F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
The biophysics and cell biology of
lipid droplets
Abdou Rachid Thiam1,2, Robert V. Farese Jr3,4 and Tobias C. Walther1
Abstract | Lipid droplets are intracellular organelles that are found in most cells, where they
have fundamental roles in metabolism. They function prominently in storing oil-based
reserves of metabolic energy and components of membrane lipids. Lipid droplets are the
dispersed phase of an oil-in-water emulsion in the aqueous cytosol of cells, and the
importance of basic biophysical principles of emulsions for lipid droplet biology is now being
appreciated. Because of their unique architecture, with an interface between the dispersed
oil phase and the aqueous cytosol, specific mechanisms underlie their formation, growth and
shrinkage. Such mechanisms enable cells to use emulsified oil when the demands for
metabolic energy or membrane synthesis change. The regulation of the composition of the
phospholipid surfactants at the surface of lipid droplets is crucial for lipid droplet
homeostasis and protein targeting to their surfaces.
Yale School of Medicine,
Department of Cell Biology,
333 Cedar Street,
New Haven, Connecticut
06510, USA.
2
Laboratoire de Physique
Statistique, Ecole Normale
Supérieure de Paris,
Université Pierre et Marie
Curie, Université Paris
Diderot, Centre National de la
Recherche Scientifique,
24 rue Lhomond, 75005
Paris, France.
3
Gladstone Institute of
Cardiovascular Disease,
1650 Owens Street,
San Francisco, California
94158, USA.
4
Departments of Medicine
and Biochemistry and
Biophysics, University of
California, San Francisco,
California 94158, USA.
Correspondence to R.V.F.Jr
and T.C.W
e-mails:
[email protected];
[email protected]
doi:10.1038/nrm3699
Published online
13 November 2013
1
Living systems are maintained by a constant flux of
metabolic energy, and lipids that are rich in reduced
hydrocarbons provide a source of energy for many
organisms. Because new energy sources are not always
available, the ability to store lipids in cells and tissues
is often crucial for survival. In addition, cells must be
able to buffer and store excess lipids in an inert form.
Thus, nearly all cells are capable of storing lipids in
partitioned reservoirs. To package lipids efficiently,
cells convert them into neutral lipids, such as triacylglycerols and sterol esters, which exclude water. These
lipids are deposited into specialized intracellular organelles termed lipid droplets (which are also referred to
as adiposomes, lipid bodies, or oil bodies)1–4. In addition to storing energy, lipid droplets provide reservoirs
of lipids (such as sterols, fatty acids and phospholipids)
for membrane synthesis. Given their diverse functions,
lipid droplets are situated at the crossroads of membrane biology and energy metabolism and are important organelles in maintaining cell homeostasis. With
their role in lipid storage, lipid droplets figure prominently in common pathologies that are linked to lipid
accumulation, including obesity, diabetes and atherosclerosis5, and in industrial applications, such as efforts
to produce triacylglycerols for food and hydrocarbons
more generally as biofuel.
Despite the almost universal presence of lipid droplets in cells of nearly all organisms, surprisingly little
is known about the molecular details of the processes
underlying their biology. However, recent advances
in understanding the cell biological properties and
functions of lipid droplets have begun to change this.
These developments have also underscored the
importance of understanding the biophysical properties of lipid droplets as they relate to cell biology. Lipid
droplets are essentially oil drops that are insoluble in
water and are dispersed in the aqueous cytosol. Many of
the fundamental processes governing lipid droplets as
organelles, and more broadly metabolism of oils in cells,
occur at the interface of the two phases. In this Review,
we focus on the analysis of lipid droplets as oil-in-water
emulsions and therefore highlight the implications of
concepts that have been derived from emulsion science
for lipid droplet biology. We first provide an overview
of relevant concepts from emulsions physics and then
review and discuss their implications for lipid droplet
biogenesis and degradation.
From emulsion physics to lipid droplet cell biology
An emulsion is a mixture of two immiscible fluids: one
is dispersed in the other in the form of drops. Examples
of emulsions include direct emulsions (that is, oil drops
in water) or inverse emulsions (that is, water drops in
oil). Lipid droplets in the cytoplasm provide a biological
example of a direct emulsion that is common to most
cells. The cytosol represents the continuous aqueous
phase, and the dispersed oil phase, the lipid droplets,
includes neutral lipids, such as triacylglycerols, sterol
esters, retinyl esters, waxes or ether lipids, depending
on the cell type and the neutral lipids they store6.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 14 | DECEMBER 2013 | 775
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Box 1 | Emulsion stability
Many emulsions are metastable (that is, thermodynamically unstable but so long-lived
to be stable for practical purposes). Key to emulsion stability is the presence of
interfacial surfactants. When an interface between oil and water is formed, a surface
tension (γ; expressed in millinewton per metre (mN/m)) is generated owing to a lack of
cohesive interactions at the interface between the molecules from each phase. The
generation of an interface of area A between the lipid droplets and the cytosol results in
an energy cost of γA. In the presence of surfactants, an emulsion evolves to minimize
the contact area between the immiscible fluids and to decrease the energy costs by
lowering the surface tension.
Stabilizing surfactants are compounds that decrease the surface tension and buffer
against thermal surface fluctuations, which would otherwise favour emulsion
destabilization. Monolayer absorbed-surfactant molecules are dynamic and can
experience lateral and transversal dilatation–compression that causes local depletion
of surfactants, and this will lead to coalescence. Having a phospholipid monolayer
proffers an elasticity to the droplet interface, which reflects the rheology of the
surfactant monolayer towards dampening such fluctuations10,11,16,17, mainly by setting
fluid monolayer with long acyl chain surfactants. Some basic principles of emulsion
physics that are relevant to lipid droplets, including surfactant properties, are
FIG. 1.
Critical micellar
concentration
The concentration at which
surfactants form micelles.
Cosurfactants
Cosurfactants are inefficient
surfactants alone and smaller
than primary surfactants.
They can fill the space between
primary surfactants to reduce
surface tension. Cosurfactants
can easily partition between
the different phases.
Surface tension
Surface tension (γ) reflects
the energy that is required to
increase the surface area of a
liquid by a unit area and is
the energy cost per unit area
generated between two
immiscible fluids. The presence
of phospholipid surfactants
minimizes γ by shielding the
interface.
Cells deal with excess lipids such as fatty acids (which
can act as detergents) by esterifying the potentially toxic
lipids to form more inert neutral-lipid oils, such as
triacylglycerols or sterol esters. The formation of these
oils occurs within membrane bilayers; however, because
bilayers are unsuited for storing large amounts of oil7,8,
an emulsion of oil droplets forms in the cytoplasm.
Specific proteins can act on cytoplasmic lipid droplets
to regulate their growth or utilization. Increasingly, we
are learning that cells have evolved a machinery to generate and utilize lipid droplet–cytosol emulsions in an
organized and regulated manner. Concepts in emulsion
science are therefore highly relevant to understand lipid
droplet cell biology. Nevertheless, this paradigm has not
yet been well integrated into the field of lipid droplet
biology.
To generate stable emulsions, surfactants such as
phospholipids are required (BOX 1). Artificially generated emulsions are most often formed with surfactants that are soluble in the continuous but not
the dispersed phase, in agreement with the Bancroft
rule (which states that the phase in which the emulsifier is soluble tends to be the continuous phase)9–11.
When the concentration of a surfactant exceeds its critical
micellar concentration, the micelles that form provide a
surfactant reservoir to buffer surface area fluctuations of
emulsion droplets and thus increase their stability.
For cellular lipid droplets, phospholipids at the surface monolayer constitute the main surfactant (FIG. 1).
In contrast to other surfactants used in artificial emulsions, phospholipids are soluble neither in the cytoplasmic aqueous phase nor in the oil phase. Therefore,
bilayer membranes (such as those of the endoplasmic
reticulum (ER)) rather than micelles serve as phospholipid reservoirs in cells. Interestingly, the phospholipid
composition differs in ER bilayers and lipid droplet
monolayers12. Specifically, lipid droplet monolayers
generally have more phosphatidylcholine and less free
cholesterol and sphingomyelin than ER membranes12.
The mechanisms leading to the differences in ER and
lipid droplet composition are unclear but are likely to
be important for understanding the biophysical properties of lipid droplets. One possibility of how this lipid
sorting may be achieved is that ER domains of distinct
lipid composition are dedicated to lipid droplet formation. Such domains could arise either by spontaneous
sorting 13 of surface lipids at sites of nascent lipid droplets or their formation could be mediated by specific
proteins. As the surface composition of lipid droplet
differs greatly, the functional implications of this variability are particularly intriguing. For example, during
lipid droplet growth or shrinkage, their surface area as
well as the amount of phospholipid surfactants change
markedly. Moreover, during those processes, intermediates of metabolic reactions, such as unesterified sterols,
diacylglycerol (DAG) or fatty acids, may accumulate
on lipid droplet surfaces. At these surfaces, they act as
fluidifiers by increasing the diffusion of phospholipids,
which results in a more even distribution of phospholipids on the monolayer, or they act as cosurfactants to
decrease surface tension (γ)14,15. This may lead to dynamic
interfacial behaviours, which in some instances favour
spontaneous lipid droplet formation.
The size of lipid droplets differs considerably between
cells and varies across different emulsion scales, from
nanoemulsion- to macroemulsion-type droplets (that is,
from 100 nm to 100 mm in diameter). For example, lipid
droplets in yeast are typically smaller than a micron,
and adipocytes often contain a single lipid droplet of
tens or hundreds of microns. Therefore, lipid droplets may be subject to different kinetic and thermodynamic destabilization mechanisms, depending on
the cell type. However, the functional implications of
the vastly different sizes of lipid droplets are poorly
understood.
Lipid droplet stability in cells
Cells have stable lipid droplets on biological timescales
because their oil phase is covered by a monolayer of
surfactant phospholipids16,17 (FIG. 1a,b). Phospholipids
are preferentially adsorbed at the interface due to
their amphiphilic property. They decrease surface tension, provide high bending elasticity to the monolayer
and thus increase lipid droplet emulsion stability 18,19,20
(FIG. 1b,c). For instance, in the presence of phospholipids,
the surface tension of lipid droplets can be reduced to
the order of 0.1–1 millinewton per metre (mN/m),
compared with an exposed interface that exhibits
γ ~ 30 mN/m21, and confers to the monolayer a higher
bending modulus, or rigidity, of the order of 1–10 kBT22,23
(where kB is the Boltzmann constant and T the temperature). This provides lipid droplets with kinetic stability
under most conditions. In addition to phospholipid surfactants, proteins on the lipid droplet surface can also
increase the interface elasticity, further contributing
to the stability of lipid droplets (FIG. 1c). However, even
with lowered surface tension and increased elasticity in
the presence of phospholipids and proteins, lipid droplet emulsions in cells are a priori only metastable and
predestined for destabilization.
776 | DECEMBER 2013 | VOLUME 14
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved
F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
a
Curvature
LPC, PtdIns,
LPA, LPE,
+
MAG
0
PC, PS
–
DAG,
Cholesterol
PA, PE, FA
c
b
γ0
TG
γ1
TG
γ0 > γ1
d
Monolayer
P0
r
Pin > P0
Laplace pressure
ΔP = Pin – P0 = 2γ/r
Figure 1 | Basic principles of emulsion physics relevant to lipid droplets. a | Surface
lipids on lipid droplets and their curvature at physiological
pH. The
curvature
of Biology
typical
Nature Reviews
| Molecular
Cell
surfactants is defined by the difference between the areas occupied by their
hydrophilic head and their lipophilic tail. Positive curvatures correspond to a
predominant hydrophilic part. Positively curved lipids, such as lysophospholipids,
phosphatidylinositol (PtdIns) and monoacylglycerol (MAG), tend to proffer a
monolayer a positive curvature and form direct micelles. Conversely, monolayers
mainly formed by diacylglycerol (DAG), phosphatidic acid (PA) or cholesterol have a
negative curvature and tend to form reverse micelles. Phosphatidylcholine (PC) is
cylindrical in shape and has no preferred curvature. Therefore, phosphatidylcholine
has no micellar preference and would generally assemble into lamella and is the main
component of bilayer membranes. b | The influence of surfactants on emulsion
stability. Increasing the amounts of surfactants at the interface of triacylglycerol
(TG)-containing droplets tends to lower the exposed surface area A (that is, favours
decreased surface tension (γ)) and increase stability. Less surfactant, or less effective
surfactants (for example, phosphatidylethanolamine (PE) versus phosphatidylcholine)
have the opposite effect. | The elasticity of surface monolayers. A loose monolayer,
such as an oil–water interface, has ‘wrinkles’ that are associated with thermal
fluctuations. The presence of phospholipids dampens the fluctuations by creating an
energy barrier to local surface deformation. They also increase the bending energy of
the monolayer: phospholipids with longer acyl chains are more efficient for dampening
fluctuations. Higher concentrations of phospholipids result in a higher barrier to
induce deformation by thermal fluctuation and prevent the formation of phospholipiddepleted areas. The presence of proteins can also increase the energy barrier to
deformation. | Laplace pressure (ΔP) is the pressure that builds up inside the lipid
droplet to counterbalance the compression effect of surface tension (which is denoted
as γ). P0
r. The Laplace
pressure of the droplet is the difference between the pressures inside and outside the
droplet and corresponds to 2γ/r. If the surface tension is similar, smaller drops have
The composition of the phospholipid monolayer
depends on the cell type but mostly comprises phosphatidylcholine, phosphatidylethanolamine and to a
lesser extent phosphatidylinositol (PtdIns) and lysophospholipids12. Each of these phospholipids has specific interfacial properties that determine its ability to
stabilize emulsions. For example, phosphatidylcholine
is cylindrical in shape and therefore provides excellent
coverage of the surface area and greatly lowers surface
tension. Phosphatidylcholine is crucially important
for lipid droplet stability. By contrast, phosphatidylethanolamine is conically shaped, has a smaller head
group and is not as good as phosphatidylcholine in stabilizing oil droplet emulsions. Some unesterified lipids,
such as sterols or DAG, may also localize to the lipid
droplet surface, where they serve as less-optimal surfactants but can fill the space between phospholipids
and act as cosurfactants. The surfactant properties of
some of the common lipids found at the surfaces of lipid
droplets are shown in FIG. 1a.
Emulsion destabilization pathways
Two physical processes destabilize emulsion droplets:
coalescence and ripening (FIG. 2). Each process aims to
minimize the interfacial surface (to reduce the energy
cost) and generally results in fewer and larger lipid
droplets.
Destabilization by coalescence. Coalescence (or fusion)
of emulsion droplets, including cellular lipid droplets,
proceeds in two steps (FIG. 2a). First, two lipid droplets need to come in close proximity, and the aqueous
film separating them is depleted. Different types of
forces, including Van der Waals, entropic or electrostatic interactions, that are mediated by biochemical
or hydrodynamic parameters can drive the drainage
of the aqueous film, which brings lipid droplets closer
together 16,24–27. In a second step, which is driven by thermal fluctuations, a pore is directly connected to the two
oil phases and links the two lipid droplets. Pore formation occurs when the droplets are so close to each other
that the thickness of the aqueous film approaches few
nanometres. The exact distance at which pore formation occurs depends on many parameters, in particular
the surface tension of the interface, which is a measure
of interface thermal fluctuations28. Finally, complete
fusion occurs as the pore expands. Of note, with fusion,
there is no shrinkage of one of the droplets, as is seen
with Ostwald ripening (discussed below).
For two triacylglycerol droplets covered with phospholipids, the timescale of pore expansion and fusion
is well below a second at room temperature. However,
pore formation does not necessarily lead to fusion; pores
can exist transiently and reseal. Whether a pore expands
and leads to lipid droplet fusion depends on the intrinsic
curvature of the surfactant, which determines intrinsic
curvature of the monolayer 9,11,17. During pore formation, the monolayer is highly bent in a small region. The
deviation of this curvature from the intrinsic curvature
of the monolayer generates an energy barrier for fusion.
Coalescence thus depends on the intrinsic curvature of
the monolayer: if the curvature of the monolayer and
that imposed by the pore formation are both either
positive or negative, the pore is stabilized, can expand
and fusion is favoured; otherwise, it closes owing to the
energy barrier caused by curvature mismatch. For example, the pore is stabilized and coalescence of oil droplets
is favoured when surface lipids such as phosphatidylethanolamine, cholesterol, DAG or fatty acids are present, which because of their small head groups favour
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 14 | DECEMBER 2013 | 777
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Intrinsic curvature
For a surfactant, it is their
spontaneous curvature
(dependent on properties such
as pH, length of acyl chains
and temperature). It reflects
the hydrophilic and lipophilic
balance of the molecules. If the
mean area of the hydrophilic
part of a surfactant is larger
than that of the hydrophobic
part, the curvature of the
molecules is considered
positive, and it tends to form
direct micelles. In the opposite
case, the curvature is negative.
Line tension
Line tension (Γ) is the energy
cost per unit length at the
boundary between different
phases. Among many
parameters, line tension is a
function of surfactant acyl
chain length and bending
modulus.
Laplace pressure
The pressure difference (ΔP)
between the inside and outside
of a curved liquid surface.
Surface tension compresses
the disperse liquid to a
spherical shape to minimize
the energy of the system.
Contraction arrests when a
relatively positive Laplace
pressure builds up inside
the drop.
bending that stabilizes the junction between the monolayer and the pore,23,29. Pore formation is modulated by
the elasticity of the monolayers that are locally deformed.
The elastic contribution of surfactants to the pore opening depends on the bending energy or rigidity of the
monolayer, which reflects the energy required to deform
an interface. The major contribution of surfactants on
pore formation is expressed in a global parameter30
termed line tension (Γ) (FIG. 2). If a pore forms between
two drops, Γ is the energy cost per unit length of the
pore diameter.
Thus, two properties determine if coalescence occurs:
surface tension and line tension of the coalescence intermediate. More specifically, coalescence is an activated
process whereby a transient pore between two objects
forms, and this leads to complete fusion of both droplets
if thermal fluctuations overcome an energy barrier that
is proportional to Γ2/γ. This means that coalescence is
favoured if surface tension is high and line tension is low,
and it is less likely to occur if line tension is high and
surface tension is low.
As pore expansion and resealing are dependent on
curvature, several interfacial lipids have a strong influence on the stability of oil-in-water emulsions and
have different properties with respect to line tension
(FIG. 2a). Unesterified cholesterol is one relevant example. In bilayer membranes (for example, at the plasma
membrane), cholesterol decreases line tension and
typically increases the kinetics of hydrophilic pore closing 31. However, it has the opposite effect at the surface
of monolayers by favouring hydrophobic pore opening,
for example in a bilayer hemifusion process32. In such
configurations, cholesterol decreases the line tension and
therefore the energy barrier for coalescence. DAG and
phosphatidic acid provide other examples of lipids that
have a strong effect on membrane dynamics. In bilayer
membranes, they facilitate the closing of the hydrophilic
pore during vesicle formation at the ‘pinching-off ’ stage,
for example as in the case of coat protein complex I
(COPI) vesicles that bud from the membrane 23,33–35.
Conversely, in the context of lipid droplets, these molecules with negative curvature would favour fusion
between lipid droplets or fusion of lipid droplets with
bilayer membranes. Other surface lipids with intrinsic positive curvature, such as PtdIns or lysophospholipids, may influence budding and pinching-off of lipid
droplets.
Lipid droplet destabilization by Ostwald ripening. The
Ostwald ripening destabilization process involves a
coarsening mechanism in which small droplets of an
emulsion disappear as bigger ones grow (FIG. 2b). For
oil droplets, this occurs when oil molecules (although
they are relatively insoluble in the aqueous phase)
transfer from one droplet to another through the continuous phase. During Ostwald ripening, molecules
from smaller droplets are transferred through the continuous phase to larger droplets (if they have a similar
surface composition). The direction of the transfer is
determined by the difference in the Laplace pressures,
2γ (1/r1 – 1/r2) between droplets with the respective
Figure 2 | Processes that govern changes in lipid droplet ▶
size. a | Coalescence and the influence of monolayer
curvature. A droplet that contains triacylglycerol (TG) and
is covered with phospholipids forms a pore with another
lipid droplet monolayer or the outer monolayer of a bilayer.
At the site of the pore, the monolayer is bent, and
monolayer curvature, depending on the types of lipids,
becomes important. If the spontaneous curvature of the
monolayer is positive (for example, in excess presence
reaction with high line tension (Γ), and the pore closes.
(for example, in excess presence of negatively curved
lipids), the curvature of the lipids matches the bending, and
the line tension is low. Therefore, the pore is stable and can
coalescence (or fusion), generating one larger lipid droplet
(see the inset). In the case of a lipid droplet and a
membrane, fusion results in a transiently stable connection
of lipid droplets with bilayers. Pore opening and fusion
occur within milliseconds. b | Ripening of lipid droplets.
During ripening, molecules from one lipid droplet diffuse
to another. The direction is determined by the difference in
the Laplace pressures of the two lipid droplets, with
triacylglycerol molecules (for example) traveling from the
smaller lipid droplet to the bigger one. In the case of
triacylglycerol and lipid droplets, diffusion might occur in
swollen micelles, which are micelles that contain small
amounts of triacylglycerol. In contrast to coalescence,
ripening takes several minutes. The volume increase of the
bigger droplet is linear over time, r3 t. Ripening also leads
shrinks while the other one grows. | Growth of lipid
droplets by de novo triacylglycerol synthesis
.
Enzymes that mediate triacylglycerol synthesis, such
1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3),
and diacylglycerol O-acyltransferase (DGAT2), can directly
localize to lipid droplets and synthesize the neutral lipids at
their surfaces. Acyl CoA synthetases localize to lipid
droplets and are likely to provide the fatty acyl CoA
substrates. As recently observed65, the volume of the
droplet increases linearly over time, r3 t. DAG,
lysophosphatidylethanolamine
radii r1 and r2. The mismatch of Laplace pressures is
driving the ripening instability. A simple analogy
is the generation of an electric current (flow of triacylglycerols) from an electrode with a high potential
(which represents a smaller lipid droplet with higher
Laplace pressure) to an electrode with a low potential (a
bigger lipid droplet with a lower Laplace pressure) when
a conductor (the aqueous phase) links them. During
ripening, the cube of the radius (r 3) of growing drops
increases linearly over time, r 3 ∞ t. The rate of growth
is proportional to the solubility of the transferred molecule in the continuous phase. Therefore, Ostwald
ripening is suppressed when the dispersed phase molecules have extremely low solubility in the continuous
phase. This is the case for both triacylglycerols and
778 | DECEMBER 2013 | VOLUME 14
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved
F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
a Coalescence
LPC, PtdIns, LPA,
LPE, MAG
PC, PS
TG
DAG, Cholesterol
PA, PE, FA
TG
Monolayer (of a lipid droplet
or as part of a bilayer)
Pore
Cytosol
>>
Γ↑
>>
Γ↓
TG
TG
Closed pore
Expanding pore
b Ripening
TG
r2
ΔP1 > ΔP2
TG
r23 ∞ t
r1
ΔP1 = 2γ/r1
ΔP2 = 2γ/r2
c Expansion by TG synthesis
FA
TG synthesis
enzymes
TG
r
TG
r3 ∞ t
sterol esters, which are almost completely insoluble in
water. Thus, it is difficult to imagine that this type of
ripening occurs between lipid droplets in vivo.
There are, however, alternative ripening pathways.
When non-ionic amphiphilic molecules, such as some
detergents, are able to form micelles in the continuous
phase, they may form swollen micelles (which are several
nm in size) that contain the dispersed phase36–39. In the
case of lipid droplets, these would be triacylglyceroland/or sterol ester-containing micelles. Under these
conditions, the micelles may carry small fractions of oil
from small drops to bigger drops. This solubilization of
triacylglycerols into micelles is analogous to the solubilization of hydrophobic protein domains into detergent
micelles. Depending on the state of the cell, the presence of small amounts of amphiphilic lipids, such as fatty
acids or DAG, may favour such processes. For example,
fatty acids form micelles in water at neutral pH, which
could mediate ripening by swollen micelles40. Similarly,
micelles that form in oil could cause tiny water phases in
lipid droplets, which possibly explains the observations
of proteins that have been found within the oil phase of
lipid droplets41.
The stability of clustered lipid droplets for long
periods of time in some cells suggests that ripening
between lipid droplets is not constitutive but may be a
triggered mechanism. For example, lipolysis may induce
lipid droplet remodelling 42,43 by ripening. In this case,
lipolysis products might form swollen micelles that facilitate ripening. In addition, surface lipid properties are
altered. Lipid droplet remodelling due to ripening would
be expected to occur on a timescale of a few minutes to
hours, and drops can be farther apart. This is completely
different to the coalescence mechanism in which drops
must be close and content mixing occurs within seconds.
In basic ripening processes, droplets can be apart
from each other and transfer occurs through the continuous phase. A particular case of ripening, permeation,
has been observed for lipid droplets and is attributed
to the action of specific proteins44–46. For this type of
ripening, droplets must be very close so that molecules
of the dispersed phase (triacylglycerols or sterol esters
for lipid droplets) avoid traveling through the continuous phase45,47,116. This often requires the formation of
channels between droplets to allow the transfer of molecules. For example, fat-specific protein p27 (FSP27;
also known as CIDEC) is required for the formation of
unilocular lipid droplets from smaller lipid droplets during adipocyte differentiation44,46,48. It has been suggested
that FSP27 creates stable channelling pores between the
droplets which allow ripening by facilitating triacylglycerol permeation from one lipid droplet to another
without crossing the aqueous phase. FSP27 might assemble at the junction between drops, which provides the
energy to keep pores open without expanding. The oil
transport occurs within a few minutes. In a permeation process, the square of the growing droplet radius
is expected11,49 to be linear over time, r 2 ∞ t. This time
frame matches that observed for FSP27-mediated lipid
droplet coarsening and lipid droplet remodelling during
lipolysis and growth in adipocytes42–44,46,48.
Nature Reviews | Molecular Cell Biology
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 14 | DECEMBER 2013 | 779
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Protein binding to lipid droplets
The lipid droplet proteome. In addition to phospholipids,
the surface of lipid droplets is decorated with proteins.
Since the identification of perilipins as lipid droplet
marker proteins that are important for the regulation
of lipid metabolism50,51, proteomic and cell biological
analyses have revealed hundreds of lipid droplet-resident
protein candidates in various cell types. However,
methodological issues confound the interpretation of
some of these studies. The sensitivity of mass spectrometry for proteomics is consistently increasing, which leads
to ever-longer growing lists of proteins that are identified
in lipid droplet fractions. Although the overlap among
these studies serves to identify a core set of lipid droplet
proteins, it is difficult to determine a priori which of the
proteins identified by mass spectrometry are genuine
lipid droplet proteins in a specific cell type and which
are low-level contaminants of the analysed lipid droplet
fraction. A case in point was the identification of histones
as lipid droplet proteins in the Drosophila melanogaster
embryo52. Although this finding was initially met with
some scepticism, unexpectedly, this association is specific, mediated by a protein receptor, and functionally
important to buffer histone levels during early development 52,53. Recent advances in quantitative proteomics
approaches (such as protein correlation profiling) that
measure the abundance and enrichment of proteins in
the lipid droplet fraction, rather than just their presence,
are likely to overcome the limitations of mass spectrometry, especially for low-abundance proteins or proteins
expressed in a specific tissue cell type54.
In repeated studies, a couple of dozen proteins have
been confirmed to be lipid droplet proteins. These proteins have various cell functions. Many of them have a role
in lipid metabolism, for example in the synthesis or trafficking of phosphatidylcholine, sterols or triacylglycerols.
Some of the proteins that are not directly involved in lipid
metabolism have a role in controlling lipid droplet surface properties. Perilipins, for example, were proposed
to protect lipid droplets from lipolysis by shielding the
triacylglycerol core of lipid droplets from lipases55.
Permeation
In the context of emulsions it is
the process by which one type
of molecule (for example,
triacylglycerol), which is
present in one compartment,
crosses a membrane barrier or
a liquid film by diffusing
through it and thus reaches
another compartment.
Protein targeting to lipid droplets. The unique properties
of the lipid droplet surface, compared with those of other
organelles, have important consequences for specific protein targeting. For bilayer membranes, the defined thickness and hydrophobicity of the fatty acid side chains in the
bilayer restricts membrane proteins to those with specific
domains, such as transmembrane α-helices and β-barrels.
Such transmembrane segments with hydrophilic regions
on either side of the bilayer cannot exist as lipid droplet
proteins, as this would place at least one of the hydrophilic
segments in the oil phase, which would be energetically
unfavourable. Instead, lipid droplet-associated proteins
must interact with surface monolayer lipids, be embedded
in the hydrophobic core or both.
Currently, targeting mechanisms for lipid droplet proteins are largely unknown. However, at least two types
of lipid droplet protein targeting signals are emerging
from a collection of studies: amphipathic α-helices and
hydrophobic hairpins4 (FIG. 3). Lipid droplet proteins
that contain one or more amphipathic α-helices, such
as cytidine triphosphate (CTP)–phosphocholine
cytidylyltransferase-α (CCTα; which is the rate limiting enzyme of phosphatidylcholine synthesis) or viperin
(an antiviral protein), are synthesized in the cytoplasm
and associate with the lipid droplet monolayer presumably through binding on the hydrophobic side of the
amphipathic helix 19,56. Other proteins, such as hepatitis C virus core protein, have amphipathic helices that
bind the ER membrane bilayer before targeting the lipid
droplet monolayer. What causes some amphipathic helixcontaining proteins to target specifically to lipid droplets and accumulate there instead of other organelles is
unknown. Currently, no specific lipid droplet lipids are
known to be involved in protein recruitment, in contrast
to other examples, such as the endocytic system, in which
phosphoinositides are involved in protein targeting.
Alterations in membrane surface tension may serve
to restrict protein targeting to lipid droplets. Whereas
bilayer membranes with relatively vast and fluid continuous surfaces have ultra-low surface tensions, lipid droplet monolayers are delimited entities that can have much
higher surface tension by exposing more triacylglycerol
to the cytosol. At higher surface tension, portions of the
oil phase that underlie the monolayer may be dynamically exposed, which could generate strongly hydrophobic patches that enable protein binding. This model is
supported by in vitro data of CCTα binding to artificially
generated emulsion droplets. Specifically, CCTα binding
to artificial lipid droplets occurs when little phosphatidylcholine is present, and thus, surface tension is likely to be
high19. CCTα binding to phosphatidylcholine-poor lipid
droplets or bilayer membranes results in the activation
of the protein19,57,58 and increases phosphatidylcholine
synthesis to balance this deficit. This provides the cell
with a homeostatic mechanism for the maintenance of
phosphatidylcholine levels. Consistent with this model,
amphipathic helices in other oil-binding proteins may
have a similar binding mode. For instance, amphipathic helices are also found in apolipoproteins that
associate with the monolayer surfaces of extracellular
lipoprotein particles, such as chylomicrons or very
low-density lipoprotein (VLDL), which can be thought
of as smaller, extracellular lipid droplets. An amphipathic helix, the carboxyl terminus of apolipoprotein
APOA-I, senses exposed triacylglycerol. It dissociates
from phosphatidylcholine-covered oil emulsions during
phosphatidylcholine monolayer compression59 (FIG. 3).
By contrast, the amphipathic helices of APOC-I bind
more efficiently to densely packed phosphatidylcholine
monolayers than sparsely covered ones60 (FIG. 3). These
examples provide precedence for proteins that detect
differences in surface properties of oil–water interfaces,
which is likely to be an important factor for determining
the targeting of lipid droplet proteins.
Another sequence motif found in many lipid droplet proteins is a hairpin of two α-helices that dips into
and out of a bilayer membrane, such as the ER, without
completely spanning it. Proteins that contain this type of
signal include specific isoenzymes of the triacylglycerol
synthesis pathway, caveolins and plant oleosins61–65.
780 | DECEMBER 2013 | VOLUME 14
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved
F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
Unfolded
helix
Amphipathic
helix-containing protein
Hairpin-containing
protein
Cytosol
Loose packing
Dense packing
LPC, PtdIns, LPA, LPE,
MAG
PC, PS
DAG, Cholesterol
PA, PE, FA
Figure 3 | Binding mode of proteins. An illustration
showing
the| binding
of amphipathic
Nature
Reviews
Molecular
Cell Biology
α-helix-and hairpin-containing proteins to lipid droplets. A loose monolayer of higher
surface tension is bound by one type of helix (for example, cytidine triphosphate (CTP)–
phosphocholine cytidylyltransferase-α (CCTα) or the carboxyl terminus of apolipoprotein
(left panel). An unfolded helix, for example of APOC-I, does not bind to lipid droplets
expelled. The protein soluble in the cytosol can now fold its amphipathic helix by
interacting, for example, with the head group of phospholipids.
These proteins are co-translationally inserted into the
ER membrane. When lipid droplets form, hairpincontaining proteins migrate to lipid droplets along membrane bridges that connect the ER membrane with the
lipid droplet-delimiting monolayer 65,66. Although membrane bridges provide a path for proteins to lipid droplets, the energy source and mechanism that lead to the
accumulation of proteins on lipid droplets are unclear.
One possibility is that this targeting is also regulated by
surface properties of the lipid droplet, with the targeted
proteins reducing surface tension of the oil–water interface, thereby providing the energy changes that result in
the accumulation of proteins.
Dewetting
The rupture of a thin film on a
substrate to form a droplet,
the counterpart to which is
spreading. It depends on the
surfactant concentration.
Dewetting of an oil droplet
within a bilayer occurs when
the monolayers of the bilayer
wet together. This process can
be favoured in emulsions by
lowering surface tension.
Contact angle
The contact angle (θ) is the
angle at which a liquid interface
meets a solid surface. It is also
applicable to the angle
between an lipid droplet and
the endoplasmic reticulum
bilayer.
Bending modulus
The bending modulus
represents the energy that is
needed to bend a monolayer
from its spontaneous
curvature.
Lipid droplet formation and growth
In eukaryotic organisms, lipid droplets form from the
ER. Although direct visualization and knowledge for
the initial stages of the formation process are lacking,
many lines of evidence support a model whereby lipid
droplets are derived from the ER. For example, most
of the enzymes involved in triacylglycerol or sterol
ester synthesis are localized to the ER (in the absence
of lipid droplets). Moreover, electron microscopy data
reveal close apposition between lipid droplets and the
ER41,51,65,67. In addition, many proteins, particularly those
containing hydrophobic hairpins, show dual localization
between the ER and lipid droplets. Recent model systems
of inducible lipid droplet formation in yeast provide more
direct evidence that newly formed lipid droplets originate
from the ER in this organism66. While this does not rule
out additional origins from other organelles under some
conditions, the available data strongly suggest that lipid
droplet formation is a function of the ER.
The steps of initial lipid droplet formation are still
unknown. Most models posit an initial lens-shaped accumulation of triacylglycerol (or sterol esters) within the
bilayer. At some point, the growing structure is predicted
to bud off the ER, forming a lipid droplet. From a physical
standpoint, we suggest a model based on a dewetting
mechanism (FIG. 4a). When a liquid is deposited on a surface, it spreads to either fully wet the surface (that is, it
forms a thin film) or it partially wets the surface (that is,
it forms a contact angle (θ) with the substrate surface).
In the extreme case of complete dewetting (for example,
water on a waxed car surface), the liquid nearly forms a
spherical drop. This dewetting process has been studied
extensively 30 and aspects of it might correspond to the
budding of a lipid droplet from a bilayer membrane, as
observed in vitro68,69. In this manner, a lipid lens would be
converted to a nascent lipid droplet by gradually decreasing the contact angle (FIG. 4a). The oil dewetting process
from a bilayer is influenced by the phospholipid composition of the bilayer membrane and the forming monolayer. Budding of lipid droplets is thermodynamically
favoured when the surface tension of the monolayer and
the bilayer are lowered47,69–73 (FIG. 4b). During lipid droplet
formation, fatty acids and DAG might be present in considerable amounts and act as cosurfactants for phospholipids, lowering surface tension and bending modulus, and
favouring lipid droplet budding 15,29,74–76. A model for lipid
droplet formation based on spontaneous emulsification,
during which surface tension is lowered and approaches
values close to zero, predicts that the size of the formed
droplet ranges from 100 to 300 nm77,78. The budding size
is a function of different parameters, which reflects the
wetting properties of the oil with the monolayer, which is
the substrate, and the local surface and line tensions7,70,73.
Modulating these parameters would lead to different
budding sizes. This model posits that lipid droplet formation occurs spontaneously when sufficient triacylglycerol
accumulates and adequate surfactants are available to
lower surface tension and elastic moduli. It also predicts
that the size of the budded lipid droplet depends on the
surfactant type. Such a purely physical process would
help to explain why no single gene products were shown
to be required for lipid droplet formation.
Theoretically, new lipid droplets can form from existing lipid droplets. If the surface tension of existing lipid
droplets is sufficiently lowered (for example, below a
threshold (~0.01 mN/m)79–81), new lipid droplets might
form spontaneously 82. In this process, the oil and surface lipids are present in a ratio that energetically favours
surface generation or the spontaneous formation of new
lipid droplets. More generally, any molecule that efficiently cycles between the triacylglycerol and aqueous
phases favours spontaneous droplet formation. For
example, particularly short-chain fatty acids or alcohols
may favour this process.
Proteins are also likely to influence the surface tension
and the budding process as already observed for bilayer
vesicle formation83,84. Perilipins, for example, bind to
regions of the ER in some but not all cells during lipid
droplet formation85, which suggests they modulate the
budding process. Additional proteins that might aid in
the formation of lipid droplets are BSCL2 (Bernardinelli–
Seip congenital lipodystrophy type 2; also known as
seipin) and FIT (fat storage-inducing transmembrane)
proteins; these ER proteins have unclear molecular functions and their deficiency dramatically alters the size of
the lipid droplet86,87.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 14 | DECEMBER 2013 | 781
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
esters or triacylglycerol exist in cells, which suggests different origins88. We speculate that some triacylglycerol
may be needed in sterol ester-containing lipid droplets
to maintain a liquid phase at physiological temperatures
(when pure sterol esters would be solid). In addition,
among triacylglycerol-containing lipid droplets, a subpopulation expands after their initial formation65. This
process is mediated by the relocalization of a subset of
enzymes that catalyse in the successive steps of triacylglycerol synthesis, including glycerol-3-phosphate
acyltransferase 4 (GPAT4), 1-acylglycerol-3-phosphate
O-acyltransferase 3 (AGPAT3) and diacylglycerol
O-acyltransferase 2 (DGAT2) (FIGS 2c,4c). Fatty acyl CoA
synthetases have been localized to lipid droplets89–91 and
are likely to be involved in the generation the fatty acyl
CoA molecules that are needed. On lipid droplets, these
enzymes together locally generate triacylglycerol, which
leads to linear volume expansion over time of these lipid
droplets specifically 65.
a Budding
Wetting
Dewetting
Liquid
Liquid
θ
Substrate
Substrate
b Tension-driven dewetting by
lowering surface tension
TG
TG
Cytosol
c Formation
TG
TG
TG
TG
TG
TG
Cytosol
LPC, PtdIns, LPA, LPE,
MAG
DGAT1
PC, PS
DGAT2
DAG, Cholesterol,
PA, PE, FA
Models for mechanisms of lipid droplet
formation.
| Illustration
of Biology
Nature
Reviews a| Molecular
Cell
dewetting transitions. A liquid that is deposited on a surface can wet the surface
completely and form a liquid film that covers it. The liquid can dewet and form a droplet
with a specific contact angle (θ) relative to the substrate. b | Wetting is controlled by
surface tension. A model showing spontaneous budding of a triacylglycerol (TG) droplet
from a bilayer based on dewetting transition. The accumulation of triacylglycerol inside a
bilayer can lead to spontaneous emulsification of a triacylglycerol-containing droplet in
dewetting state that is energetically favoured. Different surface lipids can modulate such
a process. c | Origins of different populations of lipid droplets. Evidence suggests that two
populations of basic lipid droplets exist. Smaller lipid droplets bud from the endoplasmic
reticulum (ER), for example from the enzymatic products of ER enzymes such as
diacylglycerol O-acyltransferase (DGAT1) (left panel). Such lipid droplets have a
characteristic size that is likely to be dependent on the surface surfactants. A second
population of cytosolic lipid droplets, called expanding lipid droplets, acquires enzymes
pathway (right panel). These enzymes can locally synthesize triacylglycerol at the lipid
It is unknown whether lipid droplet formation occurs
in a similar manner for different neutral lipids (for example, triacylglycerol, sterol esters or retinyl esters). Different
types of lipid droplets with either preferentially sterol
Lipid droplet disappearance and shrinkage
Cells break down triacylglycerol or sterol esters from lipid
droplets to generate metabolic energy and to liberate
lipids for membrane synthesis.
Mechanisms of lipolysis. Most information on the process
of lipid droplet breakdown, known as lipolysis, has been
derived from studies in adipocytes. In adipocytes, fatty
acids are released from cells as fuel for tissues, such as skeletal muscle and the heart. At lipid droplets, the sequential
hydrolysis of triacylglycerol, is catalysed by the sequential
action of three lipases92 (FIG. 5a). Adipose triglyceride lipase
(ATGL; also known as PNPLA2) removes a fatty acid
preferentially from the sn2-position of triacylglycerol93
to yield DAG. Subsequently, hormone-sensitive lipase
(HSL) hydrolyses DAG to monoacylglycerol (MAG) and
a fatty acid. In the final step, MAG is hydrolysed by MAG
lipase (MGL) to glycerol and a fatty acid. Sterol esters are
also hydrolysed by HSL. However, other carboxylesterase
lipases (such as CES1 or CES3) have been suggested to
catalyse this reaction. The relative contribution of these
enzymes in vivo is still under debate94,95.
ATGL and HSL are constitutively localized to lipid
droplets. In addition to their lipase domains, they have
hydrophobic stretches that mediate lipid droplet targeting. In HSL, the first 300 amino acids are responsible for
lipid droplet binding 96. A hydrophobic stretch in ATGL is
located in its C terminus and mediates its association with
lipid droplets. It is unknown how lipases, such as ATGL,
access the oil phase of lipid droplets and triacylglycerol
substrate. Intriguingly, the localization of ATGL to lipid
droplets requires the ADP-ribosylation factor 1 (ARF1)–
COPI machinery97,117. From the canonical function of
ARF1–COPI proteins in retrograde vesicular trafficking
from the Golgi to the ER, these proteins might mediate
transport of ATGL in vesicles from a donor membrane
to lipid droplets. Alternatively, as ARF1–COPI have
been observed on lipid droplets, these proteins might
act directly at their surfaces, altering the surface properties and indirectly regulating ATGL targeting. ARF1–
COPI proteins form nanolipid droplets (~60 nm) from
782 | DECEMBER 2013 | VOLUME 14
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved
F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
a Lipolysis
8
-5
GI
ATGL
FA
C
TG
OH
HSL
DG
FA
FA
MG
OH
MGL
O
H
b Lipophagy
OH OH
HO
Hydrolic
enzymes
Lysosome
TG
TG
TG
TG
Autolysosome
Autophagosome
TG
FA
Figure 5 | Consumption and utilization of lipid droplets. a | Schematic representation of lipolysis. Adipose triglyceride
Reviews CGI-58.
| Molecular
Cell Biology
lipase (ATGL) catalyses the first step of lipolysis and is recruited onto lipid dropletsNature
by the cofactor
Triacylglycerol
(TG) hydrolysis by ATGL leads to the release of free fatty acids (FA) and mainly 1,3-diacylglycerol (DAG). Hormone-sensitive
lipase (HSL), which is also bound to lipid droplets, hydrolyses DAG into free fatty acids and monoacylglycerol (MAG).
of the lipid droplet decreases over time, probably linearly. As a result, the surface phospholipids and proteins become
more crowded. Mechanisms must exist to facilitate the catabolism or removal of these surface components. b | Model
showing lipid droplet autophagy (lipophagy). Lipophagy has also been proposed to regulate lipid droplet degradation and
triacylglycerol utilization. An autophagosome forms in the cytosol and encapsulates a lipid droplet. It subsequently fuses
with a lysosomal organelle that contains enzymes that hydrolyse triacylglycerol from the encapsulated lipid droplet and
proteases that degrade lipid droplet proteins.
a phospholipid monolayer interface21, which supports a
possible function for these proteins in regulating the
surface properties of the lipid droplets.
Lipolysis is strictly controlled in adipocytes (reviewed
in REFS 98–100. Hormonal stimulation (for example, through β-adrenergic receptors) mediates cyclic
AMP (cAMP)- and protein kinase A (PKA)-dependent
phosphorylation and activation of HSL. These kinases
also phosphorylate perilipin 1 on lipid droplets.
Phosphorylation of perilipin 1 releases an interaction
with the lipid droplet-associated protein CGI-58, which
becomes available to activate ATGL on lipid droplets. The
mechanism of CGI-58-dependent activation of ATGL is
unknown but could be mediated by alterations in the surface properties of lipid droplets, making triacylglycerol
substrates available for the lipase. This hypothesis might
explain why ATGL specifically requires a cofactor to gain
access to triacylglycerol. By contrast, the substrates for
other lipases, such as HSL and MGL (DAG and MAG,
respectively), partition well into a monolayer. MAG partitions more to the monolayer than DAG, which is triacylglycerol-soluble, and this may explain why MGL does
not need to bind lipid droplets and instead exists in the
cytosol (FIG. 5a).
Unlike in adipocytes, much less is known about
triacylglycerol mobilization in other tissues. In cells
with high energy demands, mobilized fatty acids can be
directly oxidized for ATP generation. For example, muscle
cells generate a lot of metabolic energy, and in these cells,
contact sites between lipid droplets and mitochondria
are important for funnelling fatty acids for oxidation.
Recently, a lipid droplet protein, perilipin 5 (also known
as OXPAT) has been implicated in establishing the close
contacts between mitochondria and the ER101. How this
protein binds to mitochondria and how the contact sites
are established or regulated are unknown.
Many tissues express ATGL, HSL and MGL, but
they are often present at much lower levels than in adipocytes. With the identification of homologous lipases
of the PNPLA (patatin-like phospholipase domaincontaining protein A) family, this suggests that other
lipases have important lipolytic roles in different cell
types. Intriguingly, a polymorphism in one of these
lipases, PNPLA3 (also known as adiponutrin), is strongly
associated with the development with hepatic steatosis
in humans102. However, it is unclear whether it is due to
the enzyme acting as a lipase, an acyltransferase or yet as
another, unidentified function.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
VOLUME 14 | DECEMBER 2013 | 783
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Buckling interface
A ‘wrinkled’ surface that forms
when a monolayer has become
rigid because of increased
phospholipid density following
compression. The wrinkles are
a way to relax the applied
stress, as they increase the
monolayer surface.
Consequences of lipolysis for lipid droplets. When triacylglycerols are hydrolysed from the core of a lipid droplet it
shrinks. Unless phospholipids are removed from the interface, their packing is likely to become increasingly dense
on the surface of shrinking lipid droplets. Highly packed
phospholipid monolayers may transition to a solid-like
phase, which leads to a buckling interface. As an example,
during ripening or permeation processes observed by
adding ‘fusogens’ to lipid droplets103, the situation for
the shrinking lipid droplet (which loses triacylglycerols)
is similar to that of lipolysis in which triacylglycerols are
removed enzymatically. Phospholipids on the shrinking
lipid droplet are compressed over time and probably form
a buckled monolayer, which appears as a black line visible by interference contrast microscopy. The compression of proteins on the surface of lipid droplets could also
lead to the appearance of such staining due to the same
buckling effect.
Little is known about how phospholipids and proteins
are removed from shrinking lipid droplets. As the surface area per phospholipid decreases during shrinkage,
excess phospholipids could be expelled from the interface,
but this mechanism is unlikely due to the extremely low
solubility of amphipathic phospholipids in either triacylglycerols or the cytosol. Several lipid droplet proteins, such
as oxysterol-binding proteins or START (StAR-related
lipid transfer) proteins, contain lipid transfer domains,
which could mediate phospholipid transport from lipid
droplets to other organelles, but evidence for such a process is lacking. Phospholipids may also be removed from
lipid droplets interfaces enzymatically, cleaving them to
more water- and triacylglycerol-soluble products, such
as fatty acids and DAG. Consistent with this possibility,
several phospholipases localize to lipid droplets104,105.
However, as yet, phospholipases have not been implicated
to be required for lipolysis, which would be expected if
this process were crucial for lipid droplet shrinkage.
Phospholipids also could be removed from lipid droplets in the form of swollen micelles. During lipolysis, large
amounts of DAG and fatty acids are transiently generated. These lipids can access the monolayer surface, thus
decreasing the surface tension of the lipid droplet. As a
result, small micelles (10–50 nm79 and below the resolution limit of light microscopy) that contain triacylglycerol
and phospholipids at their surface, as well as fatty acids
and/or DAG, could form from lipid droplets and diffuse
in the cytosol. During lipolysis, reverse micelles that
contain water, phospholipids at their surface and fatty
acids and/or DAG might form inside the oil. No evidence supports or contradicts the formation of small
micelles. However, lowering of surface tension on lipid
droplets and spontaneous emulsification during lipolysis
might explain the fission of lipid droplets observed in
Schizosaccharomyces pombe during cell division106.
In addition to phospholipids, proteins need to be
removed from shrinking lipid droplets. For proteins
that bind to them via amphipathic helices, changes in
the surface lipids might alter the affinity, and some proteins might dissociate. For other proteins, such as those
with hairpin topology, their removal from lipid droplets
presents a process that is likely to require energy for
extraction of the hydrophobic regions. Of note, several
proteins that function in proteasome-mediated protein
degradation have been localized to lipid droplets107–109.
It remains to be determined whether these proteins function in lipid droplet protein turnover. At least one such
protein, UBX domain-containing protein 8 (UBXD8;
also known as FAF2), has a function on lipid droplets
that are distinct from protein turnover. UBXD8 interacts
with p97 (also known as VCP) to block the interaction
between CGI-58 and ATGL, thus inhibiting lipolysis107.
In addition to lipolysis, autophagy has been proposed
to mediate lipid droplet turnover 110 (FIG. 5b). In hepatocytes, autophagy proteins are observed in the vicinity
of lipid droplets. In livers of mice with dysfunctional
autophagy, triacylglycerol accumulates, and the amount
of autophagic intermediates depends on the metabolic
states of cells. Similarly, autophagy is required for normal lipid droplet metabolism in other cell types, such as
neurons and stellate cells111,112. Thus, a specialized form
of macroautophagy, termed lipophagy, might mediate the
traffic of lipid droplets to lysosomes where lipid droplet
lipids and proteins are degraded (FIG. 4b). The relative
contribution of lipolysis and lipophagy for lipid droplet
turnover in different tissues is unclear. It is also unclear
how lipid droplets are recognized by the autophagic
machinery or how their autophagy is regulated.
Non-canonical lipid droplet functions
The unique structural features of lipid droplets provide
cells with variable amounts of an interface between two
phases (that is, the lipid droplet surface) and a bulk
organic phase. Several processes seem to have evolved
to capitalize on these unique features of lipid droplets.
For example, their surfaces might temporarily store
hydrophobic proteins destined for degradation, such
as the transmembrane domain-containing HMG-CoA
(3-hydroxy-3-methylglutaryl CoA) reductase113. The surface of the lipid droplet more generally might function
as a sequestration platform for proteins that otherwise
might be toxic for cells, such as histones52. They also seem
to serve as platforms for the transient storage of some
viral proteins that contain amphipathic helices, such as
the core protein of hepatitis C virus, which can be used
subsequently for viral assembly 114.
The bulk organic phase might also provide space
to store and synthesize large and bulky lipid metabolism intermediates that would otherwise disrupt bilayer
membranes. For example, several enzymes that are
involved in the synthesis of dolichol, which can contain ~100 carbons in its isoprenoid chain, have been
found at lipid droplets115. However, the significance of
the localization of these enzymes to lipid droplets is
not understood. The hydrophobic cores of lipid droplets are also likely to provide an important reservoir for
hydrophobic drugs, which partition into this phase, and
fat-soluble vitamins (vitamin A, vitamin D, vitamin E
and vitamin K). Indeed, such partitioning has inspired
the design of triacylglycerol emulsion droplets as carriers for various drugs and motivated research in the
phase behaviour of mixtures of triacylglycerol, water,
phospholipids and derivatives.
784 | DECEMBER 2013 | VOLUME 14
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved
F O C U S O N O R g a N E l l E b I O g E N E S I S a N d h O m EROESVta
SIS
IEW
Conclusions
The basic principles governing lipid droplet biology are
still ill-defined. For example, the mechanisms involved
in the formation of lipid droplets, protein targeting to
lipid droplets and the consequences of their shrinkage
for the organelle are only beginning to be defined. To this
date, we have barely identified the key proteins and lipids
involved in these reactions and are only beginning to
reveal the mechanisms by which they act. For many of
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Walther, T. C. & Farese, R. V. Jr. Lipid droplets and
cellular lipid metabolism. Annu. Rev. Biochem. 81,
687–714 (2012).
Brasaemle, D. L. & Wolins, N. E. Packaging of fat: an
evolving model of lipid droplet assembly and
expansion. J. Biol. Chem. 287, 2273–2279 (2012).
Fujimoto, T. & Parton, R. G. Not just fat: the structure
and function of the lipid droplet. Cold Spring Harb.
Perspect. Biol. 3, a004838 (2011).
Thiele, C. & Spandl, J. Cell biology of lipid droplets.
Curr. Opin. Cell Biol. 20, 378–385 (2008).
Krahmer, N., Farese, R. V. Jr & Walther, T. C.
Balancing the fat: lipid droplets and human disease.
EMBO Mol. Med. 5, 905–915 (2013).
Bartz, R. et al. Lipidomics reveals that adiposomes
store ether lipids and mediate phospholipid traffic.
J. Lipid Res. 48, 837–847 (2007).
Zanghellini, J., Wodlei, F. & von Grunberg, H. H.
Phospholipid demixing and the birth of a lipid droplet.
J. Theor. Biol. 264, 952–961 (2010).
Khandelia, H., Duelund, L., Pakkanen, K. I. &
Ipsen, J. H. Triglyceride blisters in lipid bilayers:
implications for lipid droplet biogenesis and the
mobile lipid signal in cancer cell membranes.
PLoS ONE 5, e12811 (2010).
Kabalnov, A. & Wennerström, H. Macroemulsion
stability: the oriented wedge theory revisited.
Langmuir 12, 276–292 (1996).
Sjoblom, J. Encyclopedic handbook of emulsion
technology (CRC Press, 2010).
Leal-Calderon, F., Schmitt, V. & Bibette, J. Emulsion
science: basic principles (Springer, 2007).
Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R. &
Fujimoto, T. The surface of lipid droplets is a
phospholipid monolayer with a unique fatty acid
composition. J. Biol. Chem. 277, 44507–44512
(2002).
Roux, A. et al. Role of curvature and phase transition
in lipid sorting and fission of membrane tubules.
EMBO J. 24, 1537–1545 (2005).
Fryd, M. M. & Mason, T. G. Advanced nanoemulsions.
Annu. Rev. Phys. Chem. 63, 493–518 (2012).
van Buuren, A. R., Tieleman, D. P., de Vlieg, J. &
Berendsen, H. J. C. Cosurfactants lower surface
tension of the diglyceride/water interface: a molecular
dynamics study. Langmuir 12, 2570–2579 (1996).
Fei, W. et al. A role for phosphatidic acid in the
formation of ‘supersized’ lipid droplets. PLoS Genet. 7,
e1002201 (2011).
Krahmer, N. et al. Phosphatidylcholine synthesis for
lipid droplet expansion is mediated by localized
activation of CTP:phosphocholine cytidylyltransferase.
Cell Metab. 14, 504–515 (2011).
Langevin, D. Rheology of adsorbed surfactant
monolayers at fluid surfaces. Annu. Rev. Fluid Mech.
http://dx.doi.org/10.1146/annurevfluid-010313-141403 (2013).
Georgieva, D., Schmitt, V., Leal-Calderon, F. &
Langevin, D. On the possible role of surface elasticity in
emulsion stability. Langmuir 25, 5565–5573 (2009).
Kabalnov, A., Tarara, T., Arlauskas, R. & Weers, J.
Phospholipids as emulsion stabilizers. J. Colloid
Interface Sci. 184, 227–235 (1996).
Thiam, A. R. et al. COPI buds 60-nm lipid droplets
from reconstituted water–phospholipid–
triacylglyceride interfaces, suggesting a tension clamp
function. Proc. Natl Acad. Sci. USA 110,
13244–13249 (2013).
Chen, Z. & Rand, R. P. The influence of cholesterol on
phospholipid membrane curvature and bending
elasticity. Biophys. J. 73, 267–276 (1997).
Chernomordik, L. V. & Kozlov, M. M. Protein–lipid
interplay in fusion and fission of biological membranes.
Annu. Rev. Biochem. 72, 175–207 (2003).
these and other biological processes that involve lipid
droplets, understanding their unique behaviour as
defined by biophysical principles is essential. Viewing
cells that contain lipid droplets as an oil-in-water emulsion is an emerging concept that allows new interpretations of lipid droplet biology. Soft matter physics
knowledge is only now being integrated into this field and
promises to rapidly expand our understanding of lipid
droplet cell biology and related physiological processes.
24. Koestler, D. C. et al. Blood-based profiles of DNA
methylation predict the underlying distribution of cell
types: a validation analysis. Epigenetics http://dx.doi.
org/10.4161/epi.25430 (2013).
25. Bremond, N., Thiam, A. R. & Bibette, J.
Decompressing emulsion droplets favors coalescence.
Phys. Rev. Lett. 100, 024501 (2008).
26. Thiam, A. R., Bremond, N. & Bibette, J. Breaking of an
emulsion under an ac electric field. Phys. Rev. Lett.
102, 188304 (2009).
27. Bremond, N. & Bibette, J. Exploring emulsion science
with microfluidics. Soft Matter 8, 10549–10559
(2012).
28. Aarts, D. G., Schmidt, M. & Lekkerkerker, H. N. Direct
visual observation of thermal capillary waves. Science
304, 847–850 (2004).
29. Leikin, S., Kozlov, M. M., Fuller, N. L. & Rand, R. P.
Measured effects of diacylglycerol on structural and
elastic properties of phospholipid membranes.
Biophys. J. 71, 2623–2632 (1996).
30. De Gennes, P.-G., Brochard-Wyart, F. & Quéré, D.
Capillarity and wetting phenomena: drops, bubbles,
pearls, waves (Springer, 2004).
31. Karatekin, E. et al. Cascades of transient pores in
giant vesicles: line tension and transport. Biophys. J.
84, 1734–1749 (2003).
32. Biswas, S., Yin, S. R., Blank, P. S. & Zimmerberg, J.
Cholesterol promotes hemifusion and pore widening in
membrane fusion induced by influenza hemagglutinin.
J. Gen. Physiol. 131, 503–513 (2008).
33. Shemesh, T., Luini, A., Malhotra, V., Burger, K. N. &
Kozlov, M. M. Prefission constriction of Golgi tubular
carriers driven by local lipid metabolism: a theoretical
model. Biophys. J. 85, 3813–3827 (2003).
34. Fernandez-Ulibarri, I. et al. Diacylglycerol is required
for the formation of COPI vesicles in the Golgi-to-ER
transport pathway. Mol. Biol. Cell 18, 3250–3263
(2007).
35. Popoff, V., Adolf, F., Brugger, B. & Wieland, F.
COPI budding within the Golgi stack. Cold Spring
Harb. Perspect. Biol. 3, a005231 (2011).
36. Kabalnov, A. & Weers, J. Kinetics of mass transfer in
micellar systems: surfactant adsorption, solubilization
kinetics, and ripening. Langmuir 12, 3442–3448
(1996).
37. Kabalnov, A. S. Can micelles mediate a mass transfer
between oil droplets? Langmuir 10, 680–684
(1994).
38. Ariyaprakai, S. & Dungan, S. R. Influence of surfactant
structure on the contribution of micelles to Ostwald
ripening in oil-in-water emulsions. J. Colloid Interface
Sci. 343, 102–108 (2010).
39. Baret, J. C. Surfactants in droplet-based microfluidics.
Lab Chip 12, 422–433 (2012).
40. Hanczyc, M. M., Fujikawa, S. M. & Szostak, J. W.
Experimental models of primitive cellular
compartments: encapsulation, growth, and division.
Science 302, 618–622 (2003).
41. Robenek, H., Robenek, M. J. & Troyer, D. PAT family
proteins pervade lipid droplet cores. J. Lipid Res. 46,
1331–1338 (2005).
42. Paar, M. et al. Remodeling of lipid droplets during
lipolysis and growth in adipocytes. J. Biol. Chem. 287,
11164–11173 (2012).
43. Ariotti, N. et al. Postlipolytic insulin-dependent
remodeling of micro lipid droplets in adipocytes.
Mol. Biol. Cell 23, 1826–1837 (2012).
44. Gong, J. et al. Fsp27 promotes lipid droplet growth by
lipid exchange and transfer at lipid droplet contact
sites. J. Cell Biol. 195, 953–963 (2011).
45. Jambunathan, S., Yin, J., Khan, W., Tamori, Y. &
Puri, V. FSP27 promotes lipid droplet clustering and
then fusion to regulate triglyceride accumulation.
PLoS ONE 6, e28614 (2011).
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
46. Sun, Z. et al. Perilipin1 promotes unilocular lipid
droplet formation through the activation of Fsp27 in
adipocytes. Nature Commun. 4, 1594 (2013).
47. Thiam, A. R., Bremond, N. & Bibette, J. From stability
to permeability of adhesive emulsion bilayers.
Langmuir 28, 6291–6298 (2012).
48. Grahn, T. H. et al. FSP27 and PLIN1 interaction
promotes the formation of large lipid droplets in
human adipocytes. Biochem. Biophys. Res. Commun.
432, 296–301 (2013).
49. Kabalnov, A. S. & Shchukin, E. D. Ostwald ripening
theory: applications to fluorocarbon emulsion stability.
Adv. Colloid Interface Sci. 38, 69–97 (1992).
50. Greenberg, A. S. et al. Perilipin, a major hormonally
regulated adipocyte-specific phosphoprotein
associated with the periphery of lipid storage droplets.
J. Biol. Chem. 266, 11341–11346 (1991).
51. Londos, C. et al. Perilipin: unique proteins associated
with intracellular neutral lipid droplets in adipocytes
and steroidogenic cells. Biochem. Soc. Trans. 23,
611–615 (1995).
52. Cermelli, S., Guo, Y., Gross, S. P. & Welte, M. A. The
lipid-droplet proteome reveals that droplets are a
protein-storage depot. Curr. Biol. 16, 1783–1795
(2006).
53. Li, Z. et al. Lipid droplets control the maternal histone
supply of Drosophila embryos. Curr. Biol. 22,
2104–2113 (2012).
54. Krahmer, N. et al. Protein correlation profiles identify
lipid droplet proteins with high confidence. Mol. Cell
Proteom. 12, 1115–1126 (2013).
55. Brasaemle, D. L. et al. Perilipin A increases
triacylglycerol storage by decreasing the rate of
triacylglycerol hydrolysis. J. Biol. Chem. 275,
38486–38493 (2000).
56. Hinson, E. R. & Cresswell, P. The antiviral protein,
viperin, localizes to lipid droplets via its N-terminal
amphipathic α-helix. Proc. Natl Acad. Sci. USA 106,
20452–20457 (2009).
57. Taneva, S., Dennis, M. K., Ding, Z., Smith, J. L. &
Cornell, R. B. Contribution of each membrane binding
domain of the CTP:phosphocholine
cytidylyltransferase-α dimer to its activation,
membrane binding, and membrane cross-bridging.
J. Biol. Chem. 283, 28137–28148 (2008).
58. Ding, Z. et al. A 22-mer segment in the structurally
pliable regulatory domain of metazoan
CTP:phosphocholine cytidylyltransferase facilitates
both silencing and activating functions. J. Biol. Chem.
287, 38980–38991 (2012).
59. Mitsche, M. A. & Small, D. M. C-terminus of
apolipoprotein A-I removes phospholipids from a
triolein/phospholipids/water interface, but the
N-terminus does not: a possible mechanism for
nascent HDL assembly. Biophys. J. 101, 353–361
(2011).
60. Meyers, N. L., Wang, L. & Small, D. M.
Apolipoprotein C-I binds more strongly to
phospholipid/triolein/water than triolein/water
interfaces: a possible model for inhibiting cholesterol
ester transfer protein activity and triacylglycerol-rich
lipoprotein uptake. Biochemistry 51, 1238–1248
(2012).
61. Abell, B. M. et al. Role of the proline knot motif in
oleosin endoplasmic reticulum topology and oil body
targeting. Plant Cell 9, 1481–1493 (1997).
62. Napier, J. A., Stobart, A. K. & Shewry, P. R. The
structure and biogenesis of plant oil bodies: the role of
the ER membrane and the oleosin class of proteins.
Plant Mol. Biol. 31, 945–956 (1996).
63. Pol, A. et al. Cholesterol and fatty acids regulate
dynamic caveolin trafficking through the Golgi complex
and between the cell surface and lipid bodies.
Mol. Biol. Cell 16, 2091–2105 (2005).
VOLUME 14 | DECEMBER 2013 | 785
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
64. Le Lay, S. et al. Cholesterol-induced caveolin targeting
to lipid droplets in adipocytes: a role for caveolar
endocytosis. Traffic 7, 549–561 (2006).
65. Wilfling, F. et al. Triacylglycerol synthesis enzymes
mediate lipid droplet growth by relocalizing from
the ER to lipid droplets. Dev. Cell 24, 384–399
(2013).
66. Jacquier, N. et al. Lipid droplets are functionally
connected to the endoplasmic reticulum in
Saccharomyces cerevisiae. J. Cell Sci. 124,
2424–2437 (2011).
67. Waltermann, M. et al. Mechanism of lipid-body
formation in prokaryotes: how bacteria fatten up.
Mol. Microbiol. 55, 750–763 (2005).
68. Shum, H. C., Lee, D., Yoon, I., Kodger, T. & Weitz, D. A.
Double emulsion templated monodisperse
phospholipid vesicles. Langmuir 24, 7651–7653
(2008).
69. Hayward, R. C., Utada, A. S., Dan, N. & Weitz, D. A.
Dewetting instability during the formation of
polymersomes from block-copolymer-stabilized double
emulsions. Langmuir 22, 4457–4461 (2006).
70. Li, Y., Kusumaatmaja, H., Lipowsky, R. & Dimova, R.
Wetting-induced budding of vesicles in contact with
several aqueous phases. J. Phys. Chem. B 116,
1819–1823 (2012).
71. Liu, Y., Lipowsky, R. & Dimova, R. Concentration
dependence of the interfacial tension for aqueous
two-phase polymer solutions of dextran and
polyethylene glycol. Langmuir 28, 3831–3839
(2012).
72. Thiam, A. R., Bremond, N. & Bibette, J. Adhesive
emulsion bilayers under an electric field: from
unzipping to fusion. Phys. Rev. Lett. 107, 068301
(2011).
73. Kusumaatmaja, H. & Lipowsky, R. Droplet-induced
budding transitions of membranes. Soft Matter 7,
6914–6919 (2011).
74. Rosen, M. J. Surfactants and Interfacial Phenomena.
3rd edn Ch. 8 303–331 (Wiley, 2004).
75. Safinya, C. R., Sirota, E. B., Roux, D. & Smith, G. S.
Universality in interacting membranes: the effect of
cosurfactants on the interfacial rigidity. Phys. Rev.
Lett. 62, 1134–1137 (1989).
76. Kozlov, M. M. & Helfrich, W. Effects of a cosurfactant
on the stretching and bending elasticities of a
surfactant monolayer. Langmuir 8, 2792–2797
(1992).
77. Gursoy, R. N. & Benita, S. Self-emulsifying drug
delivery systems (SEDDS) for improved oral delivery of
lipophilic drugs. Biomed. Pharmacother. 58,
173–182 (2004).
78. Pouton, C. W. Lipid formulations for oral
administration of drugs: non-emulsifying, selfemulsifying and ‘self-microemulsifying’ drug delivery
systems. Eur. J. Pharm. Sci. 11, S93–S98 (2000).
79. Sadurni, N., Solans, C., Azemar, N. & GarciaCelma, M. J. Studies on the formation of O/W nanoemulsions, by low-energy emulsification methods,
suitable for pharmaceutical applications. Eur.
J. Pharm. Sci. 26, 438–445 (2005).
80. De Gennes, P. G. & Taupin, C. Microemulsions and the
flexibility of oil/water interfaces. J. Phys. Chem. 86,
2294–2304 (1982).
81. Langevin, D. Microemulsions. Accounts Chem. Res.
21, 255–260 (1988).
82. Yamada, A. et al. Spontaneous transfer of
phospholipid-coated oil-in-oil and water-in-oil microdroplets through an oil/water interface. Langmuir 22,
9824–9828 (2006).
83. Stachowiak, J. C. et al. Membrane bending by
protein–protein crowding. Nature Cell Biol. 14,
944–949 (2012).
84. Zimmerberg, J. & Kozlov, M. M. How proteins produce
cellular membrane curvature. Nature Rev. Mol. Cell
Biol. 7, 9–19 (2005).
85. Wolins, N. E., Brasaemle, D. L. & Bickel, P. E.
A proposed model of fat packaging by exchangeable
lipid droplet proteins. FEBS Lett. 580, 5484–5491
(2006).
86. Cartwright, B. R. & Goodman, J. M. Seipin: from
human disease to molecular mechanism. J. Lipid Res.
53, 1042–1055 (2012).
87. Fei, W., Du, X. & Yang, H. Seipin, adipogenesis and
lipid droplets. Trends Endocrinol. Metab. 22,
204–210 (2011).
88. Hsieh, K. et al. Perilipin family members preferentially
sequester to either triacylglycerol-specific or
cholesteryl-ester-specific intracellular lipid storage
droplets. J. Cell Sci. 125, 4067–4076 (2012).
89. Poppelreuther, M. et al. The N-terminal region of
acyl-CoA synthetase 3 is essential for both the
localization on lipid droplets and the function in fatty
acid uptake. J. Lipid Res. 53, 888–900 (2012).
90. Brasaemle, D. L., Dolios, G., Shapiro, L. & Wang, R.
Proteomic analysis of proteins associated with lipid
droplets of basal and lipolytically stimulated 3T3-L1
adipocytes. J. Biol. Chem. 279, 46835–46842 (2004).
91. Fujimoto, Y. et al. Involvement of ACSL in local
synthesis of neutral lipids in cytoplasmic lipid droplets
in human hepatocyte HuH7. J. Lipid Res. 48,
1280–1292 (2007).
92. Young, S. G. & Zechner, R. Biochemistry and
pathophysiology of intravascular and intracellular
lipolysis. Genes Dev. 27, 459–484 (2013).
93. Eichmann, T. O. et al. Studies on the substrate and
stereo/regioselectivity of adipose triglyceride lipase,
hormone-sensitive lipase, and diacylglycerolO-acyltransferases. J. Biol. Chem. 287,
41446–41457 (2012).
94. Ouimet, M. & Marcel, Y. L. Regulation of lipid droplet
cholesterol efflux from macrophage foam cells.
Arterioscler. Thromb. Vasc. Biol. 32, 575–581
(2012).
95. Ghosh, S., Zhao, B., Bie, J. & Song, J. Macrophage
cholesteryl ester mobilization and atherosclerosis.
Vascul Pharmacol. 52, 1–10 (2010).
96. Holm, C., Osterlund, T., Laurell, H. & Contreras, J. A.
Molecular mechanisms regulating hormone-sensitive
lipase and lipolysis. Annu. Rev. Nutr. 20, 365–393
(2000).
97. Soni, K. G. et al. Coatomer-dependent protein
delivery to lipid droplets. J. Cell Sci. 122,
1834–1841 (2009).
98. Zechner, R., Kienesberger, P. C., Haemmerle, G.,
Zimmermann, R. & Lass, A. Adipose triglyceride lipase
and the lipolytic catabolism of cellular fat stores.
J. Lipid Res. 50, 3–21 (2009).
99. Zimmermann, R., Lass, A., Haemmerle, G. &
Zechner, R. Fate of fat: the role of adipose triglyceride
lipase in lipolysis. Biochim. Biophys. Acta 1791,
494–500 (2009).
100. Lass, A., Zimmermann, R., Oberer, M. & Zechner, R.
Lipolysis — a highly regulated multi-enzyme complex
mediates the catabolism of cellular fat stores.
Prog. Lipid Res. 50, 14–27 (2011).
101. Wang, H. et al. Perilipin 5, a lipid droplet-associated
protein, provides physical and metabolic linkage to
mitochondria. J. Lipid Res. 52, 2159–2168 (2011).
102. Romeo, S. et al. Genetic variation in PNPLA3 confers
susceptibility to nonalcoholic fatty liver disease.
Nature Genet. 40, 1461–1465 (2008).
103. Murphy, S., Martin, S. & Parton, R. G. Quantitative
analysis of lipid droplet fusion: inefficient steady state
fusion but rapid stimulation by chemical fusogens.
PLoS ONE 5, e15030 (2010).
786 | DECEMBER 2013 | VOLUME 14
104. Nakamura, N., Banno, Y. & Tamiya-Koizumi, K.
Arf1-dependent PLD1 is localized to oleic acid-induced
lipid droplets in NIH3T3 cells. Biochem. Biophys. Res.
Commun. 335, 117–123 (2005).
105. Gubern, A. et al. Lipid droplet biogenesis induced by
stress involves triacylglycerol synthesis that depends
on group VIA phospholipase A2. J. Biol. Chem. 284,
5697–5708 (2009).
106. Long, A. P. et al. Lipid droplet de novo formation and
fission are linked to the cell cycle in fission yeast.
Traffic 13, 705–714 (2012).
107. Olzmann, J. A., Richter, C. M. & Kopito, R. R. Spatial
regulation of UBXD8 and p97/VCP controls ATGLmediated lipid droplet turnover. Proc. Natl Acad. Sci.
USA 110, 1345–1350 (2013).
108. Spandl, J., Lohmann, D., Kuerschner, L.,
Moessinger, C. & Thiele, C. Ancient ubiquitous protein
1 (AUP1) localizes to lipid droplets and binds the
E2 ubiquitin conjugase G2 (Ube2g2) via its G2
binding region. J. Biol. Chem. 286, 5599–5606
(2011).
109. Klemm, E. J., Spooner, E. & Ploegh, H. L. Dual role of
ancient ubiquitous protein 1 (AUP1) in lipid droplet
accumulation and endoplasmic reticulum (ER) protein
quality control. J. Biol. Chem. 286, 37602–37614
(2011).
110. Singh, R. et al. Autophagy regulates lipid metabolism.
Nature 458, 1131–1135 (2009).
111. Thoen, L. F. et al. A role for autophagy during hepatic
stellate cell activation. J. Hepatol. 55, 1353–1360
(2011).
112. Kaushik, S. et al. Autophagy in hypothalamic AgRP
neurons regulates food intake and energy balance.
Cell. Metab. 14, 173–183 (2011).
113. Hartman, I. Z. et al. Sterol-induced dislocation of
3-hydroxy-3-methylglutaryl coenzyme A reductase
from endoplasmic reticulum membranes into the
cytosol through a subcellular compartment resembling
lipid droplets. J. Biol. Chem. 285, 19288–19298
(2010).
114. Herker, E. et al. Efficient hepatitis C virus particle
formation requires diacylglycerol acyltransferase-1.
Nature Med. 16, 1295–1298 (2010).
115. Cornish, K., Wood, D. F. & Windle, J. J. Rubber
particles from four different species, examined by
transmission electron microscopy and electronparamagnetic-resonance spin labeling, are found to
consist of a homogeneous rubber core enclosed by a
contiguous, monolayer biomembrane. Planta 210,
85–96 (1999).
116. Juengst, C., Klein, M. & Zumbusch, A. Long-term live
cell microscopy studies of lipid droplet fusion dynamics
in adipocytes. J. Lipid Res. http://dx.doi.org/10.1194/
jlr.M042515 (2013).
117. Beller, M. ,Thiel, K. , Thul, P. J., Jäckle, H. Lipid
droplets: a dynamic organelle moves into focus. FEBS
Lett. 584, 2176–2182 (2010).
Acknowledgements
The authors thank N. Bremond, F. Wilfling and F. Pincet for
critical discussions on the manuscript and G. Howard for editorial assistance. Work on lipid droplets in the Walther and
Farese laboratories is supported by the National Institutes of
Health (NIH) grants R01GM097194 (to T.C.W.) and
RO1GM099844 (to R.V.F). A.R.T. is a fellow of the Marie
Curie Budding and Fusion of Lipid Droplets (BFLDs)
International Outgoing Fellowships (IOF), within the 7th
European Community Framework Program grant to a Partner
University Funds exchange grant between the Yale and Ecole
Normale Supérieure laboratories.
Competing interests statement
The authors declare no competing interests.
www.nature.com/reviews/molcellbio
© 2013 Macmillan Publishers Limited. All rights reserved