growth for all samples
(consequently, these plates were not scored.) Plate C is –Leu, -Trp, -His at 50mM 3-AT,
20
21. and Plate D is –Leu, -Trp, -His at 100mM 3-AT. The 50mM and 100mM 3-AT plates
were used in scoring because these two concentrations displayed the most dramatic
differences between the samples.
Figure 8: Mapping MEX-5 Fragments against Full-length PIE-1
Figure 9. Mapping MEX-5 Fragments against Full-Length MEX-5
21
22. Figure 10. Mapping PIE-1 Fragments against Full-length MEX-5 and PIE-1
While Figure 8 shows the N-terminus of MEX-5 to be indispensable in MEX-5 -
PIE-1 interaction, the C-terminus is somewhat dispensable in the MEX-5 – PIE-1
interaction. A small truncation of the MEX-5 C-terminus (as seen in the 1-447aa
fragment) still yields a positive interaction with full-length PIE-1 although the interaction
is not as strong as it is with full-length MEX-5, and further truncation of the protein to a
1-344aa fragment does not correlate with an additional reduction in interaction strength.
The C-terminus of MEX-5 is therefore dispensable, but still contributes to the interaction.
As soon as a small portion of the N-terminus is removed, however, 86-468aa (86-STOP)
MEX-5 fails to interact at all with full-length PIE-1.
The data in Figure 9 show that both terminal regions are dispensable in MEX-5 –
MEX-5 interactions and that the zinc-fingers are indispensable. While there is a
noticeable decrease in interaction strength when full-length MEX-5 is truncated to
1-447aa, the 1-344aa truncation interacts even more strongly with full-length MEX-5
than does the untruncated protein. Although only one patch colony was clearly readable
for the 86-468aa fragment, the fact that the 244-468aa fragment interacted as strongly
22
23. with full-length MEX-5 as the untruncated protein leads us to believe that the other
86-468aa fragment patches would also have displayed a positive interaction.
No clear patterns emerged from the results displayed in Figure 10. Both
homophilic binding of PIE-1 and its interaction with MEX-5 were dependent on the N-
terminus, C-terminus, and the second zinc-finger.
Table 2. Full-length Protein Interactions
Fused to DNA Binding Domain
Fused to DNA Binding Domain
Fused to DNA Binding Domain
Fused to DNA Binding Domain
PIE-1 MEX-5 MEX-6 MEX-1
cDNA
Fused to
Activation
Domain
PIE-1 ++ ++ +/- +++Fused to
Activation
Domain
MEX-5 +/- ++ +/- -
Fused to
Activation
Domain
MEX-6 - +++ +++ ++
Fused to
Activation
Domain
MEX-1
cDNA
+/- +/- ++ +++
KEYKEY
Very strong growth +++
Strong growth (same as positive controls) ++
Growth +
Very weak growth +/-
Background (from transfer only) -
When full-length proteins were assayed against each other, all four proteins in
Table 2 interacted strongly with themselves. Additionally, MEX-5 interacted more
strongly with PIE-1 than did MEX-6 in both directions, while MEX-6 interacted more
strongly with MEX-1 than did MEX-5 in both directions. The interactions between both
PIE-1/MEX-1 and MEX-5/MEX-6, however, were drastically different depending on
which protein was fused to the AD.
23
24. Introduction to Tissue Culture Experiments
After a successful round of yeast-two-hybrid experiments, we decided to move to
tissue culture for four reasons:
1. While the yeast-two-hybrid assay enables us to tell whether or not an interaction
occurred, differences in the strength of each interaction are difficult to detect
unless they are relatively large.
2. Yeast-two-hybrid does not provide a way to quantify results.
3. Our positive controls, while displaying strong growth relative to the negative
controls, were weaker than the positive controls that came with the kit.
4. RNase experiments can be conducted in tissue culture cells to determine whether
the protein-protein interactions are RNA-dependent or RNA-independent.
In these experiments, HEK293 human embryonic kidney cells (a popular choice in C.
elegans labs) were co-transfected with full-length proteins MEX-5, MEX-6, PIE-1, and
PAR-1. Co-immunoprecipitation and western blotting were then used to assay for
protein-protein interactions.
24
25. Figure 11. Schematic of the tissue culture experiments.
Figure 12. Schematic of anti-v5 Western probe.
25
26. Figure 13. Schematic of anti-myc Western probe.
Proteins of interest, which had been fused to either v5 or myc protein tags, were
exposed to dynabeads that had previously been treated with anti-v5 antibodies. As Figure
11 depicts, the protein with the v5 tag (Protein 1) binds to the anti-v5 antibody of the
dynabead, pulling it out of solution. If there is an interaction between Protein 1 and
Protein 2, both proteins will be pulled out of solution and stick to the dynabeads during
the co-immunoprecipitation process. Once the proteins that originally stuck to the
dynabeads have been eluted from the beads and loaded into gels, each protein migrates
through the gel at a unique speed that is dependent on its composition.
During the Western protocol, these gels (containing bands of proteins) are
transferred onto membranes that are subsequently treated with either anti-v5 antibody
(Figure 12) or anti-myc antibody (Figure 13), followed by a compatible secondary
26
27. antibody that allows for the complex’s detection. Probing the membrane with anti-v5
enables the assessment of Protein 1 expression levels, since this v5 tagged protein should
interact with the anti-v5 dynabeads if it is present. Alternatively, probing the membrane
with anti-myc shows whether or not the two proteins interacted - whether Protein 1
effectively “pulled-down” Protein 2.
27
28. Materials and Methods for Tissue Culture Experiments
Maintenance of HEK293 Cells
Cells were grown in 100mm Petri dishes in approximately 10mL of media,
DMEM (Cellgro Mediatech)+ Pen-Strep (Invitrogen) + FBS (Cellgro Mediatech), and
incubated at 37° C and 5% CO2. Cells were split either 1:5 or 1:10 when they were
70-90% confluent. Typically, it took about 3 days for the cells to reach ~80% confluency.
Transfection of HEK293 Cells
Transfection reaction tubes were prepared in 100µL aliquots, each containing 1µg
of each plasmid, 6µL of X-treme GENE HP DNA Transfection Reagent (Roche), and
were brought up to 100µL with serum-free media. Upon addition of the X-treme GENE
HP DNA Transfection Reagent (added last), the tubes were allowed to incubate for 15
minutes at room temperature inside the tissue culture hood. Following incubation, the
solution was then gently added to a well of cells that were seeded approximately 24 hours
beforehand and had been placed in fresh media 2-3 hours before the transfection to
ensure an optimum growth environment. Transfected cells were then incubated at 37° C
for 24 hours in a tissue culture incubator.
28
29. Co-IP Preparation
Part I: Cell Preparation
After incubating for 24 hours, transfected cells were removed from the incubator
and placed immediately on ice. The cells were then washed x2 with 1µL of cold PBS, and
then 500µL of cold MCLB were added to each well. (All MCLB used in these
experiments was comprised of: 50mM Tris pH 8.0, 100mM NaCl, 2mM DTT, 0.5%
NP-40, + protease and phosphatase inhibitors). Next, lysate buffer was pipetted gently to
detach the cells from the bottom of the wells, and then the lysate was transferred to a
1.5mL Eppendorf tube. The tubes were spun down for 30 seconds, and the supernatants
were transferred to new Eppendorf tubes. 50µL of the supernatants were removed at this
step and stored at -80° C as the “Load/Bound” sample.
Part II: Dynabead Preparation
On ice, 10µL of protein G Dynabeads were prepared per co-IP reaction (for 8 co-
IPs, 80µL of G Dynabeads were prepared). Starting with all of the G Dynabeads in a
single Eppendorf tube, they were washed 2x with 500µL of cold MCLB, using a
magnetic rack to separate the beads from the solution. The G Dynabeads were then
brought up in MCLB (50µL MCLB per co-IP reaction). Anti-v5 antibody was then added
to this tube (1µL anti-v5 antibody per co-IP reaction). 45µL of this solution were then
aliquoted into new Eppendorf tubes, each tube of 45µL now ready for co-IP.
29
30. Co-IP
450µL of co-IP-prepared lysate were added to their respective co-IP-prepared G
Dynabead tubes. The tubes were then slowly rotated at 4° C for 2 hours. Next, the tubes
were placed immediately in magnetic racks that were submerged in ice, and 50µL of the
supernatants were collected and stored at -80° C as the “Unbound” sample. After
removing the supernatants from the beads, each tube of beads was washed 3x with
800µL cold MCLB, and then frozen on dry ice and stored at -80° C.
Gel Loading Preparation
For co-IP Samples.
After the frozen samples had thawed slightly on ice, 40µL of 1.5x LDS were
added to each tube. The tubes were then boiled in a heat block for 10 minutes, spun down
for 20 seconds, and placed immediately back in the magnetic racks (submerged in ice).
The supernatant was then removed from each sample and transferred to a new Eppendorf
tube, at which point the supernatant was treated with an additional 40µL of 1.5x LDS
(bringing the total volume to 80µL). Again, the tubes were boiled, spun down, and the
supernatant was transferred to a new tube. 20µL of DTT were then added to each tube,
and the tubes were spun for 3 minutes at 4° C (14000rpm). Tubes were then kept on ice
until it was time to load the gels.
30
31. For “Load” Samples.
After the frozen samples had thawed slightly on ice, 25µL of 4x LDS and 25µL of
DTT were added to each tube, bringing the total volume of each tube to 100µL. After
boiling in a heat block for 10 minutes, the tubes were spun down for 3 minutes at 4° C
(14000rpm), and then kept on ice until it was time to load the gels.
Western
Running the Gels.
10µL of each sample were loaded per lane in a NuPAGE 4-12% Bis-Tris Gel
(1.5mm, 15 wells.) The gels were all run at 200V for 40 minutes.
Transfer.
Proteins were transferred from the gels to PVDF membranes (Millipore) at 100V
for 1 hour (or overnight) at 4° C. Membranes were prepared by first soaking them for 1
minute in meOH, followed immediately by 3-5 minutes in H2O and then at least 3
minutes in transfer buffer. (The transfer buffer was comprised of 100mL of 10x transfer
buffer, 800mL H2O, and 100mL methanol. The 10x transfer buffer was made with 144.1g
glycine, 30.3g Tris base, and brought to 1L with H2O).
31
32. First Incubations.
After the transfer step, membranes were blocked with PBST+milk at room
temperature for 30 minutes (or overnight 4° C). Membranes were then incubated in 3mL
of PBST+milk containing the primary antibody, anti-v5, at concentration 1:5000 for 1
hour at room temperature (or overnight at 4° C). After washing the membranes 3x with
PBST (5 minutes per wash), they were incubated in the secondary antibody αIgG2A
(HRP goat anti-mouse) at concentration 1:2500 for 1 hour at room temperature.
Membranes were then washed again 3x with PBST.
First Detection.
After the final 5-minute wash with PBST, the membranes were placed on saran
wrap and incubated at room temperature for 1 minute in HRP detection solution (HyGLO
quick spray by Denville Scientific.) Membranes were then wrapped in a clean sheet of
saran wrap and exposed for anywhere from a second to 5 minutes depending on the
visibility of the bands.
Stripping.
Membranes were stripped with a reprobing buffer (3.125mL 1M Tris pH 7.0,
10mL 10% SDS, 350uL beta mercaptoethanol, brought up to 50mL with H2O) at 45° C,
shaking at 70rpm for 30 minutes. The membranes were then washed 3x with PBST, this
time for 10 minutes per wash to ensure the removal of the reprobing buffer.
32
33. Second Incubations.
The same procedure was followed as in the first round of incubations, substituting
α-myc at concentration 1:1000 as the primary antibody and IgG1 (HRP goat anti-mouse)
at concentration 1:6000 as the secondary antibody.
Second Detection.
The same procedure was followed as in the first detection.
33
34. Results From Tissue Culture Experiments
Figure 14. Load sample probed with anti-v5.
34
35. Figure 15. IP sample probed with anti-v5.
Figure 16. Load sample probed with anti-myc.
35
36. Figure 17. IP sample probed with anti-myc.
As expected, when the load sample was probed with anti-v5 (Figure 14) there was
not much to be seen on the membrane because the proteins were present in low
concentrations in this sample. However in the IP sample (Figure 15 - which was exposed
to the dynabeads), the proteins were concentrated, and so treatment of this membrane
with anti-v5 showed strong bands - an indication that the proteins were adequately
expressed. Figure 15 confirmed the relatively even expression of the myc-tagged proteins
and also demonstrated that the controls worked, since the last four lanes contained no
myc-tagged proteins. Finally, Figure 16 depicts the interaction between the v5-tagged and
myc-tagged proteins. The two strongest interactions were v5-MEX-5 with myc-MEX-5
(lane 1) and v5-MEX-5 with myc-MEX-6 (lane 6). The next strongest interaction was v5-
MEX-6 with myc-MEX-5 (lane 2), followed by v5-MEX-6 with myc-MEX-6 (lane 7).
36
37. Finally, v5-PIE-1 pulled down more myc-MEX-5 (lane 3) compared to v5-PIE-1 and
myc-MEX-6 (lane 8)
37
38. Discussion
From the yeast-two-hybrid experiments, we see that as soon as a small portion of
the N-terminus of MEX-5 is removed (Figure 8), 86-468aa (86-STOP) MEX-5 fails to
interact at all with full-length PIE-1. Therefore, we conclude that the N-terminus is
required for MEX-5 – PIE-1 interaction, although it is not known whether the N-terminus
interacts directly with PIE-1 or if this region is critical in the proper folding of the
protein, which in turn configures an unknown region of MEX-5 to bind PIE-1.
Based on our results on Figure 9, it seems likely that the zinc-fingers contain the
interaction domain(s) for MEX-5 – MEX-5 interactions. It is also possible, however, that
two interaction domains exist in the termini: one in the N-terminus and one in the C-
terminus. If this is the case, perhaps the zinc-fingers are crucial for the correct folding of
the protein rather than directly participating in the interaction. Because zinc-fingers are
typically involved in nucleic acid binding (and not protein-protein binding), it would be
interesting to do experiments with just zinc-finger fragments.
Although no clear patterns emerged in Figure 10, these data partially support
previous findings that PIE-1 zinc-fingers are not required for interactions with full-length
MEX-5. The second zinc-finger, however, was found to be critical to the interaction.
Overall, these results suggest that multiple regions are likely required in order to properly
fold the interaction domain into its correct orientation, rendering truncation experiments
somewhat futile. A better approach would be to use point mutations, which would avoid
the disruption of the protein’s tertiary conformation.
38
39. When full-length protein-protein experiments were performed (see Table 2), all
four proteins displayed strong interactions with themselves, suggesting that these proteins
are either capable of dimerization or forming larger complexes. Two of the protein
pairings, MEX-5/MEX-6 and PIE-1/MEX-1, had drastically different growth results
depending on which protein was fused to the AD versus the DBD. This is probably due to
the way these proteins bind to the AD and DBD, perhaps sterically inhibiting the binding
sites that either the AD or DBD use to bind each other.
Table 2 also shows that MEX-5 exhibits a stronger interaction with PIE-1 than
does MEX-6 in both directions, especially when PIE-1 is fused to the activation domain.
This finding makes sense given that PIE-1 both segregates and is degraded normally in
MEX-6 knockout mutants, while 58% of MEX-5 knockout mutants are defective for PIE-
degradation. The fact that 100% of mex-5;mex-6 double mutants are defective for both
PIE-1 segregation and degradation, however, implies that MEX-6 does in fact play a role
in the regulation of PIE-1 (Schubert, 2000.) It is important to note, however, that this
result may be due to a difference in expression levels of MEX-5 and MEX-6 rather than
their inherent capacity to bind PIE-1.
Interestingly, when we look at how MEX-5 and MEX-6 interact with MEX-1, it
appears that MEX-6 interacts more strongly with MEX-1 than does MEX-5 in both
directions. This may suggest that MEX-6 plays a larger role than MEX-5 in the regulation
of MEX-1. Since MEX-1 is known to be required for the proper localization of PIE-1,
perhaps MEX-6 exerts its control over PIE-1 in a less direct fashion than MEX-5.
39
40. Figure 18. Sequence Alignment of MEX-5 and MEX-6 using ApE
A sequence alignment of MEX-5 and MEX-6 (Figure 18) shows that the majority
of the homology between these two proteins occurs in the zinc-fingers and C-terminus,
while the amino acid sequences in the N-termini are significantly more varied. The
increased variance in the N-termini supports our findings that the N-terminus of MEX-5
is crucial to its interaction with PIE-1, and that MEX-5 interacts more strongly with
PIE-1 than MEX-6 regardless of whether the protein is fused to the activation domain or
the DNA binding domain.
Tissue culture experiments confirmed that both MEX-5 and MEX-6 interact
strongly with themselves (refer back to Figure 17), producing similar findings to the
yeast-two-hybrid results in Table 2. Furthermore, the tissue culture data (Figure 17) do
seem to support a stronger interaction between MEX-5 and PIE-1 than between MEX-6
40
41. and PIE-1, as was found previously in the yeast-two-hybrid experiments. It is important
to note, however, that the tissue culture experiments have not yet been successfully
repeated. Future experiments would include a repeat of the tissue culture panel included
in this thesis, followed by RNase experiments to determine whether the proteins were
interacting with each other directly or if instead they only appeared to be interacting with
one another because they both bound the same fragment of RNA.
41
42. BIBLIOGRAPHY
Fields, S., and Song, O., 1989, A novel genetic system to detect protein-protein
interactions, Nature, v. 340, p. 245-246.
Gietz, D.R, and Schiestl, R.H., 2008, High-efficiency yeast transformation using the
LiAc/SS carrier DNA/PEG method, Nature Protocols, p. 31-33.
Griffin, E. E., Odde, D. J., and Seydoux, G., 2011, Regulation of the MEX-5 gradient by
a spatially segregated kinase/phosphatase cycle, Cell, v. 146, p. 955-968.
Guedes, S., and Priess, J. R., 1997, The C. elegans MEX-1 protein is present in germline
blastomeres and is a P granule component, Development, v. 124, p. 731-739.
Invitrogen, 2005, ProQuest Two-Hybrid System: A sensitive method for detecting protein-
protein interactions.
Lyczak, R., Gomes, J.E., and Bowerman, B., 2002, Heads or tails: cell polarity and axis
formation in the early Caenorhabditis elegans embryo, Developmental Cell, v. 3, p.
157-166.
QIAGEN, QIAprep Miniprep Handbook, May 2004.
Reese, K.J, Dunn, M. A., Waddle, J.A., and Seydoux, G., 2000, Asymmetric segregation
of PIE-1 in C. elegans is mediated by two complementary mechanisms that act through
separate PIE-1 protein domains, Molecular Cell, v. 6, p. 445-455.
Schubert, C. M., Lin, R., Vries, C.J., Plasterk, R. H. A., and Priess, J. R., 2000, MEX-5
and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos,
Molecular Cell, v. 5, p. 671-682.
Tenlen, J. R., Molk, J. N., London, N., Page, B. D., and Priess, J. R., 2008, MEX-5
asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent
phosphorylation, Development, v. 135, p. 3665-3675.
TGR BioSciences, HEK 293 Cells, http://www.tgrbio.com/cancer-cell-lines-primary-cell-
cultures/cell-models-hek-293-cells.html, (cited on November 2, 2012).
Worm Classroom, A short history of C. elegans research and A model experimental
system: properties of C. elegans, Laboratory for Optical and Computational
Instrumentation at the University of Wisconsin-Madison., http://
www.wormclassroom.org/short-history-c-elegans-research (cited on September 16,
2012).
42
