Caenorhabditis remanei As The Perfect “Aging” Organism? Genetic Variation
For Lifespan, Oxidative Stress Response, And In The Insulin Signaling
Pathway
Rose Reynolds, Richard Jovelin, Jennifer Comstock, Tyrel Love, Patrick Phillips
University of Oregon, Eugene, Oregon, USA
There is a growing consensus that one of the most promising methods for advancing
our understanding of complex biological processes will be to examine how those processes
function in a natural system. For the genetic basis of longevity and human disease, this
understanding may be particularly advantageous, yielding information on natural allelic
or gene expression differences that produce individuals that are healthier longer, without
negative pleiotropic effects. The model nematode Caenorhabditis elegans has served as
one of the most powerful systems for uncovering conserved mechanisms through which
the aging process can be manipulated in the laboratory. Although C. elegans is a well
developed model system with flexible genetic and genomic tools, recent studies have
shown it is conspicuously lacking in natural genetic diversity on local and world-wide
scales, suggesting that it will serve as a poor model for studying variation in aging within
natural populations. The closely related soil nematode C. remanei, which is dioecious
(outcrossing) and readily collected in nature, is an ideal complement to C. elegans as a
natural system. The C. remanei genome has been sequenced and is awaiting assembly.
Its genome is comparable in size to C. elegans, but experiences levels of linkage
disequilibrium comparable to Drosophila melanogaster. Significantly, C. remanei is
amenable to the same laboratory and genetic manipulations as C. elegans. In addition,
this species has both males and females, making it a better model for human aging. We
have found significant within-population genetic variation for lifespan and oxidative
stress. In addition, we have found significant levels of molecular genetic variation within
a known aging pathway, the insulin and insulin-like growth factor signaling (IIS) pathway.
This variation, and the tractability of Caenorhabditis systems, makes C. remanei the
ideal model system for studying the genetic complexities likely to characterize naturally
existing individual differences in aging.
Contact: reynolds@uoregon.edu
Lab: Phillips
62 Poster Topic: 01 Aging
Screen for signaling pathways involved in control of aging by the
reproductive pathway in C. elegans
Monika Suchanek, Cynthia Kenyon
UCSF, San Francisco, CA, USA
In C. elegans, germline ablation extends lifespan by triggering the nuclear translocation
of the DAF-16/FOXO transcription factor in the intestine, providing a unique experimental
paradigm for elucidating the molecular nature of signaling pathways involved in control
of aging.
To identified signaling pathways involved in control of aging I screened a signaling
component of C. elegans genome for RNAi clones that interfere with germline signaling
in C. elegans, utilizing both the subcellular localization of DAF-16-GFP in the intestine,
as well as the intensity of fluorescence of a DAF-16 downstream target gene (SOD-3-
GFP) in glp-1 background. Screening of 1650 RNAi clones has led to identification of
183 genes that affected both DAF-16 localization as well as levels of SOD-3 expression.
Careful re-screening of those hits with independent RNAi clones positively verified
136 of those genes. All of those genes were assayed for effects on the life span of the
germline-defective glp-1 worms. 106 of them significantly shortened glp-1 lifespan,
suggesting their importance in the germline signaling pathway. The most interesting of
those genes were further assayed for effect on lifespan of wt worms, and those which
shortened lifespan of glp-1 worms more than lifespan of wt worms were selected for
further (ongoing) analysis.
Contact: monika.suchanek@ucsf.edu
Lab: Kenyon
Poster Topic: 01 Aging 63
Genetic Screens for New Genes Involved in C. elegans Aging
Simona Tescu, Ala Berdichevsky, Bob Horvitz
HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA
Studies of C. elegans and other organisms have identified molecular pathways
important in the control of aging. Research in this field has largely been focused on genes
that normally function to reduce longevity (a loss of gene function extends lifespan),
primarily because of the relative ease of identifying long-lived mutants and of the
difficulties in distinguishing accelerated aging from sickness for short-lived mutants.
We are seeking genes that function to delay the aging of C. elegans. Loss-of-function
mutations in these genes should result in accelerated aging. We performed a genetic
screen for mutants that appear to age prematurely, based on their early accumulation
of intestinal autofluorescence. We screened 20,000 mutagenized haploid genomes and
identified 25 strains that exhibit premature accumulation of gut autofluorescence. We
characterized these isolates for lifespan and other aging-related characteristics, including
behavioral decline and tissue deterioration.
Three of our mutants are alleles of the gene kat-1, which encodes a conserved
ketoacyl-CoA thiolase involved in the last step of fatty acid β-oxidation. kat-1 maps to
chromosome II and appears to be important for normal C. elegans lifespan and aging.
Loss of kat-1 function results in an early increase in intestinal autofluorescence and fat
accumulation, a shortened lifespan and an early decline in pumping and locomotion. We
are also characterizing two other highly fluorescent mutants isolated from the screen,
n5150 and n5153, which map to chromosome IV and fail to complement each other for
increased intestinal autofluorescence. Both n5150 and n5153 accumulate more fat than
does the wild type, as shown by Nile Red staining.
To understand how KAT-1 functions in the control of aging we are seeking suppressors
of the early autofluorescence of kat-1 mutants. We screened 20,000 mutagenized haploid
genomes and identified 31 strains with wild-type intestinal autofluorescence. Some of
these mutants suppress only the intestinal autofluorescence, while others suppress both
the intestinal autofluorescence and the increased fat accumulation of kat-1 worms. We
are currently mapping these suppressors and characterizing their effects on lifespan and
other aging-related characteristics.
Contact: tescu@mit.edu
Lab: Horvitz
64 Poster Topic: 01 Aging
Analysis of the Role of MicroRNAs in the Control of C. elegans Aging
Konstantinos Boulias, Ezequiel Alvarez-Saavedra, Bob Horvitz
HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA
MicroRNAs (miRNAs) constitute a class of small (20-24 nt) non-coding RNAs
found in C. elegans, Drosophila, plants, mammals and other organisms. Studies over
the past few years indicate that miRNAs are critical regulators of gene expression in
diverse biological processes, including developmental timing, cell-fate specification,
cell proliferation and differentiation. The first miRNAs discovered were lin-4 and let-7,
which control the timing of developmental processes in C. elegans.
Since aging can be regarded as a temporally-regulated developmental process, it is
plausible that miRNAs also control aging. The genetic basis of C. elegans aging has been
studied extensively, and a number of genes that define conserved regulatory pathways that
affect lifespan have been characterized. To identify miRNAs that might function in the
regulation of the aging process, we screened our collection of miRNA mutants for those
abnormal in aging. We analyzed deletion alleles of 95 miRNA genes for abnormalities in
lifespan and the response to heat stress. This analysis identified mir-71 as a miRNA gene
that is required for normal lifespan and stress responses, since worms lacking mir-71
are short-lived and hypersensitive to heat shock and oxidative stress. We are currently
investigating the possible role of mir-71 in the control of C. elegans aging.
Contact: boulias@mit.edu
Lab: Horvitz
Poster Topic: 01 Aging 65
SAGE Protocol Combined With Illumina Flow-cell Sequencing (Tag-Seq)
Improves Detection Of Rare Transcripts Associated With Longevity In C.
elegans
Peter Ruzanov, Donald Riddle
Michael Smith Laboratories, University of British Columbia
Serial Analysis of Gene Expression (SAGE) allows extraction of short sequences
(tags) from polyadenylated transcripts, which are concatenated, cloned, sequenced and
matched to the transcriptome for estimating gene expression levels. We used a new
method (Tag-Seq) developed by Martin Hirst, Marco Marra and others at Canada’s
Michael Smith Genome Sciences Centre to improve detection of rare transcripts in
long-lived mutants of C. elegans nematode. Tags were generated using the enzyme
Mme I and subjected to parallel sequencing of more than 2 million tags per library in
an Illumina flow cell, increasing yield of tag sequences 10-fold, while reducing overall
cost by 3-fold. Tag-Seq detected transcripts on average from 11,000 of the 20,000
C. elegans genes, as compared to our previous SAGE libraries (about 100,000 tags
each), which detected about 5,000 transcript species in a library. Detection of abundant
transcripts for ribosomal proteins increased from a mean of 129 tags per gene in SAGE
libraries to over 4,700 in Tag-Seq libraries. Tags for the less abundant transcriptional
regulators increased from a mean of 3 to a mean of 96 tags per gene. With construction
of multiple replicate libraries, we were able to conduct a much more detailed analysis of
transcription profiles for long-lived dauer larvae and adults. Similar to our earlier SAGE
study, we compared the expression data for daf-2 (insulin/IGF-1 receptor) and daf-2(+)
adults and dauer larvae. We identified genes for which the expression was consistently
changed in long-lived worms. The lists appear to show a strong biological relevance as
we observed several ‘signature’ genes among those up-regulated in dauer larvae: cki-1,
aak-2, daf-16 and sod-3 (a well known target of the daf-16 transcription factor) among
the genes consistently up-regulated in daf-2 adults. Having analyzed data for animals
grown in liquid culture or on agar plates, we were able to distinguish between changes
in gene expression associated with altered genotype rather than the environment.
Contact: pruzanov@gmail.com
Lab: Riddle
66 Poster Topic: 01 Aging
Protein Turnover in Long-Lived Insulin/IGF-1-mutants of Caenorhabditis
elegans
Geert Depuydt, Jacques Vanfleteren, Bart Braeckman
Ghent University, Ghent, Belgium
In recent years the importance of protein metabolism in the aging process has become
more eminent. For example, autophagy, the process of lysosomal degradation of macro-
molecules, seems to play a crucial role in both dietary restriction- and insulin/IGF-
mediated lifespan extension in C. elegans (1,2). These findings suggest an important,
yet unresolved role of protein turnover in senescence. It is generally believed that high
protein turnover rates (i.e. high synthesis and degradation rates) is beneficial to the animal
because it removes and replaces damaged protein molecules. As progressive accumulation
of molecular damage in proteins (and other macromolecules) is considered as the major
cause of aging, a decline in these turnover rates is thus expected to contribute to the
aging process.
Mutants that carry a mutation in the gene daf-2 of the insulin/IGF-1 signaling pathway
show a robust increase in life span and, according to the protein turnover hypothesis, are
expected to show enhanced protein turnover. In accordance with this, it was shown that
intact autophagy is necessary for the longevity phenotype of daf-2 mutants (2). In order
to measure the bulk protein degradation as a function of age we conducted a series of
pulse-chase experiments on two sterile long-lived strains (glp-4;daf-2(e1370) and glp-
4;daf-2(m577)) compared to glp-4 daf-16 and glp-4 daf-16;daf-2(e1370) control strains.
35
S was monitored in the TCA-insoluble and TCA-soluble fraction of the worms as well
as in the medium.
We found that protein degradation rates declined drastically with age in the control
strains while in the two long-lived strains, degradation rates seemed to be low over their
whole life span, even tending to rise slightly at advanced age. However, label in the
TCA-soluble fraction was much higher in the long-lived strains compared to the controls,
irrespective of age. This may indicate that intracellular recycling of aminoacids (e.g. by
autophagy) may be strongly upregulated in the daf-2 mutants, resulting in high protein
turnover rates that may not be detected by classical pulse-chase experiments.
Reference List
1. Jia, K. L. and Levine, B. Autophagy Is Required for Dietary Restriction-Mediated Life Span Extension in C-Elegans. Autophagy. 2007; 3:597-599.
2. Melendez, A.; Talloczy, Z.; Seaman, M.; Eskelinen, E. L.; Hall, D. H., and Levine, B. Autophagy Genes Are Essential for Dauer Development and
Life-Span Extension in C-Elegans. Science. 2003; 301(5638):1387-1391.
Contact: Geert.Depuydt@ugent.be
Lab: Braeckman
Poster Topic: 01 Aging 67
Daf-16 Regulates Fat Storage of Nematode Bidirectionally under the
Control of PUFA
Makoto Horikawa, Kazuichi Sakamoto
University of Tsukuba, Tsukuba, Japan
DAF-16, a FoxO homolog of nematode, induces an expression of fatty acid
metabolic genes fat-7 and elo-2, resulting in the accumulation of fat storage. However,
functional interaction among daf-16, fatty acid metabolism and fat storage remains to
be clarified.
Recently, we clarified that introduction of fat-2-RNAi and fat-6-RNAi decreased
fat accumulation of nematode. RNAi of fat-2, fat-6 and elo-2 raised transcriptional
activity of daf-16, particularly, RNAi effect of fat-2 which was oleic acid desaturase
was remarkable. This result contradicts a past report that daf-16 was associated with an
increase of fat storage.
Therefore we examined functional interaction of daf-16 and fat storage. Induction of
dauer formation at 25 degrees centigrade of daf-2 mutant (e1370) resulted in the increase
of fat storage in L2 larva, and the decrease of fat storage in L4 larva. In daf-16 deficit
mutant (mgDF50), fat storage level which reduced by RNAi of fat-2, fat-7 and elo-2,
was restored to an equal level to N2-RNAi (-). Furthermore, transcriptional activity of
daf-16 was suppressed by addition of linoleic acid or alpha-linolenic acids to fat-2 RNAi
worm, and fat storage recovered at the same time.
These results suggest that daf-16 acted to an increase of fat storage in Dauer larva, and
a suppression of fat storage in adult worm. In addition, functional interaction between
daf-16 and fatty acid metabolism may undergo some controls by possibly PUFA (Poly-
unsaturated fatty acids).
Contact: alkerus@yahoo.co.jp
Lab: Sakamoto
68 Poster Topic: 01 Aging
Functional Genomic Analysis of the DAF-2 Insulin-like Pathway
Yongsoon Kim, Patrick Hu
University of Michigan, Ann Arbor, MI, USA
DAF-2 insulin–like signaling regulates dauer arrest and lifespan via a conserved
PI 3-kinase/Akt pathway that inhibits the FoxO transcription factor DAF-16 via
phosphorylation and subsequent exclusion from the nucleus. Forward genetic screens
designed to identify components of DAF-2 signaling are saturated but have failed to
identify known pathway components such as the 14-3-3 protein FTT-2, which binds
to phosphorylated DAF-16/FoxO and sequesters it in the cytoplasm. RNAi does not
phenocopy mutations in most known dauer-constitutive genes, limiting its usefulness
in studying dauer regulation.
We have recently discovered a novel endocrine pathway, the EAK pathway, that
functions in parallel to AKT-1 to inhibit nuclear DAF-16/FoxO activity. To improve the
penetrance of RNAi of dauer-constitutive genes in the daf-2 pathway, we performed
RNAi of known dauer-constitutive genes in eri;eak double mutant backgrounds. In
contrast to wild-type backgrounds, RNAi of daf-2 and ftt-2 in eri-3;eak-4 double mutants
caused high penetrance dauer arrest at 25°C. RNAi of daf-7/TGFβ, daf-9/CYP27A1,
and daf-11/guanylyl cyclase did not cause strong dauer arrest phenotypes at 25°C,
indicating specificity for daf-2 pathway components. A genome-wide RNAi screen for
gene inactivations that cause dauer arrest at 25°C in eri-3;eak-4 double mutants has
identified 62 candidate rds (regulator of DAF-16/FoxO) genes, 44 of which are conserved
in mammals. We are performing secondary screens on these rds candidates to elucidate
their potential role in the regulation of DAF-16/FoxO, dauer arrest, and lifespan.
Contact: pathu@umich.edu
Lab: Hu
Poster Topic: 01 Aging 69
Differentiating direct and indirect transcriptional targets of AGE-1/PI3K
signaling during dauer development in C. elegans
Wendy Iser, Shailaja Rao, Kevin Becker, Cathy Wolkow
NIA, NIH
In C. elegans, an insulin/IGF-I-like signaling pathway regulates dauer arrest and
adult longevity. One major component of this pathway is age-1, which encodes a PI3K
p110 catalytic subunit. Mutations in age-1 cause constitutive dauer arrest and extend
the adult lifespan. These age-1 phenotypes are completely suppressed by loss-of-
function mutations in daf-16, which encodes a FOXO transcription factor. Thus, AGE-1/
PI3K signaling antagonizes DAF-16/FOXO and allows larvae to bypass dauer arrest.
Conditions that reduce AGE-1/PI3K signaling relieve this inhibition and allow DAF-16/
FOXO to direct the expression of dauer and prolongevity genes, including sod-3 and
several hsps. AGE-1 expression within a variety of tissues is sufficient to rescue dauer
arrest and longevity in age-1 mutants. In contrast, DAF-16 regulates sod-3 expression
cell-autonomously (Libina et al., 2003). We have interpreted these findings in a model
proposing that AGE-1 signaling regulates both endocrine and cell-autonomous outputs
(Iser et al., 2007). These AGE-1 outputs may function collaboratively to direct the
organismal responses of dauer arrest and adult longevity.
To determine whether endocrine and cell-autonomous AGE-1 outputs regulate
distinct transcriptional targets, we conducted a microarray analysis of gene expression
in animals with tissue restricted age-1 expression. These animals lacked genomic age-1
activity, but carried integrated trangenes directing either intestine- or neuron-specific
expression of a rescuing age-1 cDNA. This analysis identified several candidate targets
of AGE-1 endocrine outputs that were rescued by either intestinal or neuronal age-1
activity, including hsp-16, cat-4 and cyp-35B1. The expression of these candidates was
examined using transcriptional GFP reporters. Both cat-4 and cyp-35B1 demonstrated
dauer-specific expression patterns, consistent with their regulation by AGE-1 signaling.
While cyp-35B1 appeared to be regulated cell-autonomously by intestinal DAF-16
activity, cat-4 expression appeared to be indirectly regulated by DAF-16. Identification
of the factors promoting dauer-specific expression of these genes may lead to the
identification of pathways that coordinate responses to insulin and IGF-I signaling in
tissues throughout the body.
Contact: wolkowca@mail.nih.gov
Lab: Wolkow
70 Poster Topic: 01 Aging
sod-5; A target gene of DAF-16 transcription factor in Caenorhabditis
elegans
Sumino Yanase1, 2, Yoshihiro Takahashi1, Naohiro Suga1, Naoaki Ishii2
1
Daito Bunka University, Higashi-matsuyama, Saitama, Japan, 2Tokai
University School of Medicine, Isehara, Kanagawa, Japan
We now report that a DAF-16 consensus binding element (DBE) sequence, which
binds the DAF-16 transcription factor, was discovered in the promoter region of sod-5
gene encoding a Cu/Zn superoxide dismutase (SOD) in the nematode Caenorhabditis
elegans (C. elegans). DAF-16 transcription factor is related to regulation of normal aging
and oxidative stress resistance. The DBE position was 192 base pairs (bp) upstream of
the start site for the transcription. In a daf-16 gene null mutant, daf-16(mgDf50) strain,
the sod-5 gene expression as well as sod-3 gene was obviously suppressed compared
with it in wild-type N2. In addition, the phenotype of lifespan in daf-16(mgDf50) animals
was reduced when compared with N2 during normal aging.
Contact: syanase@ic.daito.ac.jp
Lab: Yanase
Poster Topic: 01 Aging 71
Identifying direct targets of DAF-16 using a modified ChIP-SAGE protocol
in C. elegans
Tseten Yeshi, Jim Lund
University of Kentucky
Aging is a complex physiological process regulated by environmental factors and
genetic mechanisms. Microarray experiments have identified in total thousands of
genes with altered expression in long-lived worms strains and as a consequence of
aging in wild type worms. The best established genetic mechanism that affects aging
is the insulin/IGF1 signaling pathway. The DAF-16 transcription factor is the effector
molecule of this pathway and has a well established role in lifespan regulation. While
published microarray studies identify 1,646 genes with altered expression when DAF-16
is activated, a bioinformatic analysis identified 316 genes with atleast one DAF-16
binding element (TTGTTTAC) within 500bp of the start codon, and a ChIP assay found
103 genes directly regulated by DAF-16. The bioinformatic predictions remain to be
experimentally validated and the ChIP assay lack genome-wide coverage.
We are characterizing the direct target of DAF-16 using a modified SAGE procedure
on ChIP DNA in C. elegans. This will identify DAF-16’s direct targets upon heat shock
induction on a genome-wide scale. DAF-16 is nuclear localized via heat shock and the
DNA bound by DAF-16 is chromatin immunoprecipitated using anti-GFP. This ChIP
DNA is then used as the input DNA for SAGE. The tags generated are cloned, sequenced
and mapped back to C. elegans genome to identify direct targets of DAF-16. This data
is valuable in separating the direct targets from the combined direct and indirect targets
of DAF-16 identified by microarray studies.
Contact: tgyeshi@uky.edu
Lab: Lund
72 Poster Topic: 01 Aging
Age-dependent Requirement for DAF-16 Activity in C. elegans Immunity
Matthew Youngman, Dennis Kim
Massachusetts Institute of Technology, Cambridge, MA, USA
The decline in immune function associated with aging, termed immunosenescence, has
been described primarily in vertebrates, with studies focusing on the adaptive immune
system. While the phenomenon of immunosenescence has been described in some detail,
the underlying mechanistic basis is not well understood. Using infection of C. elegans
with the human opportunistic pathogen P. aeruginosa as a model for host-pathogen
interactions, we have initiated studies to define changes in host defense mechanisms
during aging. Here we demonstrate an age-dependent enhanced susceptibility to pathogen
in worms. In a manner directly correlated with their age, older worms are killed by
infection more rapidly than late larval stage animals. Infection of adult worms with a
GFP-expressing strain of P. aeruginosa reveals that aging worms accumulate pathogenic
bacteria throughout the intestine at an accelerated rate. To determine the basis of host
defense during aging, we are testing whether signaling pathways known to play a role
in the innate immunity of larval stage C. elegans also confer resistance to pathogens
later in life. Previous studies have shown that mutations in the insulin-like receptor
DAF-2 that lead to derepression of insulin signaling confer resistance to pathogen in
L4 worms. Our results suggest that the insulin signaling pathway is required for host
defense in an age-dependent manner. Loss of DAF-16 function exacerbates the enhanced
susceptibility to pathogen of older adult animals. Accordingly, overexpression of
DAF-16 confers resistance to pathogen in adult worms, further suggestive of a role for
DAF-16 in host defense during aging. Quantitative analysis of DAF-16 transcriptional
target expression suggests that DAF-16 activity increases throughout aging. Our data
indicate that enhanced basal expression of a subset of immune effectors resulting from
an age-dependent increase in DAF-16 activity is necessary but not sufficient to confer
wild-type levels of resistance to pathogen in older worms. Further studies are underway
to investigate the role of other innate immune signaling pathways during aging and to
identify specific immune effectors required for host defense later in life.
Contact: mjy@mit.edu
Lab: Kim
Poster Topic: 01 Aging 73
The Novel Non-Essential Longevity Gene misc-1 Controls Mitochondrial
Morphology and Apoptosis
Marco Gallo, Donald Riddle
Department of Medical Genetics, The University of British Columbia,
Vancouver, BC, CANADA
We identified misc-1 as a novel longevity gene. It is mainly expressed in the pharynx and
putatively encodes mitochondrial 2-oxoglutarate carrier. MISC-1 & its human orthologue
share 72% identity & 82% similarity at the amino acid level. RNA interference (RNAi)
against misc-1 increases fer-15 average life-span by 20% & daf-2;fer-15 mean life-span by
42%. Animals carrying the knock-out allele, misc-1(tm2793), had an average life-span only
7% longer than wild-type. These effects on life-span do not seem to require daf-16 or skn-1.
misc-1 seems to behave as most genes encoding mitochondrial proteins, for which levels of
gene expression intermediate between wild-type & knock-out result in the largest extension in
life-span. However, unlike most of these genes, misc-1 down-regulation results in increased
life-span only if the RNAi treatment is applied after larval development.
We employed a transgenic line carrying a mitochondrially-targeted form of Green
Fluorescent Protein (GFP) to study the effects of this gene on mitochondrial morphology.
misc-1 RNAi conveyed a mitochondrial fragmentation phenotype in adult N2.
Mitochondrial fragmentation has been associated with apoptosis in C. elegans &
mammalian systems. We therefore performed SYTO12 staining to detect apoptotic bodies
in the germ-line of knock-out animals. Germ-line apoptosis in this strain was two-fold
higher than in N2 control. These results were confirmed using a ced-1::gfp strain in an
RNAi experiment.
We then subjected the misc-1 knock-out worms to different stressors. Knock-out animals
were more sensitive than N2 to juglone, which induces production of reactive oxygen
species. However, they were not more sensitive than controls to thermal stress (both at
35°C & 37°C). Knock-out worms were more resistant than wild-type to 1 mM NaN3, a
mitochondrial poison.
Interestingly, misc-1 is not an essential gene, although it is predicted to be the only C.
elegans gene encoding a 2-oxoglutarate carrier. misc-1(tm2793) animals are phenotypically
indistinguishable from N2, have the same developmental timing & rate of pharyngeal pumping
(as assessed by feeding assays employing fluorescent microspheres). MISC-1 is therefore
an excellent candidate drug target. One model to explain the results obtained thus far is that
reduction of misc-1 activity positively affects life-span by stimulating a purifying selection
in the germ line that conveys healthier mitochondria to the developing embryo.
Contact: mgallo@interchange.ubc.ca
Lab: Riddle
74 Poster Topic: 01 Aging
Regulators of Mitochondrial Morphology Affect Intracellular pH
David Johnson, Keith Nehrke
University of Rochester, Rochester, (NY)
Within living cells mitochondria constantly change shape and size through the
processes of fission and fusion. Mitochondrial dynamics are important for numerous
cellular processes such as apoptosis, energy production, and cell signaling, and
mitochondrial morphology is tightly regulated. Misregulation of mitochondrial dynamics
in humans results in diseases such as Charcot Marie Tooth syndrome and autosomal
dominant optic atrophy. Specifically, mitochondrial fusion in C. elegans is regulated by
the mitochondria associated GTPases encoded by the genes fzo-1 and eat-3. Loss-of-
function mutations in either gene have been shown to cause high levels of mitochondrial
fragmentation in the muscle cells of C. elegans (Kanazawa et al., PLoS Genet. 2008 Feb
29;4(2)). Here, using a combination of RNAi and deletion mutants, we show that the loss
of either fzo-1 or eat-3 is sufficient to reduce intracellular pH (pHi) in both muscle and
intestinal cells. We also show that these genes are epistatic to one another and that they
function cell autonomously to maintain pH homeostasis. Furthermore, failure to rescue
using dichloroacetate suggests that the reduction in pHi is not simply a result of lactic
acidosis. Finally, since intestinal pHi oscillates during defecation (Pfeiffer et al., Curr
Biol. 2008 Feb 26;18(4):297-302), we have examined how morphology regulates dynamic
changes in pHi in live worms. These results implicate mitochondrial morphology as a
possible regulator of pHi and provide new insight into the pathology of human diseases
caused by the altered mitochondrial dynamics in humans.
Contact: david_johnson@urmc.rochester.edu
Lab: Nehrke
Poster Topic: 01 Aging 75
Interrelation Between Compartment Specific Aggregation And Protein
Trafficking
Janine Kirstein, Richard Morimoto
Northwestern University
Neurodegenerative diseases such as polyglutamine repeat disease, Lewy bodies in
Parkinson disease and amyloid fibrils in Alzheimer’s disease share the accumulation
and inclusion of protein aggregates as pathologic feature. The aberrant accumulation
of aggregated proteins affects vital cellular processes. The proposed underlying
mechanisms of the cellular toxicity range from specific protein-protein interactions to the
sequestration of essential proteins by aggregation prone proteins such as polyQ. PolyQ
expansions disrupt the global balance of the protein quality control (PQC) system in
that the aggregation of a single protein species is sufficient to affect the folding state of
metastable proteins (Gidalevitz et al., 2006).However, a previous study demonstrated that
also the cellular environment plays a critical role for the aggregation. PolyQ aggregation
is abolished when its expression is targeted to the ER or the mitochondrion (Rousseau et
al., 2004).The restriction of polyQ aggregation to the cytoplasm and nucleus supports the
hypothesis that the deleterious effects could be due to compartment specific components
interfering with the aggregation of cytotoxic proteins.
Therefore, the overall question I want to address is whether polyQ aggregation in the
cytoplasm affects the functionality of proteins localized in the organelles.
Contact: j-kirstein@northwestern.edu
Lab: Morimoto
76 Poster Topic: 01 Aging
The Relation Between Superoxide Dismutase Activity and Oxidative stress
In Caenorhabditis elegans
Patricia Back1, Filip Matthijssens1, Ryan Doonan2, Joshua McElwee2, David
Gems2, Bart Braeckman1, Jacques Vanfleteren1
1
Ghent University, Ghent, Belgium, 2University College London, London, UK
The oxidative damage theory of aging states that the accrual of molecular damage
from reactive oxygen species (ROS) is the primary cause of aging. To verify this theory
we have manipulated the superoxide dismutase (SOD) activity in C. elegans by genetic
intervention or administration of the SOD-mimetic EUK-8. Subsequently, we determined
the ROS production and oxidative damage. Superoxide production was measured in
isolated mitochondria of these strains by the specific oxidation of dihydroethidium to
2-hydroxyethidium.
Knockout of the main Cu/Zn-SOD isoform (SOD-1) shortens lifespan and reduces
SOD-activity. We found that this deletion does not lead to measurable increases in
ROS production or oxidative damage in isolated mitochondria. Although Cu/Zn-SOD
is mainly found in the cytosol we also localized it in the mitochondrial intermembrane
space by immunoblotting. Loss of mitochondrial Mn-SOD does not appear to affect
ROS production substantially. We therefore conclude that knockout of sod-genes does
not necessarily increases oxidative stress.
It was previously reported that treatment with EUK-8 increases SOD activity in C.
elegans, but the effects on lifespan have been disputed. To our surprise we found that
EUK-8 increases the ROS production in C. elegans. In addition, we show that intervention
that elevates SOD activity levels in E. coli can result in more superoxide production.
Thus, artificial increase in SOD-activity can paradoxically enhance oxidative stress and
oxidative damage.
Contact: patricia.back@ugent.be
Lab: Vanfleteren
Poster Topic: 01 Aging 77
Stress Resistance Phenotypes as an Indicator of Lifespan Potential in
Caenorhabditis elegans
George Chaffins, Jim Lund
University of Kentucky
The free radical theory of aging indicates that one of the primary causative agents
of aging is oxidative stress. During the lifetime of an organism the damage inflicted by
oxidative stress leads to a gradual deterioration of vitality and the eventual death of the
organism. A number of genes that affect lifespan also affect resistance to multiple types
of stress, usually including oxidative stress. We have screened for genes with oxidative
stress phenotypes and tested them for Age phenotypes.
Over five hundred C. elegans genes were screened for altered paraquat resistance. Of
the genes screened, seventeen genes were found to have paraquat and Age phenotypes.
Two genes were found to have increased lifespans: hsp-12.3 and srt-28. srt-28 was
sensitive to paraquat but long-lived, while hsp-12.3 was both resistant to oxidative stress
and long-lived. Typically, small HSPs that are knocked down in expression have reduced
stress resistance and lifespan. However, hsp-12.3 had significant lifespan extension
and stress resistance. srt-28 is a putative chemosensory receptor that has been found
to be expressed mostly in the neurons of the worm. srt-28 may function in detection
and signaling of oxidative stress. Oxidative stress phenotypes are being tested both on
plates and in liquid culture. Genes that have enhanced or reduced stress resistance will
be assayed for Age phenotypes.
Contact: georgechaffins@uky.edu
Lab: Lund
78 Poster Topic: 01 Aging
Disparate Effects of the Five C. elegans Superoxide Dismutases on Dauer
Formation, Stress Resistance and Aging
Ryan Doonan1, Joshua McElwee1, Filip Matthijssens2, Glenda Walker1, Patricia
Back2, Jacques Vanfleteren2, David Gems1
1
University College London, London, UK, 2Ghent University, Ghent, Belgium
The superoxide free radical (O2-) can act as a secondary messenger in cellular signaling
or as a chemical weapon in immune defense, and might also influence aging, either
accelerating it by causing molecular damage, or slowing it by hormetic stimulation of
stress responses. Cytosolic, extracellular and mitochondrial O2- pools are detoxified by
different superoxide dismutase (SOD) isoforms. sod-1 and sod-5 encode cytosolic Cu/
Zn SOD, sod-4 extracellular Cu/Zn SOD and sod-2 and sod-3 mitochondrial Mn SOD.
We have used reverse genetic approaches to investigate the role of each class of SOD
and, by inference, each O2- pool, on C. elegans life history traits, particularly lifespan.
We have also characterized the expression of each sod gene in detail, using real time
PCR, Western blotting, enzyme activity assays and GFP reporter studies.
We report that sod-1 and sod-2 function in reproductive growth, whereas sod-3 and
sod-5 function is largely restricted to the diapausal dauer larva stage. We tested the effect
of deleting each sod gene on lifespan. Only sod-1(0) reduced lifespan, slightly and to a
similar degree in wild-type and daf-2 mutant backgrounds. Moreover, over-expression
of sod-1 slightly increased adult lifespan. Additional over-expression of catalase did not
further increase lifespan. Interestingly, sod-4(0) enhanced daf-2 mutant longevity, and
also the daf-2 dauer constitutive phenotype. This could imply that extracellular SOD
generates H2O2 which enters the cell and activates receptor tyrosine kinase signaling, as
does occur in mammals. sod-2; sod-3 animals, with no Mn SOD, were hypersensitive
to induced mitochondrial oxidative stress (hyperoxia) yet normal lived under normoxic
conditions. This strongly implies that intra-mitochondrial superoxide does not contribute
to aging in C. elegans.
Our results imply that each O2- pool exerts a different effect on lifespan: the
extracellular pool promotes longevity, the cytosolic pool promotes aging, and the intra-
mitochondrial pool has no effect at all.
Contact: david.gems@ucl.ac.uk
Lab: Gems
Poster Topic: 01 Aging 79
Effects of Superoxide on Physiology, Lifespan and the Redox Proteome of
C. elegans
Daniela Knoefler, Simon Maisel , Ursula Jakob
University of Michigan, Ann Arbor, Michigan, USA
Reactive oxygen species (ROS) like superoxide (O2-) and hydrogen peroxide (H2O2) are
continuously generated during cellular respiration. Tightly regulated antioxidant machineries
prevent the accumulation of excess ROS, which can otherwise damage lipids, proteins and
DNA and severely impair integrity and functionality of cells. The free radical theory of aging
has postulated that the accumulation of oxidative damage in macromolecules is cause for the
observed age-related decline of cells and tissues. Excessive ROS are also thought to be involved
in inflammatory processes such as atherosclerosis and rheumatoid arthritis, degenerative
processes like Alzheimer’s and Parkinson’s disease as well as cardiovascular diseases, diabetes
and cancer.
Aim of our study is to explore the role of superoxide in the aging process of our model
system C. elegans. To begin to understand how superoxide affects physiological processes
and behaviour in C. elegans and to identify the proteins that might be superoxide-sensitive,
we treated synchronized L4 larvae with a short bolus of the superoxide generating herbicide
paraquat. Then, we assayed progeny production, movement and life span of C. elegans at
different temperatures. We discovered that short-term treatment of C. elegans with sublethal
paraquat concentrations caused a substantially reduced progeny production within the first two
days of their reproductive phase, after which slightly higher progeny production was observed in
the paraquat treated animals as compared to the control group. Surprisingly, while the mobility
of the animals was not immediately affected by the paraquat stress treatment, paraquat-treated
worms began to show age-dependent movement defects at much earlier time points than non-
treated animals. This shortened motility span coincided with a substantially shortened mean
life span of paraquat-treated C. elegans. These results provide first evidence that a short-term
exposure to severe superoxide stress at very early stages in the life time of an organism might
be sufficient to significantly affect life quality and life span of the organism.
We then used our recently developed mass spectrometric approach OxICAT to determine
proteins, whose thiol groups are substantially affected by the paraquat treatment. We identified
about 30 peptides that were prone to oxidation in paraquat-treated worms. Preliminary analysis
revealed that among others, the 40S ribosomal protein S2 and the Serine/ threonine-proteine
kinase Akt-1 were substantially thiol-oxidized as compared to the non-treated control group.
We will now use these proteins as markers to investigate if and when during its lifetime, C.
elegans becomes exposed to severe and potentially life span determining superoxide-stress.
Contact: knoefler@umich.edu
Lab: Jakob
80 Poster Topic: 01 Aging
Peroxiredoxin-2 Plays a Crucial Role in the Oxidative Stress Resistance of C.
elegans
Maike Thamsen, Caroline Kumsta, Ursula Jakob
University of Michigan, Ann Arbor, MI, USA
Aging is a complex physiological process and numerous aging theories have been
proposed. One of the leading models is the free radical theory of aging, which suggests
that the accumulation of reactive oxygen species (ROS) is causally linked to aging and cell
death. Aging cells have been found to accumulate proteins with oxidative modifications,
including side chain carbonylation and thiol modifications. It is this oxidative damage to
specific cellular proteins that is often proposed to constitute one of the major mechanisms
that link oxidative stress to age-associated loss of critical physiological functions.
We have now started to monitor and visualize the effects of oxidative stress treatment
of C. elegans using 2D gel electrophoresis. This analysis revealed a number of proteins
that show substantially increased levels of oxidative protein modifications. One of these
proteins is the highly abundant PRDX-2, a 2-Cys Peroxiredoxin, which is responsible for
the detoxification of peroxides. We found that short-term exposure of synchronized L4
larvae to sublethal concentrations of H2O2 leads to the rapid over-oxidation of PRDX-2’s
catalytic cysteine. This reversible overoxidation leads to the inactivation of PRDX-2’s
peroxidase activity and apparently turns PRDX-2 into a molecular chaperone. Prdx-2
knockout worms show substantial delays in oxidative stress recovery and reveal a
significantly shortened lifespan at 15°C. To dissect the peroxidase function of PRDX-2
from its chaperone activity, we began to analyze the role of PRDX-2 in sestrin knockout
worms. Sestrins are a class of proteins, which have recently been shown in human
colon carcinoma (RKO) cells to selectively reduce the overoxidized form of 2-Cys
peroxiredoxins. Preliminary evidence suggests that sestrin knockout worms accumulate
PRDX-2 in the overoxidized form, indicating that sestrin might be the dedicated PRXD-2
sulfinic acid reductase in C. elegans. Interestingly, both prdx-2 and sestrin knockout
worms reveal similar phenotypes, indicating that it is indeed the peroxidase activity of
PRDX-2 that plays the major lifespan determining role in C. elegans.
Contact: mthamsen@umich.edu
Lab: Jakob
Poster Topic: 01 Aging 81
Loss of Superoxide Dismutase Increases Sensitivity to Oxidative Stress but
does not Decrease Lifespan
Jeremy Van Raamsdonk, Siegfried Hekimi
McGill University, Montreal, Quebec, Canada
The free radical theory of aging postulates that aging results from oxidative damage
to macromolecules which accumulates over time. It has been proposed that the main
sources of this oxidative stress are reactive oxygen species (ROS) generated in the
mitochondria. In order to safely eliminate ROS, cells produce a number of detoxifying
enzymes including superoxide dismutases (SODs) which convert superoxide to
hydrogen peroxide. In C. elegans there are five different SODs. SOD1, SOD2 and
SOD4 are the primary cytoplasmic, mitochondrial and extracellular SODs respectively.
In addition, worms have inducible expression of SOD3 in the mitochondria and SOD5
in the cytoplasm. In yeast, flies and mice, elimination of either SOD1 or SOD2 results
in decreased lifespan or lethality, while the loss of extracellular SOD does not affect
lifespan. In this study, we examine the effect of individual deletions in each of the five
C. elegans SOD genes on lifespan. Unlike other model organisms, we find that none of
the SOD deletion mutants show decreased lifespan compared to wild-type N2 worms.
To ensure that elimination of individual SODs has a functional effect on the worms, we
assessed sensitivity to oxidative stress using paraquat or juglone. With both compounds
we found that sod-1 and sod-2 worms have increased sensitivity to oxidative stress, while
sod-3, sod-4 and sod-5 worms survive as well as wild-type. Thus, our results clearly
show that increasing sensitivity to oxidative stress through deletion of SODs does not
result in decreased lifespan. We are currently using real-time PCR to determine whether
the normal lifespan in the SOD mutants is at least partially explained by compensatory
upregulation of one of the other sod genes. In addition, we will present lifespan results
for worms lacking combinations of two SODs and the sod-1; sod-2; sod-4 triple mutant,
which is viable despite lack all three of the primary SOD enzymes.
Contact: jeremy.vanraamsdonk@mcgill.ca
Lab: Hekimi
82 Poster Topic: 01 Aging
Functional characterization of PNC-1 in aging and stress
Juan Carmona1, Shaday Michan1, Anne Hart2, David Sinclair1
1
Paul F. Glenn Laboratories for the Biological Mechanisms of Aging,
Department of Pathology, Harvard Medical School, Boston, MA, USA,
2
Massachusetts General Hospital Cancer Center, Charlestown, MA &
Department of Pathology, Harvard Medical School, Boston, MA, USA
In yeast cells, Pnc1 (nicotinamidase) is an evolutionarily conserved enzyme in the
NAD+ salvage pathway and a master regulator of aging, as (1) expression is induced
by environmental stresses and (2) is necessary and sufficient for lifespan extension by
caloric restriction and stress. Yeast Pnc1 is thought to control the sirtuin family of NAD+-
dependent deacetylases, enzymes previously implicated in aging and stress. Given this
work, we hypothesized that PNC-1 in C. elegans may play a role in longevity and stress
protection, and that specific tissues may be important for these biological effects.
To study the enzymatic activity of PNC-1 in vitro, recombinant protein was
expressed in bacterial cells, purified, assayed, and shown biochemically to have robust
nicotinamidase activity. Moreover, site-directed mutagenesis of a conserved residue in
the active site of PNC-1, as predicted by yeast Pnc1 crystallographic data, abolishes
nicotinamidase enzymatic function. PNC-1 over-expressing transgenic animals were
generated, employing a ubiquitous driver, to study the biological effects of over-
expression in vivo. Using previously described dietary assays, PNC-1 over-expressing
animals were tested for longevity and found to outlive controls. To assess a conserved
role in stress responses, PNC-1 over-expressing animals were subjected to an established
salt stress paradigm, to which they were remarkably resistant, suggesting that PNC-1 may
play an important role in integrating nutritional signals and environmental conditions
to determine lifespan.
Lastly, to pinpoint sites of endogenous expression, a pnc-1 promoter fragment was
cloned into a green fluorescent protein reporter system; green expression was observed in
the pharynx and specific neurons. Also, epistasis studies are in progress to address how
pnc-1 acts in relation to known regulators of aging. Collectively, all of these approaches
will better define conserved pro-survival pathways across different organisms, which
will contribute significantly to our basic understanding of the biology of aging.
Contact: carmona@fas.harvard.edu
Lab: Sinclair
Poster Topic: 01 Aging 83
Proteomic Study of Age-Related Protein Aggregation
Della David, Jonathan Trinidad, Alma Burlingame, Cynthia Kenyon
University of California, San Francisco
Misfolded or oxidatively-damaged proteins are not only functionally impaired but are
also prone to aggregate. Protein aggregation is a hallmark of several neurodegenerative
diseases. The main known risk factor for sporadic neurodegenerative disorders is aging.
Although protein aggregation in the context of neurodegenerative diseases has been
studied extensively, little is known about protein aggregation during normal aging.
Interestingly, in long-lived Caenorhabditis elegans mutants, both protein quality control
and disposal are modulated. Therefore, we hypothesize that the removal of misfolded or
oxidatively-damaged proteins is impaired during the process of normal aging, resulting
in the accumulation of aggregated proteins.
Using tandem mass spectrometry and chemical labeling, we compared aggregation-
prone proteins extracted from young and middle-aged adult C. elegans. We identified
proteins prone to aggregate with age in the somatic and the reproductive tissues. Specific
categories of proteins, such as secreted proteins, were over-represented implying that
not all proteins have the same propensity to aggregate during aging.
Overall these results suggest that protein aggregation is an inherent part of aging. With
functional assays, we plan to investigate whether aggregation is harmful to the organism
and has an impact on neurodegenerative-disease aggregation-related pathology.
Contact: della.david@ucsf.edu
Lab: Kenyon
84 Poster Topic: 01 Aging
Identification of the rate of motor activity decline as a lifespan predictor by
machine vision
Ao-Lin Hsu1, 2, Zhaoyang Feng3, Meng-Yin Hsieh2, X. Z. Shawn Xu1, 4
1
Department of Molecular & Integrative Physiology, University of Michigan,
Ann Arbor, MI, USA, 2Department of Internal Medicine, Division of Geriatric
Medicine, University of Michigan, Ann Arbor, MI, USA, 3Department of
Pharmacology, Case Western University, Cleveland, OH, USA, 4Life Sciences
Institute, University of Michigan, Ann Arbor, MI , USA
One challenge in aging research concerns identifying physiological parameters or
biomarkers that can reflect the physical health of an animal and predict its lifespan.In
C. elegans, a model organism widely used in aging research, motor deficits develop in
old worms.. Here we employed machine vision to quantify worm locomotion behavior
throughout lifespan. We confirm that aging worms undergo a progressive decline in
motor activity, beginning in early life. Importantly, the rate of motor activity decline
rather than the absolute motor activity in the early-to-mid life of individual worms in
an isogenic population inversely correlates with their lifespan, and thus may serve as
a lifespan predictor. Long-lived mutant strains with deficits in insulin/IGF-1 signaling
or food intake display a reduction in the rate of motor activity decline, suggesting that
this parameter might also be used for across-strain comparison of healthspan. Our work
identifies an endogenous physiological parameter for lifespan prediction and healthspan
comparison.
Contact: aolinhsu@umich.edu
Lab: Hsu
Poster Topic: 01 Aging 85
A New Transporter Acts As A Common Mediator In Multiple Lifespan
Pathways
Laurent Mouchiroud1, Laurent Molin1, Prasad Kasturi1, Damien Roussel2, Claude
Duchamps2, Marc Billaud1, Florence Solari1
1
Université Claude Bernard Lyon1, CNRS UMR5201, Lyon, France,
2
Université Claude Bernard CNRS UMR5123, Villeurbanne, France
In a screen for genes that extend lifespan in a daf-18 dependent manner, we identified
a new gene whose mutation increases average lifespan by 40%. Inactivation of this gene
during larval development or adulthood is sufficient to increase the average lifespan.
We then tested for its genetic interaction with genes in the well-characterized lifespan
pathways. Our results show that the extension of lifespan is daf-16, sir-2, pha-4 and
aak-2 dependent. Furthermore we found that like mitochondrial mutants that extend
lifespan, mutants for this gene are both resistant to hydrogen peroxide and sensitive to
paraquat.
This gene encodes a transporter with several homologues in mammals that are
involved in the transport of different metabolites and expressed in diverse tissues. We
are now investigating its expression pattern and biochemical specificity for metabolites.
In conclusion this study should allow us to define a physiological mechanism shared by
insulin, caloric restriction and mitochondrial pathways for lifespan regulation.
Contact: laurent.mouchiroud@sante.univ-lyon1.fr
Lab: Mouchiroud
86 Poster Topic: 01 Aging
The Role of Arrestin in IGFR Signaling using C.elegans as a Model System
Aimee Palmitessa, Jeff Benovic
Thomas Jefferson University, Philadelphia, PA, USA
Arrestins are multi-functional proteins that play an important and expanding role
in the regulation of numerous GPCR signaling pathways. Loss of non-visual arrestin
in most organisms results in embryonic lethality, hindering attempts to gain insight
into mechanisms regulating the in vivo function of this protein. Previous studies have
demonstrated that the C.elegans genome encodes for a single arrestin (ARR-1) and that
the loss of ARR-1 is not lethal. We are using C.elegans as a model system to understand
the in vivo consequences of arrestin function, specifically the role of ARR-1 in IGFR
signaling pathways (a non-classical GPCR signaling pathway). The IGFR (DAF-2)
insulin-like signaling pathway in C.elegans has been shown to regulate development,
longevity, metabolism and response to stress. arr-1 mutant animals display phenotypes
similar to DAF-2 mutant animals including defects in longevity, development, dauer
formation and sensitivity to oxidative stress. Using transgenic animlas, arr-1 double
mutant strains and RNAi treated animals, we show that ARR-1 positively regulates DAF-2
signaling in vivo. Our data indicates that ARR-1 is downstream of DAF-2 and upstream
of DAF-18(PTEN) and DAF-16(FOXO). ARR-1 also binds to MPZ-1, a multi-PDZ
domain containing protein that interacts with DAF-18. We observe that the interaction
between ARR-1 and MPZ-1 is critical for the arr-1 mutant longevity phenotype. Overall,
our data suggests that ARR-1 plays a role in regulating DAF-2 signaling. Using this
combination of genetic and biochemical methods we hope to further expand the role of
arrestin in IGFR/insulin-like signaling using C.elegans as a model in vivo system.
Contact: aimeep_72@yahoo.com
Lab: L
Poster Topic: 01 Aging 87
Expression of Seven Transmembrane Receptors in C. elegans and their
Regulation by Exposure to Blueberry Extract
Anne Logie, Mark Wilson, Cathy Wolkow
National Institute on Aging, Baltimore, MD, USA
A complex polyphenol mixture from blueberries has been shown to increase lifespan
and thermotolerance in Caenorhabditis elegans. Microarray analysis identified a number
of seven transmembrane chemoreceptor genes that changed expression in blueberry
treated animals compared to untreated controls. Seven transmembrane chemoreceptors
have an important role in detecting chemical cues within the environment that stimulate
important biological functions in C. elegans. To identify cell types that may be affected by
blueberry polyphenol, we used molecular cloning techniques to characterize the expression
patterns of the 10 most strongly responsive seven transmembrane chemoreceptor genes
in worms. Three of eight blueberry responsive seven transmembrane chemoreceptors
expressed at detectable levels in neuronal tissue of transgenic worms.
Contact: logieac@nia.nih.gov
Lab: Wolkow
88 Poster Topic: 01 Aging
Telomeric Length and Lifespan in Caenorhabditis elegans
Moon Cheol Park, Daechan Park, Junho Lee
Research Center of Functional Cellulomics, Institute of Molecular Biology and
genetics, Department of Biological Sciences, Seoul National University, Seoul,
Korea
Telomeres are multifunctional elements that protect chromosomal ends. Telomere
length affects lifespan and stress resistance. In our previous report, we found that the
increase of telomere length extended organismic lifespan.
To investigate the effect of the telomere length on global gene expression profile, we
performed microarray experiments using RNA from long telomere-containing worms and
short telomere-containing worms. We identified genes that showed expression difference
between control and long telomere-containing worms. We performed real-time RT PCR
to confirm microarray data. We examined the expression patterns of these genes and
measured lifespan of transgenic worms and RNAi worms.
Contact: leo0717@snu.ac.kr
Lab: Lee
Poster Topic: 01 Aging 89
Screening natural product prototypes for activity using C. elegans
John Peloquin
DiamondV
Diamond V Mills has produced innovative and effective natural fermentation products
for more than 64 years with our complex proprietary fermentation processes. Recently,
Diamond V’s Innovation Program has generated a number of new product prototypes we
must evaluate for biological activity and efficacy. Discovery and development of novel
health and nutrition products for animal and human markets involves many economic
and logistical challenges. Perhaps most importantly, common vertebrate models (in
vitro & in vivo) are less than optimal when screening multiple prototypes in the early
stages of product development. Because of these challenges, Diamond V developed a
medium-throughput screen using Caenorhabditis elegans to help identify promising new
product prototypes. Previously, C. elegans was shown to be an excellent whole-animal
model for host-pathogen interactions (Moy et al. 2006) and to show the influence of
natural products on aging (Wilson et al. 2005). As in these two studies, we presently
rely on lifespan alteration as an indicator of biological activity when comparing our
prototypes to appropriate controls. We further investigate the most promising of these
prototypes in targeted vertebrate models (in vitro and in vivo) to better understand their
biological and health effects. Our C. elegans model has helped Diamond V increase the
efficiency and cost-effectiveness of the early stages of new product development. With
our worm-based assay, we can reduce our use of vertebrate animals, ensure efficacy
of our products, and increase the efficiency and cost-effectiveness of our new product
development process.
Contact: jpeloquin@diamondv.com
Lab: Peloquin
90 Poster Topic: 01 Aging
Changing of the protein folding capacity during aging process in C. elegans
Min Zheng, F. Ulrich Hartl
Max-Planck Institute of Biochemistry, Martinsried, Germany
We are employing C.elegans as a model for investigating how the functional capacity
of the protein folding machinery changes during the aging process. Transgenic animals
expressing firefly luciferase as a chaperone-dependent reporter protein have been
constructed. Our results show that luciferase expressed in the C.elegans muscle cells is
functionally active as determined by luminescence assay. Initial experiments suggest that
the specific activity of luciferase expressed in muscle cells decreases when comparing
young adult animals with 12 days old worms at 25ºC, consistent with the notion that
general chaperone capacity decreases during aging.
Contact: zheng@biochem.mpg.de
Lab: Hartl
Poster Topic: 01 Aging 91
Aggregation Suppressor Screen as a Tool to Study PolyQ Induced Toxicity
Maria Catarina Silva1, 2, Monica Beam1, Happy Thakkar1, Susan Fox1, Margarida
Amaral2, Richard Morimoto1
1
Northwestern University, Evanston IL, USA, 2Universidade de Lisboa
Faculdade de Ciencias, Portugal
The hallmark characteristics of protein conformational disorders are the visual
appearance of protein aggregates and associated with this, cellular toxicity. However,
whether aggregates or inclusion bodies are pathogenic, incidental, or perhaps even
protective is still controversial, with recent findings suggesting that discrete oligomeric
species are better correlated with toxicity. To clarify the events that lead to toxicity, we
took advantage of the tools offered by C. elegans as a model system for human disease, to
address the relationship between polyglutamine (polyQ) biochemical states and toxicity.
We performed a genome-wide RNAi screen for suppression of polyQ aggregation,
and subsequently scored each modifier gene for effects on toxicity. In addition to the
expected modifiers that suppressed both aggregation and toxicity, some had either no
effect or enhanced toxicity. These results, in turn, provided an opportunity to examine
the oligomeric states of polyQ proteins using a number of biochemical and biophysical
assays including fluorescence correlation spectroscopy (FCS). FCS could not uncover any
consistent shifts in patterns of oligomeric species that could suggest a clear correlation
with toxicity. This reveals that polyQ toxicity is not due to the appearance of a single
dominant toxic species. Our results are consistent with a proposal of multiple oligomeric
species and a distributed process of aggregation-induced toxicity.
Contact: d-lima@northwestern.edu
Lab: Morimoto
92 Poster Topic: 02 Age-Related Disease
Deciphering The Chaperone Code For Protein Homeostasis Using C. elegans
Models
Cindy Voisine, Michael Schieber, Kai Orton, Richard Morimoto
Northwestern University, Evanston, (IL), USA
Misfolded and aggregation-prone proteins challenge the capacity of molecular
chaperone and clearance machines to restore cellular homeostasis. Molecular chaperones
are universally expressed in all organisms and assist in protein synthesis, folding,
and translocation and are essential for relieving cellular stress. We are conducting a
comprehensive analysis of the chaperone potential encoded within the genome of the
metazoan C. elegans to identify chaperone networks that maintain protein homeostasis
in vivo. Using a bioinformatics approach, we have compiled a list of approximately 200
putative chaperones and co-chaperones corresponding to ten gene families with multiple
members. We hypothesize that key chaperones are required to maintain homeostasis
under various types of chronic stresses while specialized networks of interactions respond
to specific proteotoxic challenges. Using established C. elegans models expressing
polyglutamine or Aβ1-42, we have identified shared and unique chaperones that regulate
protein homeostasis. RNAi analysis targeted against individual chaperones suggests that
only a small subset alters homeostasis in both models. These candidates make up the
major cytoplasmic chaperone machinery, including Hsc70, Hsp90 and subunits of the
chaperonin complex. However, unique chaperone and co-chaperone proteins are required
for homeostasis in each model. Furthermore, combinatorial RNAi studies are revealing a
larger network of genetic interactions between individual chaperones that collaborate to
maintain protein homeostasis. Our studies are defining a dynamic network of chaperones
that respond to the chronic expression of aggregation-prone disease proteins.
Contact: c-voisine@northwestern.edu
Lab: Morimoto
Poster Topic: 02 Age-Related Disease 93
Identification of Regulators Governing Lipid Homeostasis in C. elegans
Veerle Rottiers1, 2, Amy Walker1, 3, Karen Jiang1, Fajun Yang1, 2, Anne Hart1, 3,
Anders Näär1, 2
1
Massachusetts General Hospital Cancer Center, Charlestown, MA, USA,
2
Department of Cell Biology, Harvard Medical School, Boston, MA, USA,
3
Department of Pathology, Harvard Medical School, Boston, MA, USA
Cholesterol and fatty acids have important functional roles in metazoans, such as
modulating membrane fluidity, serving as signaling molecules, and providing energy
storage in the form of triacylglycerides. Abnormal cholesterol and fat levels have been
linked to prevalent diseases, including atherosclerosis, obesity, type 2 diabetes and
hypertension underscoring the importance of understanding fully how cholesterol and
lipid homeostasis are regulated and maintained.
The mammalian SREBP transcription factors are critical regulators of adipocyte
differentiation and cholesterol and fatty acid homeostasis. We have previously shown in
vitro that SREBP activates gene expression by recruiting CBP/p300 acetyltransferases
and the ARC/Mediator co-activators and that this mechanism of activation is conserved
in C. elegans. Depletion of SBP-1/SREBP and MDT-15/ARC105 results in a “clear”
phenotype, sterility, lethality and slow growth. Triacylglycerides and poly-unsaturated
acids are strongly decreased and stearic acid is increased when sbp-1 and mdt-15 are
knocked down by RNAi. Moreover, the expression of fat-6 and fat-7, the homologs of the
stearoyl-CoA desaturase genes, is dramatically decreased upon depletion of both sbp-1
and mdt-15. fat-6 and fat-7 are involved in synthesis of oleic acid from stearic acid and,
indeed, dietary supplementation with oleic acid markedly improved the phenotypes of
sbp-1, mdt-15 and fat-6/fat-7 RNAi knockdown animals. This suggests a central role of
oleic acid and stearoyl CoA desaturases in SBP-1 function and lipid homeostasis.
To identify novel conserved regulators of fatty acid desaturation and lipid homeostasis
we are performing 2 large scale RNAi screens. The first is an “Oleic Acid Auxotrophy”
screen: based on our data suggesting a critical role of oleic acid regulation in C. elegans
physiology we are currently screening through all the RNAi clones that cause clear,
sterile, lethal and slow growth phenotypes in the C. elegans genome (~1,300 genes)
in search of clones that are rescued upon dietary addition of oleic acid. The second,
genome-wide screen, derives from our finding that the fat-7 stearoyl CoA desaturase is
a key target of central lipid regulators including SBP-1. In this screen, we will identify
genes whose loss enhances or reduces the expression of a FAT-7 promotor::GFP fusion
reporter. We expect to identify key fat regulators that are conserved across species and
may be relevant in human diseases.
Contact: veerle@libertsweb.com
Lab: Näär
94 Poster Topic: 02 Age-Related Disease
In vivo dynamics of human ataxin-3 aggregation and neurotoxicity in C.
elegans neuronal cells is modulated by Q-length and age
Andreia Teixeira-Castro1, Richard Morimoto2, Patricia Maciel1
1
Life and Health Sciences Research Institute (ICVS), School of Health
Sciences, Univ. of Minho, Braga, Portugal, 2Department of Biochemistry,
Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL,
USA
Machado-Joseph disease, like other polyglutamine (polyQ) diseases, is a late onset
neurological disorder characterized by the appearance of misfolded protein species,
aggregates, neuronal dysfunction and cell death. Although the mechanism(s) underlying
the formation of ataxin-3 (AT3) neuronal inclusions are poorly understood, it is becoming
increasingly evident that proteolysis of full-length AT3 is a biological relevant event in
the disease since it occurs and affects aggregation both in vitro and in vivo.
In this study, we developed a new model for AT3 pathogenesis in Caenorhabditis
elegans, in which we observed that expression of the full-length human pathogenic
AT3 alone did not cause aggregation in live neuronal cells. In contrast, expression of a
C-terminal fragment of mutant AT3 resulted in protein aggregation, suggesting that the
aggregation-prone fragment was behaving as “seed” capable of initiating the nucleation
events. Moreover, we studied the dynamics of the sequestration process of full-length
pathogenic and wild-type AT3 into polyQ aggregates and observed that this process occurs
in an age-dependent manner and that there is a tight correlation between aggregation
and neuronal toxicity onset. We are currently using this model to address the molecular
mechanisms of the ageing-dependence of the aggregation and neurological phenotypes,
which could provide clues to the late onset of the human disease.
Contact: accastro@ecsaude.uminho.pt
Lab: Maciel
Poster Topic: 02 Age-Related Disease 95
Genome-wide RNAi Screen Identifies Suppressors of pha-4
Trisha Brock, Susan Mango
Huntsman Cancer Institute, Salt Lake City, UT
The Forkhead box A (FoxA) family of transcription factors play key roles in
digestive tract development as well as metabolism and homeostasis. In C. elegans the
FoxA transcription factor, PHA-4, is required for pharynx development (Gaudet and
Mango, 2002). More recently a post-embryonic role for PHA-4 has also been described
in mediating dietary-restriction-based lifespan extension (Panowski et al., 2007). The
timing, location, and level of PHA-4 expression are vital for both these functions and
pha-4 is tightly regulated; however, little is known about how this regulation is achieved
for PHA-4 or any of the FoxA proteins.
To discover genes that interact with PHA-4 we conducted an RNAi screen for
suppressors of lethality caused by the loss of pha-4. Our initial screen using an RNAi
library of almost 17,000 clones (Kamath et al., 2003) identified 250 potential suppressors
including all four of the previously identified pha-4 suppressors. This list of suppressors
includes genes involved in signaling, transcription, and metabolism along with many
genes with unknown functions. In fact the six strongest suppressors of pha-4, called
sap-1-6, are all unknown. Five of these six are conserved in humans. We are currently
working to characterize these suppressors in terms of expression and their effect on
pha-4 levels and activity. These genes may be conserved regulators of FoxA and play
important roles controlling this transcription factor in embryonic development as well
as post-embryonically.
Gaudet J. and Mango S.E. (2002) Regulation of Organogenesis by the Caenorhabditis elegans FoxA
Protein PHA-4. Science 295, 821-825.
Kamath R.S., Fraser A.G, Dong Y., Poulin G., Durbin R., Gotta M., Kanapin A., Le Bot N., Moreno S.,
Sohrmann M., Welchman D.P., Zipperlen P., and Ahringer J. (2003) Systematic functional analysis of
the Caenorhabditis elegans genome using RNAi. Nature 421, 231-237.
Panowski S.H., Wolff S., Aguilaniu H., Durieux J., and Dillin A. (2007) PHA-4/Foxa mediates diet-
restriction-induced longevity of C. elegans. Nature 447, 550-555.
Contact: trisha.brock@hci.utah.edu
Lab: Mango
96 Poster Topic: 03 Environmental Stress
The Role of O-GlcNAc Modification in Insulin and Nutrient Signaling in
Caenorhabditis elegans
Michelle Mondoux, John Hanover, Michael Krause
NIDDK/NIH, Bethesda, MD, USA
Type II diabetes mellitus (T2DM) is a major health concern, affecting over 20 million
Americans. MGEA5 has been identified as a susceptibility locus for the disease in the
Mexican-American population (Lehman et al. 2005). The gene encoded by the MGEA5
locus is OGA-1, a glucosaminidase that removes the post-translational O-GlcNAc
(O-linked N-acetyl glucosamine) modification from serine and threonine residues on
over 500 proteins in the cell. The action of OGA-1 counteracts the action of OGT-1,
the O-GlcNAc transferase. UDP-GlcNAc pools are regulated in part by glucose levels
and thus these proteins may act as cellular nutrient sensors. In addition to the human
genetic link to T2DM, transgenic mice that overexpress OGT in muscle develop insulin
resistance (McClain et al. 2002), as do cells in culture treated with an inhibitor of OGA
(Vosseller et al. 2002). Unlike post-translational phosphate modifications on serines and
threonines, which are regulated by hundreds of phosphatases and kinases, there is only
a single OGA enzyme and a single OGT enzyme in animals, so it is not surprising that
both these genes are essential in mammals. However, although both proteins are highly
conserved in the nematode C. elegans (63% and 88% identity), knockouts of oga-1 and
ogt-1 are viable, making it a good model system to study the mechanism of O-GlcNAc
modification and its effect on insulin resistance and nutrient signaling.
Previous work in our laboratory (Forsythe et al. 2006) demonstrated that in a daf-2
(e1370) mutant background, the absence of OGT-1 results in fewer dauers, indicating that
these worms are insulin hypersensitive. The absence of OGA-1 in this background leads
to more dauers, modeling insulin resistance. We are currently investigating how OGT-1
and OGA-1 interact with the insulin pathway. The oga-1 and ogt-1 mutants are viable
and have no obvious phenotypes, so we are also currently testing phenotypes that could
be used in a screen. One potential assay would exploit the role of glucose in this pathway.
ogt-1 (ok430) mutants are sensitive to glucose (DC Love and JA Hanover, unpublished
results); these worms have shorter lifespans and smaller brood sizes compared to wild
type when challenged with glucose. We have recently obtained a new allele, ogt-1
(ok1474) and find that this glucose sensitivity phenotype is not allele-specific and that
brood size decreases as glucose concentration increases.
Contact: mondouxm@mail.nih.gov
Lab: Krause
Poster Topic: 03 Environmental Stress 97
The transcriptional response to osmotic stress overlaps with transcriptional
responses to infection and is regulated by the GATA transcription factor
elt-2
Yana Miteva, Anne-Katrin Rohlfing, Sridhar Hannenhalli, Todd Lamitina
University of Pennsylvania
Proper cellular responses to osmotic dyshomeostasis are important for numerous
physiological and pathophysiological processes. While many responses to osmotic
disruption are regulated at the level of transcription, the global suite of genes regulated by
osmotic stress in animals are poorly defined. Using the nematode C. elegans, we profiled
gene expression changes following a time course of exposure to hypertonicity. 330 genes
that exhibit robust and highly significant transcription changes were identified, with over
50 genes exhibiting significant changes within 15 minutes of exposure to hypertonicity.
Osmotically regulated gene expression does not overlap with other types of stress induced
gene expression (heat shock, hypoxia, ethanol, heavy metals, or xenobiotics) but does
show a significant overlap with genes regulated by pathogen infection. We found that
48% (160/330) of osmotically regulated genes are also transcriptionally regulated in at
least one model of infection. Like pathogen regulated genes, osmotically regulated genes
are enriched for the GATA transcription factor binding motif and RNAi knockdown of the
GATA factor elt-2 blocked osmotically induced expression of the glycerol biosynthetic
enzyme gpdh-1 and decreased survival in worms exposed to hypertonic, but not isotonic,
environments. However, inhibition of the nsy-1/sek-1/pmk-1 p38 MAP kinase signaling
pathway that is required for induction of pathogen regulated gene expression did not
effect osmotically regulated gene expression, suggesting that the signaling mechanisms
controlling infection and osmotically regulated gene expression are molecularly distinct.
Together, these data support a model in which the GATA factor elt-2 functions downstream
from multiple signaling pathways as a co-factor for stress-inducible gene expression.
Contact: lamitina@mail.med.upenn.edu
Lab: Lamitina
98 Poster Topic: 03 Environmental Stress
Dauer pheromone component ascr#2 is produced in response to starvation
by wildtype worms, but not daf-9 mutants
Rabia Malik1, Kristen Seim1, Chirag Pungaliya1, Andreas Ludewig1, Nichole
Liachko2, Frank Schroeder1
1
Boyce Thompson Institute and Cornell University, Ithaca, NY, USA, 2Cornell
University, Ithaca, NY, USA
C. elegans secrete a synergistic blend of derivatives of the dideoxysugar ascarylose,
which promote entry into dauer diapause, an alternate non-feeding larval stage that is
highly stress-tolerant and can persist for many months (Butcher et al., 2007). It has been
speculated that this dauer-inducing signal (the “dauer pheromone”) is secreted in response
to unfavorable environmental conditions; however, concrete evidence that any of the
ascarosides are produced in response to stress has been lacking. Here we show that of
the two major components of the dauer pheromone, ascr#2 and ascr#3, ascr#2 is strongly
induced by starvation, whereas production of ascr#3 remains largely unaffected. Content
of ascr#2 and ascr#3 in supernatant and worm pellet from starved and non-starved liquid
cultures was assessed using Differential Analyses by 2D-NMR Spectroscopy (DANS)
(Schroeder et al., 2007). These NMR-spectroscopic analyses showed that non-starved
wildtype (N2) and hlh-13(tm2279) cultures produced large quantities of ascr#3, but did not
produce detectable quantities of ascr#2. On the other hand, starved cultures consistently
produced large amounts of both ascr#2 and ascr#3. For wildtype worms (N2) and hlh-
13(tm2279) mutants, it appears that production of ascr#2 is at least 100-fold upregulated
in response to starvation. In contrast, daf-9(m540) and daf-9(dh6):daf-12(rh411,rh61)
did not produce ascr#2 under either starved or non-starved conditions. Ascr#3 production
appeared unchanged in these two mutant strains. Our results fit well with the observation
that ascr#2 is the more potent dauer-inducer, whereas ascr#3 is more active as a sex
pheromone (Srinivasan et al., 2008). Additional studies suggest that ascr#2 also affects
thermotolerance of adult worms specifically under starvation conditions.
Contact: rm379@cornell.edu
Lab: Schroeder
Poster Topic: 03 Environmental Stress 99
Expression of an HSP::GFP Reporter is Heritable in Isogenic Populations
of C. elegans: Is There an Epigenetic Component to Conditioned
Thermotolerance?
Jim Cypser, Pat Tedesco, Deqing Wu, Sang-Kyu Park, Thomas Johnson
University of Colorado, Boulder, (CO), USA
Life extension mutants of C. elegans display higher expression of the HSP-16 heat shock
protein. Moreover, greater expression of an hsp-16::GFP transcriptional fusion transgene
after hormetic heat conditioning is a strong predictor of increased thermotolerance and
improved survival (Rea et al, 2005; Nature Genetics 37: 894 - 898). Surprisingly, the
expression level of this transgene is reflected in progeny even when all animals are
isogenic, i.e. descended from a single ancestor removed only seven generations. We
are therefore investigating the possibility that conditioned thermotolerance requires
epigenetic factors. Preliminary results suggest that the histone deacetylase sir-2.1 is
required for conditioned thermotolerance. Support for this work was provided by the
National Institutes of Health, grants RO1 AG16219 and KO2 AA00195 to TEJ.
Contact: jrcypser@colorado.edu
Lab: Johnson
100 Poster Topic: 03 Environmental Stress
Genome-wide RNAi screening identifies degradation of protein damage as
a novel hypertonic stress response that prevents protein aggregation
Keith Choe, Kevin Strange
Vanderbilt University, Nashville, (TN), USA
Protein misfolding and aggregation disrupt cellular homeostasis and are associated
with over 20 human diseases. Hypertonic stress is widely assumed to cause protein
misfolding in animal cells, but this has never been demonstrated in vivo and nothing is
known about the fate of proteins presumed to be damaged. To begin addressing these
problems, we screened ~19,000 C. elegans genes by RNAi feeding and identified 49
that are essential for survival during acute hypertonic stress. Almost half of these genes
(22/49) encode proteins that function to transport and degrade damaged proteins, including
components of the ubiquitin-proteasome system and lysosomes. We used a fluorescent
aggregation-prone protein reporter to demonstrate that hypertonic stress causes rapid
protein aggregation in vivo, and that many of the genes that are essential for survival
of hypertonic stress also function to prevent protein aggregation. Western analysis
demonstrated that hypertonic stress also increases high molecular weight ubiquitin
conjugates, suggesting a global change in the ubiquitination state of proteins. Our studies
demonstrate that hypertonic stress induces protein damage in intact animal cells, and
are the first to identify ubiquitin-directed degradation by lysosomes and proteasomes as
essential for survival under hypertonic conditions. These findings provide novel insights
into fundamental mechanisms of hypertonic stress resistance and identify hypertonicity
as a stressor that may contribute to the development of toxic protein aggregates in tissues
and species that are normally exposed to extreme hypertonicity.
Contact: keith.p.choe@vanderbilt.edu
Lab: Strange
Poster Topic: 03 Environmental Stress 101
Investigating Anoxia-Induced Prophase Arrest In C. elegans
Vinita Hajeri, Pamela Padilla
University of North Texas
Cellular oxygen deprivation plays an important role in several human diseases,
including distinctive hypoxic micro-regions of neoplastic and stromal cells enclosed
in most advanced tumors. Furthermore, these hypoxic tumor cells are resistant to most
therapeutic treatments like radiotherapy and chemotherapy. Hence using the C. elegans
embryo as a model to study oxygen deprivation will help in identifying conserved
cellular responses. The C. elegans embryo survives oxygen deprivation (anoxia
hsd-3 > hsd-2.
Contact: dhavalsp@u.washington.edu
Lab: Li
134 Poster Topic: 06 Dauer Development
Nuclear Receptors and Cytochrome P450 Enzymes Play Roles in C. elegans
Dauer Recovery
Kirsten Crossgrove, Peter Sackett, Dylan Nass, Brenda Garland, Adam
Hockensmith
UW-Whitewater, Whitewater, WI
Environmental cues play important roles in the development of both free-living and
parasitic nematodes. For example, in C. elegans, low food, high crowding and high
temperature signal L1 larvae to take an alternative developmental pathway, forming first
L2d and then dauer larvae instead of L2 and L3 larvae. In response to food, less crowding
and lower temperatures, dauer larvae resume reproductive development and molt to form
L4 larvae. A great deal is known about the genetic pathways involved in dauer formation,
but dauer recovery is not as well characterized. Microarray analysis identified a group of
genes whose expression patterns suggest roles in gene regulation during dauer recovery
(Wang and Kim. 2003. Development. 130: 1621-1634). Specifically, these genes are
transiently induced during dauer recovery, peaking approximately two hours after the
introduction of food. The transiently expressed genes include ten nuclear receptor genes
and four cytochrome P450 genes. Nuclear receptors are transcription factors with known
roles in coordinating transcriptional cascades during development, while cytochrome
P450 enzymes have roles in hormone synthesis. We are analyzing these fourteen genes
to see whether they function in dauer recovery. As part of our analysis, we are using
quantitative Real Time PCR to confirm the expression patterns shown by microarray
analysis. The genes we have assayed to date have peaked in expression two hours after
exposure of dauers to food, as expected. To test the role of these genes in dauer recovery,
we are using dsRNAi in daf-2(e1370) and daf-7(e1372) worms. We present data showing
that both nuclear receptor and cytochrome P450 genes are required for dauer recovery.
Contact: crossgrk@uww.edu
Lab: Crossgrove
Poster Topic: 06 Dauer Development 135
Quantitative Approach In Dissecting The Dauer Commitment Decision
Oren Schaedel, Paul Sternberg
California Institute of Technology, Pasadena, (California) USA
The decision to become a dauer larva is dependent on environmental conditions,
which are signaled via several pathways. Dauer frequency increases as conditions shift
from favorable (e.g. high food concentration and low population density) to unfavorable
(e.g. low food concentration and high population density). Dauer formation is regulated
by daf-9 and daf-12, which encode a cytochrome P450 and a nuclear hormone receptor,
respectively. Lipophilic products of DAF-9 work cell non-autonomously, inhibiting
DAF-12 from executing dauer programs.
We hypothesize that DAF-12 is regulated by environmental signals and its abundance
sets a threshold for DAF-9 mediated lipophilic inhibition. In this view the daf-9/
daf-12 ratio prior to dauer/L3 commitment might be an a priori indicator to life history
decisions. To test this hypothesis, we are constructing a quantitative model based upon
defined environmental conditions and our determination of daf-9 and daf-12 expression
levels.
Contact: orens@its.caltech.edu
Lab: Sternberg
136 Poster Topic: 06 Dauer Development
Iowa State University ADVANCE Program’s Collaborative Transformation:
Advancing Women Faculty in STEM Fields
Susan Carlson, Sharon Bird, Bonnie Bowen, Diane Debinski, Carla Fehr, Sandra
Gahn, Florence Hamrick, Kristen Constant, Charles Glatz, Fred Janzen, Lisa
Larson, Jan Thompson, Jo Anne Powell-Coffman
Iowa State University
The goal of the NSF-funded ISU ADVANCE program is to investigate the effectiveness
of a multilevel collaborative effort to produce institutional transformation that results
in the full participation of women faculty in science, technology, engineering and math
fields in the university. Our approach focuses on transforming departmental cultures,
practices, and structures, as well as university policies.
Here, we summarize the findings of the Collaborative Transformation Project with the
3 departments that participated in the first round (Departments of Genetics, Development,
and Cell Biology; Ecology, Evolution, and Organismal Biology; and Material Science and
Engineering) (Bird and Hamrick 2008). A 3-step process for departmental transformation
includes (1) focus groups to discuss aspects of department culture, practice and structure,
(2) needs assessment meetings tailored to meet the needs of individual departments, and
(3) collaborative problem solving sessions involving department faculty and ADVANCE
program leaders. The findings include 6 major issues common to all 3 departments.
Some issues do not specifically address gender or race, but these issues can have a
disproportionate effect on underrepresented groups. Each of the 3 focal departments has
developed strategies for change that address the issues identified in their departments.
Examples of these strategies will be presented.
Bird, Sharon R. and Florence A. Hamrick. 2008. ISU ADVANCE Collaborative
Transformation Project: First Round Focal Department Synthesis Report. Ames, IA:
Iowa State University ADVANCE Program.
http://www.advance.iastate.edu
Contact: japc@iastate.edu
Lab: Powell-Coffman
Poster Topic: 07 Teaching 137
What’s New on WormAtlas and WormImage?
Laura Herndon, Zeynep Altun, Carolyn Norris, Chris Crocker, Tylon Stephney,
David Hall
Albert Einstein College of Medicine
We are updating the WormAtlas website to give it a fresh look. This is the first major
revamping of WormAtlas since its launch in 2002. These changes will start from the
front page and then be implemented throughout the Handbook and many other portions
of the website. Here we will explain the principles of the new organization, and show
you how to find your favorite features. We hope you will find the site simpler to navigate
and we expect it will be more intuitive for beginners.
Inside the WormAtlas website, there will be several major changes. First will be
an improved adult hermaphrodite handbook; it will include several completely
revised chapters and a new one covering the nervous system. Second will be
the launch of a handbook for anatomy of the worm embryo. Third will be the
addition of more data to our slice-by-slice worm viewer, Slidable Worm. Lastly,
we will be adding many new Neuron pages for the male nervous system in
order to highlight new synaptic patterns emerging from the Wired Worm project
conducted together with Scott Emmons.
The WormImage website, which houses thousands of unpublished electron
micrographs and related data, is expanding steadily. It now presents much more data
from mutant animals, particularly for genes affecting the nervous system. We continue
to rely heavily on MRC datasets, but we are also adding more from the Riddle and
Hall lab files, among others. We encourage more laboratories to share their own best
archival TEM and SEM images so that this resource can continue to grow and serve the
C. elegans community. We are very grateful to many labs that have already contributed
ideas, advice and experimental results that are featured on these websites.
This work is supported by NIH RR12596.
Contact: lherndon@aecom.yu.edu
Lab: Hall
138 Poster Topic: 07 Teaching