Inactive and couch potato mutants of Drosophila:
Locomotor activity vs metabolism
Sherry Lee
Dr. Jadwiga Giebultowicz
Dr. Natraj Krishnan
HHMI Summer Program
Circadian Rhythm
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

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Endogenous
Period (per) and Timeless (tim) genes
Entrained by exogenous cues – light
Drosophila – crepuscular
Dark
Light
Dark
Days
jneurosci.org/content/vol26/issue9/images/large/zns0080614060002.jpeg
Feedback Loop
From Dr. Jadwiga Giebultowicz
Hypoactive Mutants
- Inactive (iav): 70 and 78
- Couch potato (cpo): E023
flybase.bio.indiana.edu
Metabolism
 Intracellular – mitochondria
 Cellular respiration converts glucose to
ATP
C6H12O6 (aq) + 6O2 (g) → 6CO2 (g) + 6H2O (l)
 Waste products
Activity Links to
ROS
Production
↑ activity =
↑ respiration =
↑ reactive oxygen
species (ROS)
www.sapphirebioscience.com
Rhythmic ROS Production –
wild-type
END
Krishnan N., et al. 2008. Biochemical and Biophysical Research
Communications
Objectives
 Rhythmic activity compared to wild-type
controls (CS)
 Rhythmic ROS and ATP productions
compared to wild-type controls (CS)
Hypothesis
Inactive and couch potato flies have less
activity  less metabolism  less ROS
and ATP production.
Activity and Longevity
 DAM activity monitor
with computer
data recording
 Longevity cages
Google images
Mitochondrial H2O2 Production
 Mitochondria isolated from whole bodies
 Hydrogen peroxide microplate assay
Google images
ATP Production
 Mitochondria isolated from whole bodies
 ATP bioluminescence
Google images
Activity Results - LD
 iav and cpo: not hypoactive
Horizontal activity vial
Fig. 1 Activity graph of three genotypes, 78 iav, 70 iav, and E023 cpo.
Activity Results - LD
Fig. 2 CS actogram
Fig. 4 70 iav actogram
Fig. 3 78 iav actogram
Fig. 5 E023 cpo actogram
Activity Results - DD
Fig. 6 CS actogram
Fig. 8 70 iav actogram
Fig. 7 78 iav actogram
Fig. 9 E023 cpo actogram
Activity Results - DD
% rhythmicity in constant darkness:
 78 iav: 50% (mean = 23.4)
 70 iav: 50% (mean = 23.6)
 E023 cpo: 80% (mean
= 27.6)
Climbing Ability
 iav and cpo: impaired negative geotropic
movement
Genotypes
% climbed above
5 cm line
78 iav
70 iav
E023 cpo
CS wildtype
7.50
47.0
83.9
89.7
iav
CS
H2O2 Production Results
 iav: more H2O2 than CS
 cpo: less H2O2 than iav
Rate of H2O2 produced (M/min/mg)
Rate of H2O2 Production at ZT8 and ZT20
0.4
78 iav
70 iav
E023 cpo
CS wild-type
0.3
0.2
0.1
0.0
8
8
20
ZT
20
Fig. 10 Rate of H2O2 production for 78, 70, and E023 mutants and CS
wild-type flies at ZT8 and ZT20.
ATP Production Results
 iav and cpo: more ATP than CS
 cpo: more ATP than iav
Rate of ATP Production at ZT8 and ZT20
ATP produced ( M/g)
0.015
78 iav
70 iav
E023 cpo
CS wild-type
0.010
0.005
0.000
8
8
ZT
20
20
Fig. 11 Rate of ATP production for 78, 70, and E023 mutants and CS
wild-type flies at ZT8 and ZT20.
Longevity Results
 iav: shorter lifespan than CS
 cpo: longer lifespan than iav
Lifespan of CS and Mutant Flies
Fraction survival
1.5
iav1
iav3621
CS wild-type
1.0
TO BE
CONTINUED
0.5
0.0
0
20
40
60
80
Age (days)
Fig. 12 Kaplan-Meyer survival curves for 78, 70, and E023 mutants and CS
wild-type flies.
Discussion
 iav compared with CS
 cpo defects
 Compare results with Hyperkinetic (Hk)
Significance
 Level of beneficial physical activity
 ROS production affects longevity
 Promotion of healthy aging
Acknowledgements
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Dr. Natraj Krishnan
Dr. Jadwiga Giebultowicz
Dr. Kevin Ahern
HHMI Summer Undergraduate Research
Program