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Poster Full Abstracts - Drosophila Models of Human Diseases
Poster board number is above title. The first author is the presenter
235
380B
CREB transcription factors and drug tolerance.
Benjamin R. Troutwine, Yan Wang, Nigel Atkinson. ICMB, University of Texas, Austin, TX.
As a means to understand the mechanisms that underlie addiction, we study the acquisition of drug tolerance in Drosophila. Tolerance is defined as a
reduced response to a drug caused by previous drug exposure, and is considered a core component of addiction. Tolerance is an adaptive process that works
to dampen effects of a drug, but can cause the user to require a larger dose to achieve the desired effect and lead the user toward addiction. Drosophila
acquire tolerance to the sedative effects of ethanol and benzyl alcohol after a single sedation, and previous work in our lab has shown that the BK-type Ca
2+
-
activated K
+
channel encoded by
slowpoke (slo)
is central to this process. Our lab has shown that
slo
is induced following sedation, that artificial induction of
slo
mimics the tolerant state, and that
slo
mutants do not acquire tolerance. Examination of the
slo
transcription control region identified three putative
CREB binding sites, indicating that CREB transcription factors play a role in the drug-induced upregulation of
slo
. It has been shown in a number of systems
that CREB signaling is important for neural processes linked to addiction; including drug reward, withdrawal and tolerance. A former member of our lab
found that after sedation CREB isoforms are differentially regulated, CREB binding at the
slo
transcription control region increased and a CRE-regulated
reporter was induced. Additionally,
Creb2
mutants failed to acquire tolerance and induction of a transgenic CREB repressor isoform
(Creb2b)
blocked the
acquisition of tolerance and
slo
induction. We are using CREB mutants, RNAi constructs, and transgenes to further examine the roles of the CREB genes in
the acquisition of tolerance. Additionally, we have generated an epitope-tagged CREB gene to further explore CREB regulation of
slo
following sedation
and assay interactions with other transcription factors.
381C
Investigating the role of MRL proteins in invasive border cell migration.
Lauren Dodgson, Eleanor Taylor, Daimark Bennett. University of Liverpool,
Institute of Intergrative Biology, Liverpool, United Kingdom.
The invasion of cancer cells into surrounding tissues plays a causal role in tumour progression and is the initial step in tumour metastasis, which clinically
is the most important process in the progression of cancer. For invasion to occur, cells must detach from the epithelium, acquire and regulate both their
motile properties and affinity for other cell types as they migrate to a new location. Several lines of evidence indicate that the Mig-10/RIAM/Lamellipodin
(MRL) family of adapter proteins transduce signals derived from growth factor receptors, via interactions with Ras GTPases and/or phospholipids, resulting
in changes in the actin cytoskeleton, increased lamellipodia protrusion, cell motility and altered cell adhesion properties.
Drosophila
encodes only one MRL
protein, encoded by
pico
, which we previously showed to have a role in the regulation of actin dynamics. Here we report on the role of
pico
in invasive
border cell migration in the
Drosophila
ovary. During oogenesis, a pair of specialised cells differentiates at the anterior end of the egg chamber and recruits
four to eight additional cells to form a border cell cluster. After detaching from their epithelial neighbours, border cells make their way to the oocyte-nurse-
cell border, guided by redundant signalling through the PDGF/VEGF receptor (Pvr) and Egfr. We will present data from live imaging approaches that reveal
the requirement for
pico
in the formation of actin-based protrusions and invasion in this system. This work points to the involvement of MRL proteins in
tumour cell invasion and metastasis and is consistent with their known role in promoting lamellipodia-like structures at the leading edge of invasive cells,
which provide the main driving force for cellular locomotion and invasion.
382A
Analyzing cancer stem cells using the Drosophila ovary.
Rebecca L. Frederick
1
, Allan Spradling
1,2
. 1) Carnegie Institution for Science, Department of
Embryology, Baltimore, MD; 2) Howard Hughes Medical Institute.
A small subpopulation of cells known as cancer stem cells has been documented to sustain a few types of cancer, and their existence has been postulated in
many other cases. Understanding the initial steps in tumorigenesis and whether a particular cancer is maintained by stem cells is critically important for
designing optimal therapeutic approaches. However, the lineage tracing methods that can decisively delineate stem cells in normal or cancerous tissue have
not been broadly applied to mammalian tumors because of technical difficulties. We have used lineage analysis in Drosophila to analyze the cellular basis of
genetically induced ovarian dysplasias. In particular, mutations in the gene lgl transform the somatic follicle cells that surround the germ-line from a
monolayered, polarized epithelium into a disorganized cellular mass. We find that lgl mutant cells display altered growth patterns even before they invade
the follicle interior, a region normally occupied by the germ cells. Instead of remaining in a compact unit like the daughters of a wild-type follicle cell, lgl
sister cells break apart and disperse over a larger region of the follicular surface. While all mutant cells do not grow equally, our results do not support the
existence of highly specialized tumor stem cells. In marker gene expression studies, we observe that lgl dysplasias comprise heterogeneously differentiated
cell populations. Our system also allows us to investigate differentiation state and epigenetic stability of normal and dysplastic cells. Our methods are
applicable to many other types of dysplasias, including cells containing mutations in human oncogene homologs, indicating that Drosophila provides a
robust system for systematically addressing the existence and genetic programming of cancer stem cells.
383B
Tumor suppressor mutations in
pebble
/
Ect2
activate Rac1 and reveal a mechanism of autoregulation.
Jin-Yu (Jim) Lu
1
, Michelle Pirruccello
2
, Ming
Wu
1
, Jose C. Pastor-Pareja
1
, Tian Xu
1
. 1) Dept. Genetics; 2) Cell Biology, Yale Sch Medicine/HHMI, New Haven, CT.
Proto-oncogene
Ect2
, the single human orthologue of
pebble
(
pbl
), is a Rho guanine nucleotide exchange factor required for cytokinesis. However,
overexpressing full-length Ect2 does not lead to transformation, and its oncogenic form has not been found in human cancer. While a mutation (Ect2
T802P
) is
identified in the C-terminus of Ect2 in human breast cancer, its potential role and regulation in cancer remain unclear. Here we report that
pbl
/
Ect2
, despite
being a proto-oncogene, also functions as a tumor suppressor. From a large-scale genetic screen, we identified multiple EMS-induced
pbl
tum
mutations that
promote tumor growth and invasion in cooperation with
Ras
V12
. Distinct from null alleles, which do not cause tumors,
pbl
tum
are partial loss-of-function
alleles with mutations affecting a highly conserved C-terminal region that is also mutated in Ect2
T802P
. Importantly, expressing the Ect2
T802P
-corresponding
mutant Pbl also causes tumor growth and invasion. Genetically,
pbl
tum
mutations decrease Rho1 signaling but abnormally activate Rac1 signaling, suggesting
indirect inhibition of Rac1 by Pbl. Blocking Rac1 or JNK signaling suppresses the tumor phenotype, indicating that
pbl
tum
mutations drive tumor growth and
invasion via the Rac1-Pak-JNK pathway. To determine the function of the C-terminus of Pbl/Ect2, where
pbl
tum
/
Ect2
mutations cluster, we identified a
conserved helical region that contains mitotic phosphorylation sites. We further discovered an intra-molecular interaction between the N- and C-terminus of
Pbl/Ect2, suggesting an autoinhibitory regulation. Significantly, phosphomimetic mutations and serum stimulation both decrease the intra-molecular