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distinguish LLDs from presecretory VLDL or VLDL precursor particles. The only
reported success in observing LLDs was by immunogold electron microscopy, where
they were identified as VLDL-sized particles in the smooth ER lacking immunode-
tectable apoB (
Alexander, Hamilton, & Havel, 1976
). Subsequently, chylomicron-
sized LLDs were detected in the ER of enterocytes lacking apoB expression,
further supporting this observation (
Hamilton, Wong, Cham, Nielsen, & Young,
1998
). No further progress has been made to provide additional evidence for the
presence of these LDs. We have developed a method to purify LLDs by subcellular
fractionation and to biochemically characterize protein and lipid properties of the
isolated LLDs, thus providing an approach to biochemically study the formation
and metabolism of LLDs. The purification of LLDs will become a powerful tool
to study events involved in the lipidation step of VLDL assembly. LLDs were
found to be heterogeneous in size, possibly reflecting the various states of synthe-
sis/turnover (
Wang, Gilham, & Lehner, 2007
). Whereas in mouse CLDs, TG
accounts for up to 80% of total lipids, phospholipid for about 15%, and cholesterol
with CE for the remaining 5%, LLDs contained on average a lower percentage of TG
(60%) and a higher percentage of phospholipid (25%) (
Wang et al., 2007
); lipid ratios
si
milar to those found in mature VLDL. Proteomic analysis of LLDs revealed
the presence of microsomal TG transfer protein (MTP), protein disulfide isomerase
(PDI), apolipoprotein E (apoE), and several other ER-resident proteins including
two members of the carboxylesterase family, carboxylesterase 3/triacylglycerol
hydrolase (Ces3/TGH, Ces1d) and carboxylesterase 1/esterase-x (Ces1/Es-x, Ces1g)
(
Wang et al., 2007
).
Despite the important functions LDs serve in many cellular processes, our
knowledge of the cell biology of LDs lacks behind that of other intracellular organ-
elles. It is generally believed that LD biogenesis in eukaryotes initiates from the ER
where TG biosynthesis takes place. However, little is known about the mechanism by
which nascent LDs accrue additional TG and grow in size after nascent formation.
This gap is in part due to the lack of good tools to visualize the flux of lipids.
Traditional lipophilic dyes such as BODIPY 493/503, Nile Red, and LD540 are ex-
cellent tools to visualize the morphology of already formed CLDs by fluorescence
microscopy; however, they do not allow tracking the dynamics of initial LD
formation and cannot distinguish different pools of LDs at the different stages of
biogenesis. Thus, a method is needed that distinguishes between preformed and
newly synthesized CLDs. Additionally, CLD formation is a rapid process that
occurs almost immediately (within 15 min) after OA addition (
Wang et al., 2010
).
Thus, real-time cell imaging is necessary to capture this short window during
CLD formation.
Many lipid analogues have been developed in the past decade, including a
variety of fluorescent fatty acid analogues, thus enabling the tracking of initial lipid
incorporation into CLDs with real-time microscopy. These analogues include, but
are not limited to, NBD-conjugated fatty acids (
Chattopadhyay, 1990
),
BODIPY-conjugated fatty acids (
Pagano, Martin, Kang, & Haugland, 1991
),
