Biology Reference
In-Depth Information
Oil-Red-O, and BODIPY. These methods have been described in detail elsewhere
(
Ashrafi et al., 2003; Brooks et al., 2009; Kimura, Tissenbaum, Liu, & Ruvkun,
1997; O'Rourke et al., 2009; Yen et al., 2010; Zhang, Trimble, et al., 2010
). Lipophilic
dye staining on fixed animals seems to reliably label lipid droplets and serve as a semi-
quantitative way to estimate fat content. The use of flow cytometry allowed a large
number of stained animals of a given genotype to be analyzed (
Klapper et al.,
2011
). However, it is technically challenging to extend these techniques for large scale
genetic and functional genomic surveys. More recently, a number of laboratories have
developed label-free lipid imaging techniques to measure fat content in live
C. elegans
animals (
Hellerer et al., 2007; Le, Duren, Slipchenko, Hu, &Cheng, 2010; Wang, Min,
Freudiger, Ruvkun, & Xie, 2011; Yen et al., 2010
). These techniques are based on the
detection of C
d
H bond vibrations in lipids that can be detected by coherent anti-
Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS). In one study,
the fat content of live animals was measured by SRS microscopy, after 272 individual
gene inactivations by RNA interference (
Wang et al., 2011
). This suggests that SRS
microscopy can be applied to whole genome surveys. However, microscopy systems
for CARS and SRS are far from common. Therefore, there is a need for the establish-
ment of complementary approaches to visualize lipid droplets.
In this chapter, we will focus on the use of lipid droplet associated proteins as
lipid droplet markers in live animals. The Perilipin family of proteins, which are stan-
dard lipid droplet markers in mammalian cells, cannot be found in
C. elegans
(
Bickel, Tansey, &Welte, 2009
). However, the
C. elegans
orthologs of a triglyceride
lipase (ATGL) and a triglyceride synthesis enzyme (DGAT-2) appear to localize to
lipid droplets (
Xu et al., 2012; Zhang, Box, et al., 2010
). We describe strategies for
generating transgenic strains that express fluorescent protein tagged lipid droplet as-
sociated proteins and how they can be visualized in two different commercial con-
focal microscopy systems. In addition, methods for examining lipid droplets and
lipid droplet associated proteins by electron microscopy are described.
3.1
TRANSGENIC EXPRESSION OF LIPID DROPLET
MARKERS IN C. elegans
Subcellular localization of proteins may be dependent on expression levels. Overex-
pression of proteins may cause mistargeting or altered turnover. Therefore, care
should be taken to express lipid droplet associated proteins at physiological levels
in order to visualize lipid droplets specifically. To achieve this in
C. elegans
, single
copy transgenes can be generated by the Mos1 single copy insertion technology
(
Frokjaer-Jensen, Davis, Ailion, & Jorgensen, 2012; Frokjaer-Jensen et al., 2008
).
Mobilization of a Mos1 transposon at a defined location of the genome causes a
DNA break, which can be repaired by a template that contains a transgene for fluo-
rescent fusion protein expression (
Fig. 3.1
). To visualize lipid droplets, we generated
single copy transgenes that express the green fluorescent protein (GFP) or mono-
meric Ruby (mRuby) red fluorescent protein, fused to a
C. elegans
ortholog of a dia-
cylglycerol acyltransferase (DGAT-2) (
Xu et al., 2012
). DGAT-2 is the terminal
