Biology Reference
In-Depth Information
“naked” pupae (a detailed description for the procedure and the setup for imaging can
be found in
Zitserman and Roegiers, 2011
). This preparation is then carefully placed
onto a cellulose membrane dish (such as the lumox dish from Sarstedt), with the dor-
sal thorax surface of the pupal epidermis abutting the cellulose membrane. Residual
water brought with the “naked” pupae during dissection helps to “glue” the dorsal
thorax onto the membrane dish. After depositing drops of water on the dish to prevent
desiccation of the “naked” pupae during imaging, the dish is then inverted with the
membrane side up for imaging under the upright Olympus two-photon microscope.
Because plenty of fat body cells reside just underneath the dorsal epidermis at the
thorax, it is convenient to locate those with CARS. Continuous label-free imaging
can be done over many hours with this setup. Chien et al. have used a similar CARS
setup to continuously observe fat body remodeling in live pupae for more than a day.
The development process is unperturbed under the long-term noninvasive label-free
CARS imaging conditions (
Chien et al., 2011
).
4.3
LD FUNCTIONS IN DROSOPHILA PHYSIOLOGY
In the past decade, more than a dozen proteomic studies have identified hundreds of
proteins associated with LDs (
Yang et al., 2012
). These include the LD structural
proteins, lipid synthesis and hydrolyze enzymes and cofactors, sequestered highly
hydrophobic proteins, and many proteins without identified functions in LD biology.
The
Drosophila
model, equipped with versatile genetic tools and conserved LD reg-
ulatory pathways, is an ideal system in which physiological and molecular/cellular
level investigations have been conducted. Several LD-associated proteins are
reviewed for their physiologic roles.
4.3.1
LD structural proteins
Fly perilipins (
Dm
PLIN1 and
Dm
PLIN2; lipid storage droplet 1 and 2) are members
of the evolutionarily conserved protein family that were first identified as mamma-
lian PAT domain proteins (
p
erilipin,
a
dipophilin/adipose differentiation-related pro-
tein, and
t
ail-interacting protein 47) (
Beller et al., 2010; Brasaemle, 2007
).
Dm
PLIN1 indiscriminately labels LDs of varying sizes whereas
Dm
PLIN2 localizes
to smaller LDs (
Bi et al., 2012
).
Dm
PLIN1 exclusively locates on LDs while
Dm
PLIN2 presents both in the cytoplasm and on LD surfaces (
Beller et al.,
2010
). Consistent with different LD preference and cellular distribution, the physi-
ological functions of
Dm
PLIN1 and
Dm
PLIN2 are also distinct.
Dm
PLIN1 has dual
functions in both promoting and preventing lipolysis whereas
Dm
PLIN2 functions to
only prevent lipolysis (
Bi et al., 2012
). More specifically,
Dm
PLIN1 recruits
d
HSL
(fly hormone-sensitive lipase) upon adipokinetic hormone (AKH) stimulation to me-
diate acute lipolysis.
Dm
PLIN1 mutant flies have larger LDs, develop obesity in
adulthood (consistent with impaired lipolysis) (
Arrese et al., 2008; Beller et al.,
2010
), but show a significantly decreased life span upon starvation challenge
