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In-Depth Information
Abstract
The powerful forward and reverse genetic tools, and emerging sets of biochemical
assays for fat metabolites, make Caenorhabditis elegans an attractive model organ-
ism for elucidating conserved mechanisms in fat storage. The ability to observe lipid
droplets in live animals at single cell resolution offers a unique advantage for study-
ing cellular fat storage in vivo . In this chapter, we describe transgenic technologies
for expressing fluorescent lipid droplet marker proteins at near-physiological levels.
Methods to visualize these markers using sensitive confocal microscopy systems are
detailed. Additional methods for visualizing lipid droplets by transmission electron
microscopy and detection of lipid droplet associated proteins by immunoelectron mi-
croscopy are described.
INTRODUCTION
Lipid droplets are ubiquitous fat storage organelles consisting of a neutral lipid core
that is enclosed by a phospholipid monolayer ( Walther & Farese, 2012 ). The delimit-
ing phospholipid monolayer can be used to readily distinguish lipid droplets by trans-
mission electron microscopy (TEM) ( Blanchette-Mackie et al., 1995; Robenek et al.,
2009; Tauchi-Sato, Ozeki, Houjou, Taguchi, & Fujimoto, 2002 ). This is because all
other intracellular organelles are bound by membranes composed of a phospholipid
bilayer instead. In Caenorhabditis elegans , lipid droplets can be found in the intes-
tine, the hypodermis, and the gonad ( Brooks, Liang, & Watts, 2009; O'Rourke,
Soukas, Carr, & Ruvkun, 2009; Yen et al., 2010; Zhang, Box, et al., 2010; Zhang,
Trimble, et al., 2010 ). The intestine spans almost the entire length of an animal
(
1 mm) and is composed of 20 cells that are endowed with a large number of ve-
sicular structures ( Altun & Hall, 2009; Leung, Hermann, & Priess, 1999 ). In the ab-
sence of specialized adipose tissues in C. elegans , the intestine is the major site of fat
storage. It is also responsible for fat absorption, synthesis, and export ( Brock,
Browse, & Watts, 2006; Grant & Hirsh, 1999; Hall et al., 1999; Kimble &
Sharrock, 1983; Van Gilst, Hadjivassiliou, Jolly, & Yamamoto, 2005 ). Since the ma-
jority of C. elegans body fat is stored in the intestine, it is not surprising that it con-
tains the highest number of lipid droplets (
200 per cell). The median diameter of
these lipid droplets is 1
m in wild-type animals.
The wealth of forward and reverse genetic tools and an emerging set of biochem-
ica l assays for fat metabolites have made C. elegans an attractive system to explore
how fat storage is regulated at the cellular and organismal levels ( Elle, Rødkær,
Fredens, & Færgeman, 2012; Mullaney & Ashrafi, 2009; Perez & Van Gilst,
2008; Watts, 2009 ). Since there is a high level of conservation in genes that act in
metabolic and intercellular signaling pathways, discovery in C. elegans may be read-
ily translated to mammalian systems. The demand for high-throughput monitoring of
lipid droplets in C. elegans has prompted the development of methods that rely on
staining of live or fixed animals with lipophilic dyes such as Sudan Black, Nile Red,
m
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