See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/222680290

Biogenesis of cytoplasmic lipid droplets: From

the lipid ester globule in the membrane to the
visible structure
Article in Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids June 2009
Impact Factor: 5.16 DOI: 10.1016/j.bbalip.2008.10.002 Source: PubMed

CITATIONS

READS

99

98

6 authors, including:
Akikazu Fujita
Kagoshima University
100 PUBLICATIONS 3,255 CITATIONS
SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate,

letting you access and read them immediately.

Available from: Toyoshi Fujimoto

Retrieved on: 14 July 2016

Biochimica et Biophysica Acta 1791 (2009) 399407

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a l i p

Review

Biogenesis of cytoplasmic lipid droplets: From the lipid ester globule in the
membrane to the visible structure
Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara, Akikazu Fujita, Toyoshi Fujimoto
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya 466-8550, Japan

a r t i c l e

i n f o

Article history:
Received 7 May 2008
Received in revised form 9 August 2008
Accepted 6 October 2008
Available online 21 October 2008
Keywords:
Lipid droplet
Biogenesis
Apolipoprotein B-100
Lipoprotein
Endoplasmic reticulum
Electron microscopy

a b s t r a c t
The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester
synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaets is
supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution
limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid
esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and
the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the
surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that
these processes may give rise to the structural and functional diversities of the CLD is discussed.
2008 Elsevier B.V. All rights reserved.

1. Introduction: cytoplasmic lipid droplets and lipoproteins

The white adipocyte contains a huge unilocular lipid droplet, often
reaching more than 100 m in diameter. The adipocyte lipid droplet
can be observed by light microscopy using classical histological stains
developed more than a century ago [1]. On the other hand, the presence of lipid droplets in nonadipocytes has not been paid much
attention, except when they occupy a large proportion of the
cytoplasm in some pathological conditions; hepatocytes in steatotic
liver and macrophages in the atherosclerotic plaque of the arterial
wall are well-known examples. Lipid droplets, however, probably exist
as normal organelles in any cell type. Although given the same name,
those in nonadipocytes are much smaller than the adipocyte lipid
droplet, i.e., generally less than a few m in diameter. These droplets
usually remain multilocular even when they proliferate extensively.
The important functions of nonadipocyte lipid droplets, other than as
a mere lipid storage depot, are rapidly becoming known [26]. The
above-mentioned lipid droplets are cytoplasmic structures, and in this
article, they will be referred to as cytoplasmic lipid droplets (CLDs) to
distinguish them from lipoproteins and their precursors that exist in
the organellar lumen.
The major part of the CLD is occupied by lipid esters; they are
mostly triacylglycerides (TG) in the white adipocyte, whereas CLDs of
most nonadipocytes contain a high proportion of cholesterol ester
(CE). In some specialized cells, retinyl esters are the major constituent
[4]. Because lipid esters are hydrophobic molecules, a special interface
Corresponding author. Tel.: +81 52 744 2000; fax: +81 52 744 2011.
E-mail address: tfujimot@med.nagoya-u.ac.jp (T. Fujimoto).
1388-1981/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2008.10.002

is necessary to keep them in a stable form in the aqueous cytoplasm. A

phospholipid monolayer, rather than the phospholipid bilayer that
makes the conventional biological membrane, serves this function.
This was proven by cryoelectron microscopy of isolated CLDs: the CLD
surface was observed as a single electron-dense line representing a
row of phosphorus atoms in the head group of phospholipids, in
contrast to the two parallel electron-dense lines that are seen in
membrane vesicles [7].
Lipoproteins are another kind of lipid particles containing lipid
esters. Very low-density lipoprotein (VLDL) and chylomicron are
made in the hepatocyte and the intestinal epithelial cell, respectively.
They are synthesized in the lumen of secretory compartments and
secreted by exocytosis. The diameter ranges from 30 to 90 nm for VLDL
and from 70 to 1000 nm for chylomicrons [2]. These lipoproteins are
also thought to have a lipid ester core and a phospholipid monolayer.
Thus, in lipoprotein-producing cells, two different lipid particles of a
similar structure exist in opposite compartments across an organelle
membrane. In mammalian cells, the last step of the triacylglyceride
and cholesterol ester synthesis is mediated by acyl-coenzyme A:
diglycerol acyltransferases (DGAT) and acyl-CoA:cholesterol acyltransferases (ACAT), respectively. These enzymes were shown to be
essential to the formation of both CLDs and lipoproteins [810].
In this article, we summarize data related to the CLD biogenesis.
Several hypotheses on the CLD formation process have been proposed,
but experimental evidence has been rather scarce. Recently we
reported a structure topologically equivalent to the lipid ester globule
intercalated between the two membrane leaets of the ER [11]. The
result suggests that the CLD biogenesis initiates by the deposition of
lipid esters in the membrane, but considering the resolution limit of

400

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

the available techniques, the CLD that we observe is likely to form only
after the nascent CLD grows to a certain size. We discuss the possibility that the growth and involution processes generate the structural
and functional heterogeneities of the CLD.
2. Molecules related to lipid particle formation
2.1. Enzymes for lipid ester synthesis
The synthesis of lipid esters is critical for CLD formation. Although
lipid esters may be transported to a distant region as a soluble
complex, CLDs are most likely generated in the vicinity of lipid estersynthesizing enzymes. In Saccharomyces cerevisiae, the enzymes
synthesizing TG, Dga1p and Lro1p and those synthesizing sterol
esters, Are1p and Are2p are all distributed in the endoplasmic reticulum (ER). But Dga1p localizes in CLDs as well, and it is thought to
synthesize TG locally [11]. Both DGAT and ACAT also exist in two
isoforms in mammalian cells. The enzymatic activities are largely
recovered from the microsome fraction [12,13], but the precise distribution of some enzyme molecules has not been dened clearly.
ACAT1 was shown to exist in the ER, whereas the distribution of
endogenous ACAT2 is less distinct [10,14]. A recent immunouorescence study showed that DGAT2, the functional ortholog of Dga1p, is
localized around CLDs [15]. The study, however, did not delineate
whether DGAT2 is embedded in the CLD surface itself or if it exists in
the ER membrane close to the CLD. The latter possibility appears more
likely at present, considering the presence of the two putative
membrane-spanning domains in the DGAT2 molecule [16] and the
absence of DGAT2 in a number of proteomic studies of CLD preparations [1723]. Thus, the ER membrane is most likely a site where
CLDs are formed, but this does not exclude the possibility that
other cellular organelles contribute to the CLD-forming process. For
example, a subset of caveolae was shown to synthesize TG in the
adipocyte [24]. The adipocyte caveolae distribute in a very high
density, and they are unique in that they contain perilipin B, are
exposed to a massive inux of fatty acids, and face a single large CLD
across a thin cytoplasm [2527]. It is not clear whether the nonadipocyte caveolae is also engaged in the TG production. But, as we
discuss later, small nascent CLDs should acquire additional lipid esters
to become larger grown-up CLDs, and the involvement of several
organelles at different steps is possible.
Results of knockout mouse studies showed that DGAT2 plays a
major role in TG synthesis for both CLD and VLDL [9]. Similarly, ACAT2
was shown to supply CE for the generation of CLD and VLDL in the
mouse liver [28]. Lipid esters synthesized by the other isoforms, i.e.,
DGAT1 and ACAT1, may also contribute to lipid particle generation,
and they might be used preferentially for either CLD or VLDL. Here,
however, it is important to recognize that lipid esters synthesized by
one enzyme, i.e., DGAT2 and ACAT2, can be utilized for the two different lipid particles that exist in the two compartments separated by
the ER membrane.
2.2. PAT proteins
Many proteins have been found to associate with the CLD either
constitutively or only in certain conditions. PAT proteins were named
after perilipin, adipocyte differentiation-related protein (ADRP; also
called as adipophilin), and tail-interacting protein of 47 kDa (TIP47).
Among PAT proteins, perilipin and ADRP are constitutively distributed
in the CLD, whereas TIP47, S3-12, and MLDP/OXPAT/PAT-1 exist
largely as soluble proteins in the cytoplasm and are recruited to CLDs
upon fatty acid loading or other stimuli [2932]. Three-dimensional
structure of TIP47 suggested that the hydrophobic interaction is
important for the binding of PAT proteins to CLDs; this contrasts with
the hydrophilic proteins that are recruited to conventional vesicular
organelles by using resident proteins and/or lipids as binding cues.

In the adipocyte at the resting condition, perilipin works as a shield

against cytosolic lipases and sequesters CGI-58, a coactivator of adipocyte triglyceride lipase [33]. When lipolysis is activated, perilipin
is phosphorylated by protein kinase A, provides a docking site for
hormone-sensitive lipases, and releases CGI-58 [34]. The -adrenergic
stimulation induces lipolysis in perilipin-rich CLDs rather than in
perlipin-poor ones, suggesting the importance of the docking function. ADRP and TIP47 appear to share the shielding function against
lipolysis at least partially [35], but whether they become a docking site
for lipases upon stimulation has not been reported. In the hepatocyte,
an increase of ADRP expression was shown to help sequester TG in
CLDs by diverting it from VLDL synthesis [36]. In the ADRP-null mouse,
however, despite a marked reduction of the cellular TG, normal VLDL
secretion was maintained, probably due to the upregulation of the
MTP protein level [37]. Moreover, the size distribution of CLD in the
ADRP-null mouse was different from that in the normal counterpart
[37]. These results may be largely explained by the protective role of
ADRP against lipases, but the possibility that this protein has additional functions cannot be excluded.
3. Initial stage of CLD biogenesis
3.1. Hypothetical models
Several hypotheses have been proposed for the nascent CLD formation. The most prevailing hypothesis supposes that lipid esters
accumulate between the two leaets of the ER membrane (or other
membranes), gradually grow into a globular shape, and are nally
pinched off from the ER to become independent CLDs (Fig. 1A) [38,39].
In model experiments, the phospholipid bilayer can accommodate
only 3 mol% of TG [40] or 2% of CE [41], and a further increase of lipid
esters induces the phase separation, which probably results in a small
globule between two membrane leaets. Using the membrane
isolated from the sunower seed, an in vitro formation of lipid ester
globules was shown by 1H-NMR analysis [42]. TG molecules made in
the microsomal membrane showed isotropic tumbling in the same
manner as in isolated CLDs. The presence of isotropically mobile lipids
was also reported in the mammalian cell membrane [43]. These
results are consistent with the presence of a lipid ester globule that is
about 25 nm in diameter [44], but it has not been settled whether the
NMR signal is derived from lipid esters that exist between the two
leaets of the membrane, from CLDs already independent from the
mother membrane, or from an alternative source [45]. Nonetheless,
the result might suggest that the approximate size of the smallest CLD
is in this range.
The above scheme supposes that the lipid ester globule bulges
toward the cytoplasm and that its surface is covered by a phospholipid
monolayer derived from the cytoplasmic leaet of the ER membrane.
As an alternative mechanism of the CLD separation was proposed that
the ester globule is detached from the ER membrane as a bicellar
structure (Fig. 1B) [46]. That is, the ester globule may be released from
the ER together with the cytoplasmic and lumenal leaets of the
membrane by making a transient pore. An important aspect of this
hypothesis is that the CLD may be able to carry a fragment of the ER
membrane as a wrinkle, which can explain the presence of integral
membrane proteins in isolated CLDs [19,21,22] and the so-called
membrane tab that was observed by electron microscopy [47]. In the
above two schemes, the lipid ester was supposed to bulge as a globule
within a relatively small area of the membrane, but it is not clear
whether such a globule formation occurs based on the lipid properties
alone. It is probable that some protein-based mechanism restricts thin
lateral spreading of the lipid ester between the two membrane
leaets.
Another hypothesis was formulated from the observation of bacteria, in which wax ester synthase/diacylglycerol acyltransferase
(WS/DGAT) is responsible for TG synthesis [48]. WS/DGAT localizes

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

401

Fig. 1. Hypothetical models of CLD biogenesis. (A) In eukaryotic cells, enzymes for the lipid ester synthesis (DGAT, ACAT) are distributed in the ER membrane, and the produced esters
are supposed to accumulate between the two membrane leaets. Model A is the most prevalent model that presumes the release of nascent CLDs by budding. The cytoplasmic leaet
of the ER membrane is thought to cover the lipid ester globule and become the surface phospholipid monolayer of the CLD. (B) In Model B, the lipid ester globule formed in the ER
membrane is hypothesized to hatch as a whole carrying both the cytoplasmic and luminal membrane leaets. A transient pore should form in the ER membrane after the hatching.
A small fragment of the ER membrane may be attached to the CLD as a bilayer membrane, which may explain the presence of transmembrane ER proteins in isolated CLD preparations
and the membrane tab observed by electron microscopy. (C) In some bacteria, a lipid ester-synthesizing enzyme, WS/DGAT, is a transmembrane protein of the plasma membrane.
The lipid ester is supposed to form as a small globule around the cytoplasmic portion of the enzyme. The model proposes that the lipid ester rst makes the oleogenous layer that
covers the cytoplasmic surface of the plasma membrane, gradually bulges to make lipid prebodies, and nally becomes a CLD in the cytoplasm. The origin of the surface
phospholipids is not known. (D) In leukocytes, membranous structures and ribosomes are observed in the interior of some CLDs. In other mammalian cells, ER chaperones,
transmembrane ER proteins, caveolins, and PAT proteins have been detected inside the CLD by immunoelectron microscopy. How these structures and proteins are integrated with
lipid esters is not understood.

in the plasma membrane and in the cytosol, and small lipid ester
globules are postulated to form around the cytoplasmic domain of the
enzyme (Fig. 1C) [49,50]. The initial small ester globules may lack
surface phospholipids, but, as they grow to make an oleogenous
emulsive layer along the cytoplasmic membrane surface and become
larger lipid prebodies, the phospholipid monolayer is acquired. Finally,
the lipid prebodies detach from the plasma membrane to become
mature CLDs. The bacterial WS/DGAT has no sequence similarity to
DGATs of eukaryotes, but the proposal that the CLD is formed on the
cytosolic surface of the ADRP-positive ER domain [51] may be
premised on a similar extramembrane mechanism. How the phospholipid monolayer is formed around lipid particles has not been
explained by this mechanism.
Still another hypothesis may be required to explain the origin of
CLDs that have membranous structures and/or various proteins in the
core (Fig. 1D). Membranes of the unit membrane appearance and
ribosomes were observed in CLDs of leukocytes [20]. Combined with
the observation of RNA in the mast cell CLD [52], protein synthesis was
speculated to occur in CLDs. Detection of ER membrane proteins, ER
chaperones, and caveolins by immunoelectron microscopy [5356]
also imply that CLDs may have a structure other than a simple mass of
lipid esters. For the biogenesis of CLDs with the internal membranous
structure, compilation of ER and/or other membranes may occur
along with the deposition of lipid esters, and a few hypothetical

models have been proposed [20,56]. Heterogeneous labeling patterns

of CLD-associated proteins in the cross-fractured core by immunoelectron microscopy [57] suggest that CLDs with different internal
structures may coexist in a cell. Although methodology used to
analyze the internal structure embedded in the lipid ester needs to be
critically evaluated, the above results may suggest the occurrence of
unique CLDs under some conditions. We suppose that the processes of
the growth and involution could give rise to the heterogeneous CLDs
(see below for discussion).
3.2. Structural association of CLD with the ER and other organelles
Consistent with the involvement of the ER in the CLD formation,
association of the CLD and ER is observed frequently [58,59]. The
cytoskeletal laments observed in the vicinity of adipocyte CLDs may
be important to stabilize the CLDER association [60], and they may be
part of the vimentin lament basket [61]. Interestingly, disruption of
vimentin laments in differentiating 3T3-L1 cells inhibited CLD
formation probably by increasing TG turnover [62]. Despite this
apparent importance, the function of vimentin appears to be
compensated by other molecules, because vimentin-null mice
develop normal adipocytes [63].
Although ER cisterns are often observed in the vicinity of CLDs,
not all CLDs are associated with the ER in thin section electron

402

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

micrographs. By live imaging, some CLDs are highly mobile while

others are not, and these two populations might correspond to free
and ER-bound CLDs, respectively [64]. But these results do not
necessarily indicate that some CLDs exist without any connection to
other organelles. Even the mobile CLDs may bind to other organelles
and move along their surface.
Close apposition between the CLD and ER, which appears different
from a mere association, was induced by overexpression of Rab18 [65].
A similar structure was observed simply by knocking down ADRP by
RNA interference, suggesting that the reduction of ADRP is responsible
for the CLDER apposition [65]. It is not known, however, whether the
absence of ADRP itself is sufcient to cause the observed structural
change or whether the recruitment of other proteins, such as TIP47
[32,66], is involved. The CLDER apposition induced by Rab18 is likely
to be related to the disposal of lipids from CLDs rather than to CLD
formation or lipid accumulation [67].
Ultrastructural analyses have shown that CLDs also associate with
mitochondria and peroxisomes [68]. The association between CLDs
and mitochondria [58,69] and the one between CLDs and peroxisomes [58,59,70] may be instrumental to supplying lipids for
-oxidation in the latter organelles and not related to the CLD formation. Notably, in the close contacts seen between CLDs and peroxisomes of Saccharomyces cerevisiae, enzymes for -oxidation are
preferentially distributed near the interface, and a portion of the
peroxisome membrane was observed invading CLDs [71]. The two
organelles were speculated to be in a hemifusion state, but the
continuity of the peroxisomal membrane leaets and the CLD surface
was not shown. A recent study showed a kiss-and-run-type
encounter between CLDs and phagosomes; this encounter may be
important to transferring arachidonic acids for the phagosomal

maturation [72]. Unfortunately, the resolution of the imaging technique used for the observation is rather low, and how close a contact
CLDs and phagosomes really form is not clear.
3.3. Lipid ester globules in the ER membrane
Despite the frequent association of the CLD and ER, morphological
evidence of the lipid ester accumulation in the ER membrane as
predicted in the popular models (Fig. 1A, B) has been scarce. An
exception may be the electron microscopic observation that the CLD
core is continuous with the middle electron-lucent layer of a thick
membranous appendage of the trilaminar appearance in cotyledons of
some plants [73]. A similar structure has not, however, been observed
in any other plant or animal cells [2]. A freeze-fracture study showed a
continuity of the cytoplasmic leaet of the ER membrane to a putative
CLD phospholipid monolayer [59], but the identity of the latter
structure, studded with many intramembrane particles, is questionable because later works showed a virtual absence of intramembrane
particles in the CLD fracture faces [7,57,74].
CLDs harboring ADRP and Apolipoprotein B-100 (ApoB) on the
complementary surface areas were observed when proteasomal or
autophagic protein degradation was inhibited in hepatoma-derived
cell lines (Fig. 2A) [75]. Subsequent analysis revealed that these CLDs
face both the cytoplasm and the ER lumen, and are topologically
equivalent to the lipid ester globule intercalated between the two
leaets of the ER membrane (Fig. 2B, C) [11]. Consistently, the CLDbound ApoB was lipidated by the action of microsomal triglyceride
transfer protein (MTP), a soluble protein in the ER [11]. The result
showed that lipid esters can exist as a globule between the two leaets
of the membrane.

Fig. 2. The ApoB-crescent structure. In cultured cells expressing ApoB and MTP (e.g., Huh7, HepG2, McA-RH7777), some CLDs bear the ApoB-positive cap-like structure, which was
termed the ApoB-crescent [75]. (A) By immunouorescence microscopy of the ApoB-crescent, ApoB and ADRP occupy complementary surfaces of CLDs. Bar, 1 m. (B) Electron
microscopy showed that the ApoB-crescent is a CLD fused with a thin ER-derived membrane cistern. The cisternal lumen harbors all the ER chaperones and secretory proteins, and it
is positive for the glucose-6-phosphatase activity [11]. The cisternal membrane opposite to the CLD shows the appearance of the trilaminar unit membrane and ends on the CLD
surface that lacks an apparent membrane. Ribosomes are shown by arrowheads. Bar, 200 nm. (C) Topologically, the CLD in the ApoB-crescent is equivalent to a lipid ester mass
intercalated between the two membrane leaets of the ER membrane.

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

The structure, which was termed ApoB-crescent, is thought to

form because anomalous lipidated ApoB binds to the lumenal surface
of the ER membrane and perturbs the normal process of CLD
separation from the ER [11]. But if the normal CLD biogenesis in
other cells begins by deposition of lipid esters within the ER membrane, one can expect that a similar CLDER fusion structure will be
observed, albeit only infrequently. For example, upon addition of oleic
acid to the culture medium of broblasts, many new CLDs are induced
to form within a few hours, but, even in such a favorable condition, the
nascent CLD or deposition of lipid esters in the membrane has not been
captured [30]. We speculate that one possible reason for the failure is
the low resolution of the techniques used for the CLD observation.
Light microscopic methods can visualize only CLDs larger than 200
300 nm in diameter, and even by electron microscopy, it is difcult to
unambiguously identify CLDs of less than 50 nm in diameter (J. Cheng
and T. Fujimoto, unpublished observation). If lipid ester globules
intercalated between the two ER membrane leaets are the direct
precursor of CLDs, the diameter of nascent CLDs must be smaller than
that and could be around 25 nm, as suggested from the 1H-NMR result
[44]. This small size may preclude unequivocal observation of these
CLDs by the above techniques. In other words, the CLD that we are
observing is formed only after the growth of the nascent CLD.
3.4. ER domains for CLD biogenesis
The ER consists of heterogeneous domains that are differentiated
both structurally and functionally [76], and CLD biogenesis may occur
in specic domains [77]. In agreement with this supposition, oleosins,
CLD-specic proteins, as well as some enzymes in the TG synthesis
pathway, were found concentrated in a restricted portion of the ER in

403

plant cells [78,79]. In animal cells as well, the enzymes for the TG
synthesis pathway were hypothesized to exist as a complex, or
metabolon, in several different ER domains; respective domains were
supposed to specialize in de novo CLD formation, CLD recycling, or
VLDL assembly in the hepatocyte [2,80]. Whether or not distribution
of endogenous DGATs is restricted to a specic ER domain has not
been shown, but this is likely because the epitope-tagged tung DGAT1
and DGAT2 were distributed in distinct ER regions when expressed in
tobacco BY-2 cells [81].
The ER region specialized for CLD formation may be also differentiated
in its lipid composition. The phospholipid composition of isolated CLD
has been characterized in several studies [7,82,83]. In both mammalian
and yeast CLDs, the phospholipids composition is largely similar to that of
ER in that phosphatidylcholine (PC) is most abundant, followed by
phosphatidylethanolamine (PE) and phosphatidylinositol. Mass spectrometric analysis, however, revealed several interesting characteristics of
mammalian CLDs [7,82]: PC and lysophosphatidylcholine are enriched
with unsaturated acyl chains, and the ether-linked form of PC and PE
exists. The unique composition suggests that the phospholipid monolayer of the CLD surface is not simply formed by a random sampling of
the bulk ER membrane and that a specialized ER subdomain may be
involved in this monolayer's formation.
Isolated CLDs also contain some free cholesterol (FC). Although FC
may also exist in the core as speculated for lipoproteins [84], lipin
labeling was observed around the adipocyte CLD [85] and in the ER
membrane of the ApoB-crescent [11], indicating that FC is enriched in
the CLD surface and/or in the membrane associated with the CLD. This
enrichment of FC is intriguing because the molar percentage of TG and
CE that can be dissolved in the phospholipid bilayer decreases in the
presence of FC [86,87]. That is, a phase separation in the membrane

Fig. 3. Mechanism of VLDL synthesis. In hepatocytes, ApoB is lipidated co-translationally and makes the pre-VLDL. The pre-VLDL then acquires additional lipids from ApoB-free lipid
particles and becomes mature VLDL in the ER or in post-ER compartments. The co-translational ApoB lipidation and the acquisition of lipids from ApoB-free lipid particles are
catalyzed by microsomal triglyceride transfer protein (MTP). The lipids that make the ApoB-free lipid particles are derived from lipid esters stored in the CLD through hydrolysis and
re-esterication. How this supply of lipids occurs is un-known, but it may require a repeated fusion between the CLD and the ER membrane. The putative fusion and separation of CLD
is supposed to occur in ER subdomains different from that of the new CLD formation.

404

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

that leads to the ester globule formation should preferentially occur in

the FC-rich domain. It is not known how a local FC enrichment could
be produced in the ER membrane, but formation of the stoichiometric
complex between FC and lysophosphatidylcholine may be involved in
the process [88].
4. VLDL synthesis; how it differs from CLD formation
VLDL is the lipoprotein particle secreted from the hepatocyte. Its
basic structure is thought to be similar to that of CLD, i.e., the lipid
ester core and the surface phospholipid monolayer. The mechanism of
VLDL synthesis has been studied extensively and discussed in many
review articles [8991]. Here, only a brief outline of VLDL synthesis is
presented for immediate comparison to that of CLD (Fig. 3). The VLDL
formation in hepatocytes starts by co-translational lipidation of ApoB,
which makes the primordial lipoprotein particle, or pre-VLDL. This
particle subsequently acquires more lipids probably by fusing with
ApoB-free lipid particles in the lumen and becomes a mature VLDL
ready for exocytosis. MTP is necessary for the formation of both preVLDL and the ApoB-free lipid particle [89,92]. Interestingly, lipids
stored in CLDs rather than the newly synthesized or imported fatty
acids are preferentially used for the above process [80]. In other
words, free fatty acids that are synthesized in the cell or incorporated
from the outer sources are rst stored in CLDs as esters and then
hydrolyzed and re-esteried to make the lipoprotein core. The rst
esterication step for storage in the CLD and the second reesterication step for VLDL formation are both catalyzed by the lipid
ester-synthesizing enzymes in the ER. It was hypothesized that the
two esterication reactions occur in separate ER domains and are
coupled with ssion and fusion between the CLD and ER [80].
It is intriguing how the same enzymes can supply the lipid ester to
VLDL and CLD that exist in different compartments across the ER
membrane. Because the lipid ester may not be transported across the
membrane bilayer easily, it is reasonable to assume that it is deposited
within the membrane rather than on either side of the membrane. In
VLDL synthesis, the phospholipid transfer activity of MTP is indispensable for the co-translational lipidation of ApoB on the lumenal side
of the ER membrane [93]. By contrast, in CLD formation that occurs on
the cytoplasmic side of the ER membrane, involvement of enzymatic
activities like MTP is not known. This suggests that the bulging of the
lipid ester globule toward the cytoplasmic side is the default process in
the absence of MTP. The presence of PAT proteins in the cytoplasm and/
or difference of lipid composition between the two leaets of the ER
membrane may be related to the directional preference.
Although both VLDL and CLD are thought to be made of a lipid ester
core and a phospholipid monolayer, the size of CLD is generally larger
and far more variable than that of VLDL. The obligatory presence of
one ApoB molecule per VLDL particle may be responsible for the small
and relatively constant size of VLDL. Interestingly, phospholipase D
was shown to participate in the formation of both VLDL and CLD
[94,95]. With regard to VLDL, phospholipase D appears to be
important for the second step of assembly, in which pre-VLDL acquires
additional lipids [95]. On the other hand, it is not clear at what stage of
CLD formation (the initial process, the later growing process, or both)
phospholipase D is required. This uncertainty is partly because the
smallest CLDs immediately after formation are not likely to be
captured by conventional techniques [96]. It is important to elucidate
how phospholipase D exerts its effect because this may be the critical
step in which both VLDL and CLD formation are regulated in the cell.
5. CLD in later stages
5.1. Growth of CLD
The size of CLD may become larger by fusion of smaller CLDs [97]
and/or accretion of additional lipid esters by other means, which may

include repeated fusion and ssion between the CLD and ER,
continuous coupling of the CLD and ER, local production of the lipid
ester in the LD, and transport of the lipid ester as soluble complexes
[96]. It is not evident whether the fusion events observed by live
imaging are frequent enough to explain the growth rate, but we need
to be aware that the fusion between small CLDs (invisible by the
current techniques) and that of small CLDs to large ones are not
counted. Although other mechanisms may also operate, here we
consider the consequences of the mutual CLD fusion.
CLDs are equipped with SNARE proteins [98], which suggests that
the molecular mechanism for their mutual fusion may be similar to
that of membrane vesicles. But the result of the fusion may be quite
different due to the basic structural difference between CLDs and
vesicular organelles: i.e., the viscous ester core covered by a
phospholipid monolayer vs. the aqueous content surrounded by a
phospholipid bilayer. When vesicles fuse each other, the volume of the
aqueous content can be adjusted in accordance with the available
membrane by exibly changing the shape and/or by transporting
water and solutes across the membrane (Fig. 4A). In contrast, under
the premises that the total volume of the lipid esters does not change
and that the CLD after fusion regains the spherical shape immediately,
the CLD fusion should cause a decrease of the surface-to-volume ratio
and thus an excess of phospholipids (Fig. 4B). The result of live
imaging indicates that the premises are correct [97].
If we suppose that the nascent CLD is as small as 25 nm in diameter
as conjectured above and that a large CLD of 1 m in diameter is
generated only by repeated CLD fusions, 64,000 small CLDs are needed
to account for the volume of the lipid ester. On the other hand, a
simple calculation predicts that phospholipids from 1600 small CLDs
are sufcient to cover the 1 m CLD and that phospholipids from
62,400 small CLDs (of 25 nm in diameter) are in excess. This
astonishing calculation presupposes that the lateral phospholipid
density is the same between small and large CLDs. This is not likely
due to the huge difference in their surface curvatures, but nonetheless,
the above calculation suggests that a considerable amount of
phospholipids needs to be processed by some mechanism when
CLDs fuse with each other. Degradation of phospholipids by
phospholipases and/or their removal by lipid transfer proteins may
be required; phospholipase D might be involved in this process [99]. If
excessive phospholipids remain in and/or around the CLD, they might
form a bilayer membrane like the membrane tab [47] (Fig. 1B).
Alternatively, some of excessive proteins and phospholipids might be
incorporated into the CLD core; the presence of membranous
structures and several proteins in the CLD core [20,56,57] may be
caused by this kind of process (Fig. 4B). In whichever mechanism that
works, however, it would be a waste for cells to dispose of a large
amount of proteins and phospholipids for every CLD fusion. Interestingly, a recent study showed that larger LDs were induced when
enzymes catalyzing the rate-limiting step of phosphatidylcholine
synthesis were knocked down [100]. The results indicate that the
phospholipid availability, or more precisely the relative phospholipidto-lipid ester ratio is an important determinant of the CLD fate. It is
intriguing to attempt to understand whether and how the protein
machinery for CLD fusion and fragmentation is regulated by the
relative lipid ratio.
5.2. Division of CLD
Division of CLDs should change the surface-to-volume ratio in the
opposite direction of the mutual fusion, and should cause a decrease
of the surface density of phospholipids and proteins (Fig. 4C). Thus,
fragmentation of CLDs that occurs upon chronic stimulation of
lipolysis in adipocytes is thought to increase the chance that cytosolic
lipases access lipid esters and facilitate lipolysis [26]. The Arf1-COPI
machinery should mediate the physiological process by promoting
budding at the CLD surface [100]. An articial fragmentation should

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

405

Fig. 4. The surface and volume changes upon fusion and division of vesicles and CLDs. (A) Vesicle fusion. The shape of the vesicle and the volume of its aqueous content can change in
a exible manner, so that an excess or deciency of phospholipids may not arise by mutual fusion (or ssion; not shown in the scheme). (B) CLD fusion. When two CLDs fuse to make
one CLD, an excess of phospholipids arises if the total volume of the lipid ester and the distribution density of phospholipids remain the same. After multiple rounds of fusion, the
amount of excessive phospholipids becomes large and might form the bilayer membrane at the surface and/or in the core of the CLD. (C) CLD division (ssion). If the total volume of
lipid esters does not change upon division, the total surface area of the divided CLDs is larger than the original CLD. This should cause a decrease in the distribution density of the
phospholipids and proteins in the surface.

occur when the CLD is disrupted by homogenization [6]. The chance

that hydrophobic lipid esters are exposed to the aqueous environment may be even greater in this case than in physiological divisions,
because most experiments are done at a cold temperature so that
phospholipids may not redistribute evenly on the disrupted CLD
surface. This should cause exposure of hydrophobic esters and nonspecic adsorption of unrelated molecules. We previously speculated
that this kind of adsorption may partly explain the apparently
contradictory results that many Rab proteins identied in puried
CLD preparations are scarcely found to locate in the CLDs in situ
[6,65,101].
5.3. Heterogeneity of CLD
The change in the surface-to-volume ratio of the CLD could be a
cause of heterogeneity in CLDs. The ratio may be modulated as a
result of fusion and division as conjectured above, but disagreement
between the lipid ester volume and the surface phospholipid/
protein layer area may also occur in other occasions, e.g., when the
increment of lipid esters and phospholipids does not proceed
cooperatively. Immunouorescence microscopy revealed that CLDs
in a cell contain perilipin, ADRP, TIP47, S3-12, and Rab18 in varying
proportions [30,32,102]. Perilipin and ADRP locate in the CLD
constitutively, but the others are exchangeable between the cytosol
and the CLD [103]. The exchangeable proteins may be readily
released from or recruited to the CLD when the surface-to-volume
ratio increases or decreases, respectively, whereas the amount of
constitutive proteins in the CLD may not change rapidly. Binding and
release of other seemingly unrelated proteins to the LD may be also
affected by the changes of the surface-to-volume ratio [5]. Repetition
of these changes may result in the compositional difference of CLD-

associated proteins, which then should modulate the functional state

of the CLD.
6. Concluding remarks
The CLD is no longer regarded as an inert lipid blob. The basic
function of storing excessive lipids as esters is operating actively to
defend cells against lipotoxicity [104], and the lipids stored in the CLD
are mobilized for specic needs preferentially over those from other
sources [2]. Many more aspects of CLD functions related to the
intracellular lipid homeostasis are likely to be revealed in the coming
years, but the importance of the CLD is not limited to the lipid world.
Its involvement in intracellular signaling, vesicular trafcking, protein
degradation, and temporal protein storage has been suggested [3,5,6].
More recently, the assembly of the hepatitis C virus particle was
shown to occur around the CLD [105]. Most of these functions are
carried out at the CLD surface, but its property may be diverse among
CLDs in a cell. As conjectured in this article, the unique architecture of
the CLD is likely to bring about the structural and functional
heterogeneities while it is born, grows, and wears. To further
understand the CLD function, it is becoming all the more important
to dene the molecular mechanism of CLD formation.
Acknowledgments
Work in our laboratory has been supported by Grants-in-Aid for
Scientic Research and the 21st Century COE Program Integrated
Molecular Medicine for Neuronal and Neoplastic Disorders of the
Ministry of Education, Culture, Sports, Science and Technology of the
Government of Japan (to YO, AF, TF), and by a grant from the Nitto
Foundation (to YO).

406

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

References
[1] L. Daddi, Nouvelle methode pour colorer la graisse dans les tissues, Arch. Ital.
Biol. 26 (1896) 143.
[2] D.J. Murphy, The biogenesis and functions of lipid bodies in animals, plants and
microorganisms, Prog. Lipid Res. 40 (2001) 325438.
[3] S. Martin, R.G. Parton, Lipid droplets: a unied view of a dynamic organelle, Nat.
Rev., Mol. Cell Biol. 7 (2006) 373378.
[4] D. Zweytick, K. Athenstaedt, G. Daum, Intracellular lipid particles of eukaryotic
cells, Biochim. Biophys. Acta 1469 (2000) 101120.
[5] M.A. Welte, Proteins under new management: lipid droplets deliver, Trends Cell
Biol. 17 (2007) 363369.
[6] T. Fujimoto, Y. Ohsaki, Cytoplasmic lipid droplets: rediscovery of an old structure
as a unique platform, Ann. N.Y. Acad. Sci. 1086 (2006) 104115.
[7] K. Tauchi-Sato, S. Ozeki, T. Houjou, R. Taguchi, T. Fujimoto, The surface of lipid
droplets is a phospholipid monolayer with a unique fatty acid composition,
J. Biol. Chem. 277 (2002) 4450744512.
[8] S.J. Smith, S. Cases, D.R. Jensen, H.C. Chen, E. Sande, B. Tow, D.A. Sanan, J.
Raber, R.H. Eckel, R.V. Farese Jr., Obesity resistance and multiple mechanisms
of triglyceride synthesis in mice lacking Dgat, Nat. Genet. 25 (2000) 8790.
[9] S.J. Stone, H.M. Myers, S.M. Watkins, B.E. Brown, K.R. Feingold, P.M. Elias, R.V.
Farese Jr., Lipopenia and skin barrier abnormalities in DGAT2-decient mice,
J. Biol. Chem. 279 (2004) 1176711776.
[10] T.Y. Chang, C.C. Chang, N. Ohgami, Y. Yamauchi, Cholesterol sensing, trafcking,
and esterication, Annu. Rev. Cell Dev. Biol. 22 (2006) 129157.
[11] Y. Ohsaki, J. Cheng, M. Suzuki, A. Fujita, T. Fujimoto, Lipid droplets are arrested in
the ER membrane by tight binding of lipidated apolipoprotein B-100, J. Cell. Sci.
121 (2008) 24152422.
[12] K.F. Buhman, M. Accad, R.V. Farese, Mammalian acyl-CoA:cholesterol acyltransferases, Biochim. Biophys. Acta 1529 (2000) 142154.
[13] R. Lehner, A. Kuksis, Biosynthesis of triacylglycerols, Prog. Lipid Res. 35 (1996)
169201.
[14] T.Y. Chang, C.C. Chang, S. Lin, C. Yu, B.L. Li, A. Miyazaki, Roles of acyl-coenzyme A:
cholesterol acyltransferase-1 and -2, Curr. Opin. Lipidol. 12 (2001) 289296.
[15] L. Kuerschner, C. Moessinger, C. Thiele, Imaging of lipid biosynthesis: how a
neutral lipid enters lipid droplets, Trafc 9 (2008) 338352.
[16] S.J. Stone, M.C. Levin, R.V. Farese Jr., Membrane topology and identication of key
functional amino acid residues of murine acyl-CoA:diacylglycerol acyltransferase-2, J. Biol. Chem. 281 (2006) 4027340282.
[17] S. Cermelli, Y. Guo, S.P. Gross, M.A. Welte, The lipid-droplet proteome reveals that
droplets are a protein-storage depot, Curr. Biol. 16 (2006) 17831795.
[18] Y. Fujimoto, H. Itabe, J. Sakai, M. Makita, J. Noda, M. Mori, Y. Higashi, S. Kojima, T.
Takano, Identication of major proteins in the lipid droplet-enriched fraction
isolated from the human hepatocyte cell line HuH7, Biochim. Biophys. Acta 1644
(2004) 4759.
[19] D.L. Brasaemle, G. Dolios, L. Shapiro, R. Wang, Proteomic analysis of proteins
associated with lipid droplets of basal and lipolytically stimulated 3T3-L1
adipocytes, J. Biol. Chem. 279 (2004) 4683546842.
[20] H.C. Wan, R.C. Melo, Z. Jin, A.M. Dvorak, P.F. Weller, Roles and origins of leukocyte
lipid bodies: proteomic and ultrastructural studies, FASEB J. 21 (2007) 167178.
[21] E. Umlauf, E. Csaszar, M. Moertelmaier, G.J. Schuetz, R.G. Parton, R. Prohaska,
Association of stomatin with lipid bodies, J. Biol. Chem. 279 (2004) 2369923709.
[22] P. Liu, Y. Ying, Y. Zhao, D.I. Mundy, M. Zhu, R.G. Anderson, Chinese hamster ovary
K2 cell lipid droplets appear to be metabolic organelles involved in membrane
trafc, J. Biol. Chem. 279 (2004) 37873792.
[23] S. Sato, M. Fukasawa, Y. Yamakawa, T. Natsume, T. Suzuki, I. Shoji, H. Aizaki, T.
Miyamura, M. Nishijima, Proteomic proling of lipid droplet proteins in
hepatoma cell lines expressing hepatitis C virus core protein, J. Biochem. 139
(2006) 921930.
[24] A. Ost, U. Ortegren, J. Gustavsson, F.H. Nystrom, P. Stralfors, Triacylglycerol is
synthesized in a specic subclass of caveolae in primary adipocytes, J. Biol. Chem.
280 (2005) 58.
[25] N. Aboulaich, A.V. Vener, P. Stralfors, Hormonal control of reversible translocation
of perilipin B to the plasma membrane in primary human adipocytes, J. Biol.
Chem. 281 (2006) 1144611449.
[26] A. Marcinkiewicz, D. Gauthier, A. Garcia, D.L. Brasaemle, The phosphorylation
of serine 492 of perilipin A directs lipid droplet fragmentation and dispersion,
J. Biol. Chem. 281 (2006) 1190111909.
[27] R.G. Parton, J.C. Molero, M. Floetenmeyer, K.M. Green, D.E. James, Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes, J. Biol. Chem. 277 (2002) 4676946778.
[28] K.K. Buhman, M. Accad, S. Novak, R.S. Choi, J.S. Wong, R.L. Hamilton, S. Turley, R.V.
Farese Jr., Resistance to diet-induced hypercholesterolemia and gallstone
formation in ACAT2-decient mice, Nat. Med. 6 (2000) 13411347.
[29] E. Tsuiki, A. Fujita, Y. Ohsaki, J. Cheng, T. Irie, K. Yoshikawa, H. Senoo, K. Mishima,
T. Kitaoka, T. Fujimoto, All-trans-retinol generated by rhodopsin photobleaching
induces rapid recruitment of TIP47 to lipid droplets in the retinal pigment
epithelium, Invest. Ophthalmol. Vis. Sci. 48 (2007) 28582867.
[30] N.E. Wolins, B.K. Quaynor, J.R. Skinner, M.J. Schoensh, A. Tzekov, P.E. Bickel,
S3-12, Adipophilin, and TIP47 package lipid in adipocytes, J. Biol. Chem. 280
(2005) 1914619155.
[31] N.E. Wolins, B. Rubin, D.L. Brasaemle, TIP47 associates with lipid droplets, J. Biol.
Chem. 276 (2001) 51015108.
[32] Y. Ohsaki, T. Maeda, M. Maeda, K. Tauchi-Sato, T. Fujimoto, Recruitment of TIP47
to lipid droplets is controlled by the putative hydrophobic cleft, Biochem.
Biophys. Res. Commun. 347 (2006) 279287.

[33] T. Yamaguchi, N. Omatsu, S. Matsushita, T. Osumi, CGI-58 interacts with perilipin

and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization
in ChanarinDorfman syndrome, J. Biol. Chem. 279 (2004) 3049030497.
[34] V. Subramanian, A. Rothenberg, C. Gomez, A.W. Cohen, A. Garcia, S. Bhattacharyya,
L. Shapiro, G. Dolios, R. Wang, M.P. Lisanti, D.L. Brasaemle, Perilipin A mediates the
reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes, J. Biol. Chem.
279 (2004) 4206242071.
[35] L.L. Listenberger, A.G. Ostermeyer-Fay, E.B. Goldberg, W.J. Brown, D.A. Brown,
Adipocyte differentiation-related protein reduces the lipid droplet association of
adipose triglyceride lipase and slows triacylglycerol turnover, J. Lipid Res. 48
(2007) 27512761.
[36] B. Magnusson, L. Asp, P. Bostrom, M. Ruiz, P. Stillemark-Billton, D. Linden, J. Boren,
S.O. Olofsson, Adipocyte differentiation-related protein promotes fatty acid
storage in cytosolic triglycerides and inhibits secretion of very low-density
lipoproteins, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 15661571.
[37] B.H. Chang, L. Li, A. Paul, S. Taniguchi, V. Nannegari, W.C. Heird, L. Chan,
Protection against fatty liver but normal adipogenesis in mice lacking adipose
differentiation-related protein, Mol. Cell. Biol. 26 (2006) 10631076.
[38] D.J. Murphy, J. Vance, Mechanisms of lipid-body formation, Trends Biochem. Sci.
24 (1999) 109115.
[39] G. Wanner, H. Formanek, R.R. Theimer, The ontogeny of lipid bodies (spherosomes) in plant cells: ultrastructural evidence, Planta 151 (1981) 109123.
[40] J.A. Hamilton, Interactions of triglycerides with phospholipids: incorporation
into the bilayer structure and formation of emulsions, Biochemistry 28 (1989)
25142520.
[41] J.A. Hamilton, D.M. Small, Solubilization and localization of cholesteryl oleate in
egg phosphatidylcholine vesicles. A carbon 13 NMR study, J. Biol. Chem. 257
(1982) 73187321.
[42] D.J. Lacey, F. Beaudoin, C.E. Dempsey, P.R. Shewry, J.A. Napier, The accumulation
of triacylglycerols within the endoplasmic reticulum of developing seeds of
Helianthus annuus, Plant J. 17 (1999) 397405.
[43] A. Ferretti, A. Knijn, E. Iorio, S. Pulciani, M. Giambenedetti, A. Molinari, S.
Meschini, A. Stringaro, A. Calcabrini, I. Freitas, R. Strom, G. Arancia, F. Podo,
Biophysical and structural characterization of 1H-NMR-detectable mobile
lipid domains in NIH-3T3 broblasts, Biochim. Biophys. Acta 1438 (1999)
329348.
[44] C.E. Mountford, L.C. Wright, Organization of lipids in the plasma membranes of
malignant and stimulated cells: a new model, Trends Biochem. Sci. 13 (1988)
172177.
[45] J.M. Hakumaki, R.A. Kauppinen, 1H NMR visible lipids in the life and death of
cells, Trends Biochem. Sci. 25 (2000) 357362.
[46] H.L. Ploegh, A lipid-based model for the creation of an escape hatch from the
endoplasmic reticulum, Nature 448 (2007) 435438.
[47] K.E. Howell, G.E. Palade, Heterogeneity of lipoprotein particles in hepatic Golgi
fractions, J. Cell Biol. 92 (1982) 833845.
[48] R. Kalscheuer, A. Steinbuchel, A novel bifunctional wax ester synthase/acylCoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol
biosynthesis in Acinetobacter calcoaceticus ADP1, J. Biol. Chem. 278 (2003)
80758082.
[49] M. Waltermann, A. Hinz, H. Robenek, D. Troyer, R. Reichelt, U. Malkus, H.J. Galla,
R. Kalscheuer, T. Stoveken, P. von Landenberg, A. Steinbuchel, Mechanism of
lipid-body formation in prokaryotes: how bacteria fatten up, Mol. Microbiol. 55
(2005) 750763.
[50] M. Waltermann, A. Steinbuchel, Neutral lipid bodies in prokaryotes: recent
insights into structure, formation, and relationship to eukaryotic lipid depots,
J. Bacteriol. 187 (2005) 36073619.
[51] H. Robenek, O. Hofnagel, I. Buers, M.J. Robenek, D. Troyer, N.J. Severs,
Adipophilin-enriched domains in the ER membrane are sites of lipid droplet
biogenesis, J. Cell. Sci. 119 (2006) 42154224.
[52] A.M. Dvorak, E.S. Morgan, P.F. Weller, RNA is closely associated with human mast
cell lipid bodies, Histol. Histopathol. 18 (2003) 943968.
[53] A.M. Dvorak, E. Morgan, R.P. Schleimer, S.W. Ryeom, L.M. Lichtenstein, P.F. Weller,
Ultrastructural immunogold localization of prostaglandin endoperoxide
synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies
in human lung mast cells, alveolar macrophages, type II pneumocytes, and
neutrophils, J. Histochem. Cytochem. 40 (1992) 759769.
[54] D. Ghosal, N.W. Shappell, T.W. Keenan, Endoplasmic reticulum lumenal proteins
of rat mammary gland. Potential involvement in lipid droplet assembly during
lactation, Biochim. Biophys. Acta 1200 (1994) 175181.
[55] P.T. Bozza, W. Yu, J.F. Penrose, E.S. Morgan, A.M. Dvorak, P.F. Weller, Eosinophil
lipid bodies: specic, inducible intracellular sites for enhanced eicosanoid
formation, J. Exp. Med. 186 (1997) 909920.
[56] M.J. Robenek, N.J. Severs, K. Schlattmann, G. Plenz, K.P. Zimmer, D. Troyer,
H. Robenek, Lipids partition caveolin-1 from ER membranes into lipid
droplets: updating the model of lipid droplet biogenesis, FASEB J. 18 (2004)
866868.
[57] H. Robenek, M.J. Robenek, D. Troyer, PAT family proteins pervade lipid droplet
cores, J. Lipid Res. 46 (2005) 13311338.
[58] A.B. Novikoff, P.M. Novikoff, O.M. Rosen, C.S. Rubin, Organelle relationships in
cultured 3T3-L1 preadipocytes, J. Cell Biol. 87 (1980) 180196.
[59] E.J. Blanchette-Mackie, N.K. Dwyer, T. Barber, R.A. Coxey, T. Takeda, C.M.
Rondinone, J.L. Theodorakis, A.S. Greenberg, C. Londos, Perilipin is located on
the surface layer of intracellular lipid droplets in adipocytes, J. Lipid Res. 36
(1995) 12111226.
[60] L.L. Luckenbill, S.S. Cohen, The association of lipid droplets with cytoplasmic
laments in avian subsynovial adipose cells, J. Cell Biol. 31 (1966) 195199.

Y. Ohsaki et al. / Biochimica et Biophysica Acta 1791 (2009) 399407

[61] W.W. Franke, M. Hergt, C. Grund, Rearrangement of the vimentin cytoskeleton
during adipose conversion: formation of an intermediate lament cage around
lipid globules, Cell 49 (1987) 131141.
[62] J.G. Lieber, R.M. Evans, Disruption of the vimentin intermediate lament system
during adipose conversion of 3T3-L1 cells inhibits lipid droplet accumulation, J.
Cell. Sci. 109 (1996) 30473058.
[63] E. Colucci-Guyon, M.M. Portier, I. Dunia, D. Paulin, S. Pournin, C. Babinet, Mice
lacking vimentin develop and reproduce without an obvious phenotype, Cell 79
(1994) 679694.
[64] P. Targett-Adams, D. Chambers, S. Gledhill, R.G. Hope, J.F. Coy, A. Girod, J.
McLauchlan, Live cell analysis and targeting of the lipid droplet-binding
adipocyte differentiation-related protein, J. Biol. Chem. 278 (2003)
1599816007.
[65] S. Ozeki, J. Cheng, K. Tauchi-Sato, N. Hatano, H. Taniguchi, T. Fujimoto, Rab18
localizes to lipid droplets and induces their close apposition to the endoplasmic
reticulum-derived membrane, J. Cell. Sci. 118 (2005) 26012611.
[66] C. Sztalryd, M. Bell, X. Lu, P. Mertz, S. Hickenbottom, B.H. Chang, L. Chan, A.R.
Kimmel, C. Londos, Functional compensation for adipose differentiation-related
protein (ADFP) by Tip47 in an ADFP null embryonic cell line, J. Biol. Chem. 281
(2006) 3434134348.
[67] S. Martin, K. Driessen, S.J. Nixon, M. Zerial, R.G. Parton, Regulated localization of
Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid
droplet catabolism, J. Biol. Chem. 280 (2005) 4232542335.
[68] F.N. Ghadially, Ultrastructural Pathology of the Cell and Matrix, Edward Arnold,
London, 1997.
[69] B.H. Stemberger, R.M. Walsh, S. Patton, Morphometric evaluation of lipid droplet
associations with secretory vesicles, mitochondria and other components in the
lactating cell, Cell Tissue Res. 236 (1984) 471475.
[70] M. Schrader, Tubulo-reticular clusters of peroxisomes in living COS-7 cells:
dynamic behavior and association with lipid droplets, J. Histochem. Cytochem.
49 (2001) 14211429.
[71] D. Binns, T. Januszewski, Y. Chen, J. Hill, V.S. Markin, Y. Zhao, C. Gilpin, K.D.
Chapman, R.G. Anderson, J.M. Goodman, An intimate collaboration between
peroxisomes and lipid bodies, J. Cell Biol. 173 (2006) 719731.
[72] H.J. van Manen, Y.M. Kraan, D. Roos, C. Otto, Single-cell Raman and uorescence
microscopy reveal the association of lipid bodies with phagosomes in leukocytes,
Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 1015910164.
[73] G. Wanner, R.R. Theimer, Membranous appendices of spherosomes (oleosomes):
possible role in fat utilization in germinating oil seeds. Planta 140 (1978)
163169.
[74] T. Fujimoto, H. Kogo, K. Ishiguro, K. Tauchi, R. Nomura, Caveolin-2 is targeted to
lipid droplets, a new membrane domain in the cell, J. Cell Biol. 152 (2001)
10791085.
[75] Y. Ohsaki, J. Cheng, A. Fujita, T. Tokumoto, T. Fujimoto, Cytoplasmic lipid droplets
are sites of convergence of proteasomal and autophagic degradation of
apolipoprotein B, Mol. Biol. Cell 17 (2006) 26742683.
[76] T. Levine, C. Rabouille, Endoplasmic reticulum: one continuous network
compartmentalized by extrinsic cues, Curr. Opin. Cell Biol. 17 (2005) 362368.
[77] J.A. Napier, A.K. Stobart, P.R. Shewry, The structure and biogenesis of plant oil
bodies: the role of the ER membrane and the oleosin class of proteins, Plant Mol.
Biol. 31 (1996) 945956.
[78] D.J. Lacey, M.J. Hills, Heterogeneity of the endoplasmic reticulum with respect to
lipid synthesis in developing seeds of Brassica napus L. Planta 199 (1996)
545551.
[79] C. Sarmiento, J.H. Ross, E. Herman, D.J. Murphy, Expression and subcellular
targeting of a soybean oleosin in transgenic rapeseed. Implications for the
mechanism of oil-body formation in seeds, Plant J. 11 (1997) 783796.
[80] G.F. Gibbons, K. Islam, R.J. Pease, Mobilisation of triacylglycerol stores, Biochim.
Biophys. Acta 1483 (2000) 3757.
[81] J.M. Shockey, S.K. Gidda, D.C. Chapital, J.C. Kuan, P.K. Dhanoa, J.M. Bland, S.J.
Rothstein, R.T. Mullen, J.M. Dyer, Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different
subdomains of the endoplasmic reticulum, Plant Cell 18 (2006) 22942313.
[82] R. Bartz, W.H. Li, B. Venables, J.K. Zehmer, M.R. Roth, R. Welti, R.G. Anderson, P.
Liu, K.D. Chapman, Lipidomics reveals that adiposomes store ether lipids and
mediate phospholipid trafc, J. Lipid Res. 48 (2007) 837847.
[83] R. Leber, E. Zinser, G. Zellnig, F. Paltauf, G. Daum, Characterization of lipid
particles of the yeast, Saccharomyces cerevisiae, Yeast 10 (1994) 14211428.

407

[84] T. Hevonoja, M.O. Pentikainen, M.T. Hyvonen, P.T. Kovanen, M. Ala-Korpela,

Structure of low density lipoprotein (LDL) particles: basis for understanding
molecular changes in modied LDL, Biochim. Biophys. Acta 1488 (2000)
189210.
[85] S. Prattes, G. Horl, A. Hammer, A. Blaschitz, W.F. Graier, W. Sattler, R. Zechner, E.
Steyrer, Intracellular distribution and mobilization of unesteried cholesterol in
adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like
surface layer structures, J. Cell. Sci. 113 (2000) 29772989.
[86] H. Gorrissen, A.P. Tulloch, R.J. Cushley, Deuterium magnetic resonance of
selectively deuterated cholesteryl esters in phosphatidylcholine vesicles,
Biochemistry 19 (1980) 34223429.
[87] H. Gorrissen, A.P. Tulloch, R.J. Cushley, Deuterium magnetic resonance of
triacylglycerols in phospholipid bilayers, Chem. Phys. Lipids 31 (1982) 245255.
[88] L.S. Ramsammy, H. Brockerhoff, Lysophosphatidylcholinecholesterol complex,
J. Biol. Chem. 257 (1982) 35703574.
[89] G.S. Shelness, A.S. Ledford, Evolution and mechanism of apolipoprotein
B-containing lipoprotein assembly, Curr. Opin. Lipidol. 16 (2005) 325332.
[90] S.O. Olofsson, J. Boren, Apolipoprotein B: a clinically important apolipoprotein
which assembles atherogenic lipoproteins and promotes the development of
atherosclerosis, J. Intern. Med. 258 (2005) 395410.
[91] E.A. Fisher, H.N. Ginsberg, Complexity in the secretory pathway: the assembly
and secretion of apolipoprotein B-containing lipoproteins, J. Biol. Chem. 277
(2002) 1737717380.
[92] A. Kulinski, S. Rustaeus, J.E. Vance, Microsomal triacylglycerol transfer protein is
required for lumenal accretion of triacylglycerol not associated with ApoB, as
well as for ApoB lipidation, J. Biol. Chem. 277 (2002) 3151631525.
[93] P. Rava, G.K. Ojakian, G.S. Shelness, M.M. Hussain, Phospholipid transfer activity
of microsomal triacylglycerol transfer protein is sufcient for the assembly and
secretion of apolipoprotein B lipoproteins, J. Biol. Chem. 281 (2006)
1101911027.
[94] D. Marchesan, M. Rutberg, L. Andersson, L. Asp, T. Larsson, J. Boren, B.R.
Johansson, S.O. Olofsson, A phospholipase D-dependent process forms lipid
droplets containing caveolin, adipocyte differentiation-related protein, and
vimentin in a cell-free system, J. Biol. Chem. 278 (2003) 2729327300.
[95] L. Asp, C. Claesson, J. Boren, S.O. Olofsson, ADP-ribosylation factor 1 and its
activation of phospholipase D are important for the assembly of very low density
lipoproteins, J. Biol. Chem. 275 (2000) 2628526292.
[96] T. Fujimoto, Y. Ohsaki, J. Cheng, M. Suzuki, Y. Shinohara, Lipid droplets: a classic
organelle with new outts, Histochem. Cell Biol. 136 (2008) 263279.
[97] P. Bostrom, M. Rutberg, J. Ericsson, P. Holmdahl, L. Andersson, M.A. Frohman, J.
Boren, S.O. Olofsson, Cytosolic lipid droplets increase in size by microtubuledependent complex formation, Arterioscler. Thromb. Vasc. Biol. 25 (2005)
19451951.
[98] P. Bostrom, L. Andersson, M. Rutberg, J. Perman, U. Lidberg, B.R. Johansson, J.
Fernandez-Rodriguez, J. Ericson, T. Nilsson, J. Boren, S.O. Olofsson, SNARE
proteins mediate fusion between cytosolic lipid droplets and are implicated in
insulin sensitivity, Nat. Cell Biol. 9 (2007) 12861293.
[99] L. Andersson, P. Bostrom, J. Ericson, M. Rutberg, B. Magnusson, D. Marchesan, M.
Ruiz, L. Asp, P. Huang, M.A. Frohman, J. Boren, S.O. Olofsson, PLD1 and ERK2
regulate cytosolic lipid droplet formation, J. Cell. Sci. 119 (2006) 22462257.
[100] Y. Guo, T.C. Walther, M. Rao, N. Stuurman, G. Goshima, K. Terayama, J.S. Wong,
R.D. Vale, P. Walter, R.V. Farese, Functional genomic screen reveals genes
involved in lipid-droplet formation and utilization, Nature 453 (2008) 657661.
[101] P. Liu, R. Bartz, J.K. Zehmer, Y.S. Ying, M. Zhu, G. Serrero, R.G. Anderson, Rabregulated interaction of early endosomes with lipid droplets, Biochim. Biophys.
Acta 1773 (2007) 784793.
[102] H.P. Moore, R.B. Silver, E.P. Mottillo, D.A. Bernlohr, J.G. Granneman, Perilipin
targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive
lipase, J. Biol. Chem. 280 (2005) 4310943120.
[103] N.E. Wolins, D.L. Brasaemle, P.E. Bickel, A proposed model of fat packaging by
exchangeable lipid droplet proteins, FEBS Lett. 580 (2006) 54845491.
[104] L.L. Listenberger, X. Han, S.E. Lewis, S. Cases, R.V. Farese Jr., D.S. Ory, J.E. Schaffer,
Triglyceride accumulation protects against fatty acid-induced lipotoxicity, Proc.
Natl. Acad. Sci. U. S. A. 100 (2003) 30773082.
[105] Y. Miyanari, K. Atsuzawa, N. Usuda, K. Watashi, T. Hishiki, M. Zayas, R.
Bartenschlager, T. Wakita, M. Hijikata, K. Shimotohno, The lipid droplet is an
important organelle for hepatitis C virus production, Nat. Cell Biol. 9 (2007)
10891097.