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Graduate Category: Physical and Life Science Degree Level: Graduate, PhD Abstract ID# 1046
A novel myosin light-chain kinase is required for ovulation in the C. elegans spermatheca
Charlotte
1Department
of Biology, Northeastern University, Boston, MA
ZC373.4 is required for proper
spatiotemporal calcium signaling
B
Figure 1: A Cartoon of human (above) and C. elegans (below) myosin light-chain kinase proteins with
conserved actin binding, calmodulin binding, and protein kinase domains. B The alignment of the kinase
domains (orange in A) for human MYLK and C. elegans ZC373.4 shows a high degree of agreement with
darker greys representing greater similarity.
C
B
whole worm
A
C. elegans reproductive system in
Figure 4: A Comparison of calcium
D
plots normalized to the average
intensity detected prior to ovulation
(F/FO). The top three plots of
ovulations representing worms that
have been fed ZC373.4 RNAi. The
bottom three plots are WT calcium
traces. It is clear that ZC373.4 is
required for normal calcium
oscillations and oocyte transit. B
Knocking down ZC373.4 in worms
causes a decrease in average pixel
intensity of the 30 frames prior to
end
ovulation. C Depletion of ZC373.4 also leads to a lower maximum calcium level compared to wild type. D Kymograms
of both WT and ZC373.4 RNAi treated worms shows the disruption in normal
calcium localization. The distal neck is on the left of each kymogram and the sp-ut
valve is on the right. Conclusions and Future Directions
ZC373.4 regulates the phosphorylation of MLC
A
before ovulation
B
15
C
*
20
Conclusions Based on these data we can conclude that ZC373.4 regulates transit.
ZC373.4 is similar to human myosin light-chain kinase and is likely phosphorylating the
regulatory MLC in the spermatheca. Additionally, ZC373.4 RNAi results in abnormal
calcium signaling during ovulation. . *
*
*
Maximum Intensity (AU)
Average Intensity (AU)
15
Ovulation requires a balance of phosphorylation
10
oocyte trapped
5
Future Directions We aim to understand the functions of the other domains of our new
myosin light-chain kinase by using CRISPR to delete portions of the C-terminal end
which shares little homology to the human MYLK. We also plan to use CRISPR to
fluorescently label ZC373.4 with mKate2 and a FLAG tag. We can then perform imaging
experiments to determine its localization along with pull downs to determine how it is
interacting with other proteins 10
5
Ser19
0
Hypothesis
ZC373.4 is the MLCK that phosphorylates the myosin light
chain and drives spermatheca cell constriction
after ovulation
m
el
-1
1
ZC
37
3.
4
Em
pt
y
el
m
ZC
37
3.
-1
4
1
0
Em
pt
y
Ser19 phosphorylation gives the
myosin complex actin binding and
ATPase capabilities out
start
Figure 2: A Time course of a worm treated with ZC373.4 RNAi. The embryo enters
and is trapped for nearly an hour while the embryo divides in the spermatheca. B
ZC373.4 was depleted using RNAi in worms that suppress the gene ubiquitously
(UN1108) and only in the spermatheca (UN1524). Both worm lines express
fln-1P::GFP to allow visualization of trapped or empty spermathecae on a plate of
freely moving worms. ZC373.4 RNAi caused trapping comparable to plc-1 and
fln-1 RNAi, both of which have been shown to be required for successful
ovulations1,2. UNC-89 is the only other known MLCK in C. elegans and does not
seem to cause trapping. Although LET-502/ROCK can phosphorylate rMLC, it does
not cause significant trapping compared to WT. Within the spermatheca
Maximum intensity is less in ZC373.4
deficient worms WT
Depletion of ZC373.4 causes oocyte trapping in the
spermatheca
Introduction
Coiled-coil tails can form
bipolar filaments B
A
Background calcium is less in ZC373.4
deficient worms ZC373.4 RNAi
A
spermathecaspecific RNAi
Erin J.
1
Cram ZC373.4 shares homology with human myosin lightchain kinase
Abstract
The C. elegans somatic gonad is essentially a single layer tube of
myoepithelial cells that coordinate oocyte maturation and ovulation
through the spermatheca, the site of fertilization. Oocyte entry into
the spermatheca triggers calcium oscillations that culminate in
coordinated actomyosin constriction and expulsion of fertilized
embryos. Scaffold proteins, phosphatases, and kinases are required
for this coordinated contraction of the actomyosin cassette. To date,
there has only been one myosin light-chain kinase (MLCK) identified
in the worm and it is not necessary in C. elegans spermathecal
actomyosin constriction. In a broad RNAi screen, we have identified a
potential MLCK required for successful ovulation, ZC373.4. This
MLCK shares key homology to human myosin light-chain kinase
(MYLK) domains. Depletion of ZC373.4 via RNAi causes oocyte
trapping in the spermatheca. This trapping phenotype is likely due to
a lack of coordination between contractions in the distal end and
opening of the spermatheca-uterine valve. To further characterize this
potential MLCK, ZC373.4 will be fluorescently tagged to identify its
localization within the worm. The protein will also be
immunoprecipitated to conduct kinase assays to analyze its kinetic
activity in vitro. This work will characterize a new MLCK in C. elegans
and further elucidate the mechanism by which cells of the somatic
gonad control ovulation and spermathecal transit.
1
Kelley ,
Figure 3: A Preliminary results show phosphorylated MLC (pS20) localizes
primarily to the apical surface of the spermatheca. (red = actin; green =
phosphorylated MLC) Scale bar = 20μm. B Quantification of empty/WT, ZC373.4,
and mel-11 RNAi conditions shows that phosphorylation levels change between
the different conditions. C Highest phosphorylation occurs when the myosin
phosphatase, MEL-11, is removed. Removal of ZC373.4 results in the least
phosphorylation. References
1.  Kariya, K. I., Kim Bui, Y., Gao, X., Sternberg, P. W., & Kataoka, T. (2004). Phospholipase C epsilon regulate
ovulation in Caenorhabditis elegans. Developmental Biology, 274(1), 201–210. doi:10.1016/j.ydbio.
2004.06.024
2.  Kovacevic, I., Orozco, J. M., & Cram, E. J. (2013). Filamin and phospholipase C-ε are required for calcium
signaling in the Caenorhabditis elegans spermatheca. PLoS Genetics, 9(5), e1003510. doi:10.1371/
journal.pgen.1003510
NIH R01 GM110268 to E.J.C. funded this work