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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
195
233B
Monosaccharide
O
-fucosylation of Notch receptor is required for Notch signaling in
Drosophila
.
Akira Ishio
1
, Tomonori Ayukawa
1
, Naoki Aoyama
1
,
Hiroyuki O.Ishikawa
1
, Tomoko Yamakawa
1
, Takeshi Sasamura
1
, Tetsuya Okajima
2
, Kenji Matsuno
1
. 1) Department of Biological Science and Technology,
Tokyo University of Science, Chiba; 2) Nagoya University Graduate School of Medicine.
Notch (N) is a transmembrane receptor with homology to epidermal growth factor (EGF)-like repeats and mediates cell-cell interactions necessary for
many cell-fate decisions. These EGF-like repeats are
O
-fucosylated by the protein
O
-fucosyltransferase 1 (
O
-fut1), which is essential for N signaling.
However, roles of monosaccharide
O
-fucose modification in N signaling became elusive, because it was proposed that
O
-fucsyltransferase activity-
independent functions of
O
-fut1, per se including N-specific chaperon function and modulation of N endocytosis, could be essential for N signaling in
Drosophila
.
In this study, we showed that monomeric
O
-fucose modification of N was essential for Delta-N but not Serrate-N signaling activity in the signal receiving
cells of imaginal organs in Drosophila. This novel function of monomeric signaling in a subset of organs. In agreement with this finding, we found that lack
of monomeric
O
-fucose modification of N caused a temperature-sensitive neurogenic phenotype in embryos. In addition, disruption of N signaling
associated with the lack of
O
-fucose modification was partly rescued by upregulation of the unfolded protein response.These results suggest that monomeric
O
-fucose modification of N has a novel role for the ligand-dependent activity of N, which collaborates with the proper folding of N.
234C
Regulation of
broad
expression by Notch signaling during the mitotic/endocycle switch in
Drosophila
follicle cells.
Dongyu Jia, Yoichiro Tamori, Wu-
Min Deng. Department of Biological Science, Florida State University, Tallahassee, FL.
During
Drosophila
oogenesis, mediated by multiple signaling pathways, the follicle cells sequentially undergo three distinct cell-cycle programs: the
mitotic cycle (stages1-6), the endocycle (stages 7-10a), and gene amplification (stages10b-14). Activation of Notch signaling at stages 6/7 and inactivation
of it at around stage 10b in follicle cells are essential for the proper two cell-cycle program switches.
broad (br)
, encoding a family of zinc-finger
transcription factors, has been known as an early ecdysone response gene that is pivotal for metamorphosis. During oogenesis, Br is first expressed
uniformly in follicle cells at stages 6/7,then expressed at high levels in two patches at the anterior dorsal region from stage 10b. To determine how the early
pattern of Br in follicle cells is established, we examined the effect of Notch signaling on Br expression. We found that loss of key components of Notch
signaling, such as
Notch, Presenilin, Nicastrin, Suppressor of Hairless
in somatic follicle cells, or
Delta
in germline cells, fails to upregulate Br expression.
Recently, Shvartsman’s Lab at Princeton University identified two non-overlapping enhancers regulate Br expression in response to EGFR signaling in
follicle cells, which are named
Early Enhancer (brEE)
and
Late Enhancer (brLE)
. Expression of brEE reporters is similar to the early pattern of Br, uniform
in follicle cells starting at stages 6/7. Consistently, we found that the
brEE
pattern is disrupted when key Notch signaling components are mutated. The facts
together suggest that Br is regulated by Notch signaling via
brEE
during the mitotic cycle-endocycle (M/E) switch. Clonal analysis and RNAi studies
revealed that follicle cells with loss of
br
function have a mild defect in entering the endocycle at stage 7. In contrast, misexpression of Br isoforms Z1, Z2,
or Z4, but not Z3, prompts follicle cells to exit the mitotic cycle earlier. Currently, we are characterizing the potential function of
br
in mediating different
Notch regulated cellular processes during the M/E transition.
235A
Xylose: A Novel Modulator of Notch Signaling.
Tom V. Lee
1
, Maya Sethi
2
, Jessica Leonardi
1,4
, Nadia Rana
3
, Robert Haltiwanger
3
, Hans Bakker
2
, Hamed
Jafar-Nejad
1,4
. 1) University of Texas Health Science Center, Houston, TX; 2) Hannover Medical School, Hannover, Germany; 3) Stony Brook University,
Stony Brook, NY; 4) Baylor College of Medicine, Houston, TX.
An important post-translational modification identified on the Notch receptors is the addition of
O
-glucose to Epidermal Growth Factor-like (EGF) repeats
by the protein
O
-glucosyltransferase Rumi, a temperature-sensitive activator of Notch signaling.
O
-glucose on EGF repeats can be extended by the addition
of two xylose residues, but the functional role of xylose residues in the regulation of Notch signaling is not known. We have recently identified the
mammalian glucoside xylosyltransferases (GxylT1 and GxylT2) and xyloside xylosyltransferase (XxylT), which add the first and the second xylose residues
to
O
-glucosylated EGF repeats of human Notch, respectively. Here, we show that addition of xylose to
O
-glucose residues negatively regulates Notch
signaling in
Drosophila
. Mutations in the sole
Drosophila
homolog of GxylT1/2,
shams
, result in temperature-sensitive mutant phenotypes compatible with
increased Notch signaling. Genetic interaction studies show that
shams
mutations suppress both
Notch
and
Delta
haploinsufficient phenotypes, but enhance
the
Abruptex
gain-of-function phenotype. Overexpression of xylosyltransferases in
Drosophila
results in Notch-related mutant phenotypes that are
dependent on their catalytic activity. Biochemical and
in vivo
analyses indicate that functionally relevant sites of xylosylation are localized to a specific
region of the
Drosophila
Notch. Finally, mass spectral analysis of Notch EGF repeats indicates that endogenous levels of the
Drosophila
GxylT (Shams) and
XxylT enzymes can only partially xylosylate the
O
-glucosylated Notch EGF repeats. Taken together, these data indicate that altering the level of xylose
occupancy on a specific group of Notch EGF repeats can modulate the strength of the
Drosophila
Notch signaling over a broad range and potentially provide
a novel tool to fine-tune the strength of Notch signaling.
236B
Investigating the role of the NHR2 domain of Neuralized in Notch Signaling.
Sili Liu
1,2
, Julia Maeve Bonner
1,2
, Gabrielle Boulianne
1,2
. 1) Stem Cell &
Developmental Biology, Hospital For Sick Children, Toronto, ON, Canada; 2) Department of Molecular Genetics, University of Toronto, Toronto, ON,
Canada.
In the Notch pathway, one critical step in initiating Notch activation is the endocytosis of the Notch ligands, Delta and Serrate in the signal-sending cell.
Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized or Mind bomb. Neuralized is comprised of a C-terminal RING domain,
which is required for Delta ubiquitination, and two Neuralized Homology repeat (NHR) domains. We have previously shown that a conserved glycine
residue in the NHR1 domain is required for Delta trafficking. Here we show that this mutation also affects binding and internalization of Serrate.
Furthermore, we show that the NHR2 domain is required for Neuralized function and that a point mutation in the corresponding glycine residue in the NHR2
domain (Gly430) abolishes its ligase activity and affects ligand internalization. Lastly, we provide evidence that Neuralized may form oligomers or
intramolecular loops through the two NHR domains. These results demonstrate that the NHR1 domain regulates the interaction of Neuralized with both