Question

Based on the article:

Question: Write a short description of the possible functions of lipid droplets.

"Expanding the roles for Lipid droplets."

Lipid droplets are the intracellular sites for neutral lipid storage. They are critical for lipid metabolism and

energy homeostasis, and their dysfunction has been linked to many diseases. Accumulating evidence suggests

that the roles lipid droplets play in biology are significantly broader than previously anticipated. Lipid

droplets are the source of molecules important in the nucleus: they can sequester transcription factors and

chromatin components and generate the lipid ligands for certain nuclear receptors. Lipid droplets have also

emerged as important nodes for fatty acid trafficking, both inside the cell and between cells. In immunity, new

roles for droplets, not directly linked to lipidmetabolism, have been uncovered, with evidence that they act as

assembly platforms for specific viruses and as reservoirs for proteins that fight intracellular pathogens. Until

recently, knowledge about droplets in the nervous system has been minimal, but now there aremultiple links

between lipid droplets and neurodegeneration: many candidate genes for hereditary spastic paraplegia also

have central roles in lipid-droplet formation and maintenance, and mitochondrial dysfunction in neurons can

lead to transient accumulation of lipid droplets in neighboring glial cells, an event thatmay, in turn, contribute

to neuronal damage. As the cell biology and biochemistry of lipid droplets become increasingly well understood,

the next few years should yield many new mechanistic insights into these novel functions of lipid

droplets.

Introduction

Lipid droplets are the sites where cells store neutral lipids, such

as triglycerides, steryl esters, and retinyl esters [1–3]. These

stored lipids can then be used in times of need to generate

energy, membrane components, and signaling lipids. Impairment

of the machinery that makes or degrades lipid droplets

has severe physiological consequences [1,4–6], demonstrating

that lipid droplets play central roles in cellular and organismal

energy homeostasis, in particular, and overall lipid metabolism

in general.

Lipid droplets also allow cells to safely sequester otherwise

toxic lipids. For example, as amphipathic molecules, overabundant

fatty acids can severely compromise membrane integrity.

Once turned into triglycerides and incorporated into lipid droplets

(Figure 1A), they are relatively inert, stable, and harmless.

This protective function is probably the reason for the abundant

accumulation of lipid droplets in many disease states characterized

by aberrant lipid supply and metabolism, such as obesity,

atherosclerosis, and fatty liver disease [1,6,7].

Lipid droplets are particularly important in tissues specialized

for energy storage or lipid turnover, such as adipose tissue,

the liver, and the intestine [2,3,8], yet they also accumulate in

skeletal muscle, the adrenal cortex, macrophages, and mammary

glands [1]. They control lipid signaling in immune cells

and are the targets of attack by pathogens [9]. Finally, they

have been observed in most cell types and occur throughout

the animal kingdom, in plants, and in unicellular organisms.

Recently, it has become apparent that lipid droplets play even

broader cellular roles than previously appreciated. For example,

they modulate the availability of proteins and signaling lipids in

the nucleus, act as hubs for fatty acid trafficking, are used by

viruses as assembly platforms, and their dysfunction in neurons

and glia may lead to neurodegeneration. This review summarizes

key recent findings into these emerging roles of lipid droplets,

with the aim of sharing these exciting developments with researchers

beyond the lipid-droplet field. Lipid droplets are still

relatively understudied organelles and, given the versatile functions

already revealed, it seems likely that further roles in new

areas of biology will be discovered.

Some Basic Concepts in Lipid-Droplet Biology

In the last few years, there has been an explosive growth in our

understanding of the structure, biogenesis, and turnover of lipid

droplets, which have been extensively covered in many excellent

recent reviews [1,5,10–15]. Among cellular organelles, lipid

droplets have a unique structure (Figure 1A): a central core of

hydrophobic (neutral) lipids is surrounded by a single layer of

amphipathic lipids and proteins (reminiscent of half a membrane

bilayer). The triglycerides in the hydrophobic core are generated

by an elaborate biosynthetic pathway (for a summary, see [10]),

with the final step being catalyzed by the acyl-CoA:diacylglycerol

acyltransferases DGAT1 and DGAT2 (Figure 1A), converting

diacylyglycerol (DAG) and fatty acids, first activated to

acyl-CoA, into triglycerides. Both enzymes are located in the

endoplasmic reticulum (ER), where triglycerides accumulate at

privileged sites that represent nascent lipid droplets [16]; mature

lipid droplets are generated by continuous growth of these

structures and finally become distinct from the ER, likely via aprocess resembling budding [10,13]. DGAT2 is only inserted

into one leaflet of the ER membrane and can therefore diffuse

onto the surface of lipid droplets, promoting triglyceride synthesis

and continued droplet growth locally [17]. The hydrophobic

core can also contain steryl esters, the synthesis of which is

catalyzed by acyl-CoA:cholesterol acyltransferases. Depending

on cell type and conditions, steryl esters or triglycerides may

predominate.

Breakdown of the droplet triglycerides can occur by two

distinct pathways. Cytoplasmic triglyceride lipases bound to

the surface of lipid droplets hydrolyze triglycerides to DAG and

fatty acids. DAG can be further broken down, in two steps, into

fatty acids and glycerol (Figure 1C). In adipose tissue and

many other cells, the bulk of triglyceride hydrolysis is catalyzed

by a single lipase, adipose triglyceride lipase (ATGL) [5]. Lipid

droplets can also be turned over by autophagy (Figure 1C):

like other cellular organelles, lipid droplets are taken up by autophagosomes,

which fuse with lysosomes to form autolysosomes.

The hydrolytic enzymes delivered from lysosomes then

break down the autophagosome content; triglycerides, in particular,

are predominantly hydrolyzed by lysosomal acid lipase

(LAL) [5]. Discovered in hepatocytes [18], autophagy of lipid

droplets (‘lipophagy’) appears to make varied contributions to

triglyceride breakdown, depending on cell type and physiological

conditions [5].

Lipid Droplets as Modulators of Nuclear Functions

Lipid droplets arise from the ER and typically reside in the cytoplasm,

often at considerable distance from the nucleus. Nevertheless,

recent studies have uncovered intimate connections

between lipid droplets and nuclear events. There is emerging

evidence for a nuclear population of lipid droplets, which has

been proposed to directly modulate lipid metabolism in the nucleus.

In addition, lipid droplets in the cytoplasm can sequester

transcription factors, enzymes, and chromatin components —

and possibly many other proteins—and thus control their availability

in the nucleus.

Nuclear Lipid Droplets

Two different groups have reported the presence of lipid droplets

inside nuclei [19,20]. Using dyes specific for neutral lipids, small

spherical structures were identified in the nuclei of cultured cells

as well as in biochemically isolated nuclei [20]. Electron microscopy

of serial sections revealed that at least some of these

structures truly reside inside the nuclear compartment [19].

Biochemical fractionation suggests that these structures differ

in their lipid composition from the lipid droplets in the cytoplasm

[20]; however, they resemble cytoplasmic lipid droplets in their

morphology and in the presence of neutral lipids and were thus

named ‘nuclear lipid droplets’. It is not yet known how these lipid

droplets arise, what proteins they associate with, or what their

functional significance is. However, it is an intriguing possibility

that they contribute to nuclear lipid homeostasis and locally

modulate the availability of signaling lipids.

Exchange of Proteins between Lipid Droplets and Nuclei

Cytoplasmic lipid droplets can also profoundly affect nuclear

events. For example, lipid droplets have been implicated in suppressing

the activity of a transcription factor by keeping it out of

the nucleus [21]. The lipid-droplet protein Fsp27, also known as

CIDEC, is expressed in adipocytes and promotes fusion between

droplets, causing the formation of a single droplet per

cell [22,23]. A yeast two-hybrid screen revealed the transcription

factor NFAT5 (nuclear factor of activated T cells 5) as a potential

Fsp27 interaction partner. NFAT5 is cytoplasmic under hypotonic

conditions and translocates to the nucleus upon hypertonic

stress to activate osmoprotective genes [24]. The physical interaction

between Fsp27 and NFAT5 was confirmed in vivo, and

Fsp27 knockdown in adipocytes led to expression of NFAT5

target genes even in the absence of hypertonic stress [21]. To

examine the underlying mechanism, Fsp27 was ectopically expressed

in the heterologous HEK293 cell line; under these conditions,

Fsp27 was observed broadly throughout the cytoplasm.

Fsp27 overexpression reduced the amount of nuclear NFAT5, as

determined both by imaging and biochemistry, and blunted the

expression of NFAT5 target genes when cells were exposed to

hypertonic stress [21]. These results suggest that Fsp27 is able

to sequester NFAT5 in the cytoplasm and interferes with its

nuclear trafficking; since endogenous Fsp27 is associated with

lipid droplets in adipocytes, this interaction would retain

NFAT5 at the droplet surface (Figure 2A), something that remains

to be demonstrated directly. It will be interesting to determinewhether the interaction between Fsp27 and NFAT5 is regulated,

for example, by signaling pathways controlling lipolysis.

In Drosophila embryos, lipid droplets are associated with large

amounts of specific histones [25] via the histone anchor Jabba

[26]. This association is first detected during oogenesis and

makes it possible for females to build up massive histone stores

in the developing eggs (Figure 2B,C). Wild-type embryos contain

enough excess histones for thousands of diploid nuclei, whereas

mutants lacking Jabba have drastically reduced histone stores

[26]; indirect evidence suggests that extranuclear histones not

bound to lipid droplets are degraded. Transplantation experiments

revealed that in the embryo droplet-bound histones

can transfer to nuclei [25] and presumably support chromatin

assembly. Surprisingly, embryos lacking this droplet-bound histone

supply develop largely normally [26,27]. This is possible

because of the intricate regulation of histone metabolism in early

embryos (reviewed in [28]), which also contain abundant levels of

histone messages deposited during oogenesis. When new synthesis

of histones in the embryo is even mildly impaired, Jabba

mutants cannot sustain development and die very early [26].

Thus, in this case, lipid droplets sequester a nuclear protein for

long-term storage. This sequestration allows the organism to

build up histone stores during oogenesis and keep them available

for when they are needed later for chromatin assembly

(Figure 2C).

Lipid droplets of early Drosophila embryos also appear

to affect histone metabolism in the short term, by buffering

the histone supply [27]. When droplets are transplanted

between embryos, the donor droplets can bind histones

from the recipient embryo, suggesting that histones can be

loaded onto droplets even in embryos. In Jabba mutants,

the synthesis of histone H2A and its variant H2Av are

imbalanced, and H2Av overaccumulates in the nuclei, an

event linked to DNA damage [27]. This nuclear overaccumulation

does not occur in wild-type embryos, presumably because

here lipid droplets can trap histones produced in excess and

prevent their unregulated entry into nuclei. Whether other

species use similar droplet-based histone buffering remains

to be determined, although histones have also been detected

on lipid droplets in housefly embryos, rat sebocytes, and

mouse oocytes [25,29,30]

The enzyme CCT1 also displays dramatic exchange between

lipid droplets and nuclei. CCT1 is one of two isoforms of

CTP:phosphocholine cytidylyltransferase, an enzyme that catalyzes

the rate-limiting step in the synthesis of the phospholipid

phosphatidylcholine. In cultured fly cells, CCT1 is usually present

in the nucleus, but, under conditions in which cells synthesize

new triglycerides and expand the hydrophobic core of droplets,

CCT1 accumulates at the droplet surface [31,32] (Figure 2D). The

presence at the droplet surface is critical to expand the droplet

surface in concert with growth of the core: droplet binding activates

the enzyme and thus leads to an increase in the cellular

phosphatidylcholine supply. Whether CCT1’s presence in the

nucleus in the basal state is functionally important remains unclear.

Nuclear accumulation is apparently not a mechanism to

prevent access to the droplet surface: fluorescence recovery

after photobleaching (FRAP) experiments revealed that CCT1

is not immobilized inside nuclei, but rapidly exchanges with a

cytoplasmic pool. And overexpression of CCT2, an isoform

exchanging between the cytoplasm and droplets, can fully

rescue the effect of CCT1 depletion on droplet growth [31].

High nuclear accumulation and consequent low cytoplasmic

pools of CCT1 might possibly modulate the kinetics of relocalization

to droplets.

Prp19 is a subunit of the NineTeen Complex, which is involved

in a number of nuclear events, including spliceosome activation

and transcription elongation [33]. In mouse adipocytes, Prp19

was also found associated with lipid droplets, and Prp19 knockdown

resulted in reduced triglyceride accumulation [34]. It is not

clear whether this dual localization to lipid droplets and to thenucleus represents distinct functions of Prp19, or whether the

two populations are connected. Initial experiments with inhibitors

of nuclear export revealed no changes in overall intracellular

Prp19 distribution [34].

Modulation of Lipid Signaling

Peroxisome proliferator-activated receptors (PPARs) are transcription

factors bound and activated by lipid ligands, including

fatty acids and their derivatives. In oxidative tissues, such as the

mammalian heart and liver, PPARa promotes the expression of

proteins involved in lipid homeostasis [35]. In principle, fatty

acids from exogenous sources or synthesized de novo might

activate PPARa directly. However, free fatty acids are typically

channeled, via activation to acyl-CoA, into specific metabolic

pathways [36] and thus are not readily available for signaling.

Studies in mice uncovered that PPARa signaling is severely

compromised in the hearts of animals lacking ATGL [37]. Given

that lack of ATGL function in the heart causes many profound

changes (such as massive lipid accumulation and mitochondrial

dysfunction), the effect on PPARa signaling might conceivably

be indirect. Yet pharmacological stimulation of the PPARa

pathway is sufficient to reverse these phenotypes, establishing

PPARa signaling as a primary defect in these mutant hearts

and suggesting a direct link between lipid droplets and PPARaactivation [37]. It was proposed that

ATGL-mediated triglyceride hydrolysis

generates the ligands for PPARa [37]

(Figure 3A). This pathway may be tissue

specific because liver-specific knockdown

of ATGL impaired the expression

of PPARa target genes in this tissue, but PPARa agonists failed

to reverse this effect [38].

Lipid Droplets as Hubs for Fatty Acid Trafficking

Lipid droplets act as a sink for overabundant fatty acids, and they

can release lipids when needed for energy production, synthesis

of membrane components, or signaling. It is becoming increasingly

apparent that lipid trafficking to and from droplets is highly

regulated in space (Figure 3A). Fatty acids from triglyceride hydrolysis

signal to nuclear receptors (as discussed above); fatty

acids released during autophagy are shuttled through lipid droplets,

as a way station before import into mitochondria for ATP

production; production of steroid hormones in flies requires lipid

exchange between the ER, lipid droplets, and mitochondria; and

within a population of cells, high accumulation of droplets in a

subset of cells has been proposed to protect the rest of the cells

from fatty acid overload.

Lipid Trafficking between Lipid Droplets and

Mitochondria

In starved mammalian cells, fatty acids fuel ATP production, via

b-oxidation in mitochondria. These fatty acids could be derived

from triglycerides (Figure 1B,C) or from various membranous

organelles. To follow the flux of fatty acids through variouscompartments, mouse embryonic fibroblasts (MEFs) were

allowed to incorporate fluorescently labeled lipids into either lipid

droplets or membranes, and their fate during starvation was

monitored by imaging and biochemistry [39]. Fatty acids present

as triglycerides in lipid droplets moved to mitochondria fairly

quickly and were readily broken down. When ATGL (Figure 1B)

was knocked down, transfer of fatty acids was dramatically

reduced and mitochondrial oxygen consumption rates dropped.

Under the starvation conditions employed, lipophagy (Figure 1C)

was not induced and autophagy made no detectable contribution

to the transfer of fatty acids from droplets to mitochondria

or to mitochondrial oxygen consumption rates.

The rapid relocalization of fatty acids to mitochondria is presumably

accomplished by direct transfer. Lipid droplets and

mitochondria indeed display close physical associations [40–

42], and direct channeling of fatty acids from their site of release

(droplets) to the site of consumption (mitochondria) would minimize

the risk of toxic effects elsewhere, such as disruption of

cellular membranes or inappropriate nuclear signaling.

Curiously, during starvation, the number and size of lipid droplets

increased and total cellular triglyceride levels went up [39].

Using fluorescently labeled phospholipids and inhibition of

autophagy pathways, this effect was traced to autophagic

breakdown of membranous organelles. Presumably, fatty acids

from phospholipid breakdown in autolysosomes are employed

to replenish triglyceride stores in droplets upon starvation

(Figure 3A).

Mitochondria can be remodeled by fusion and fission [43],

allowing them to form highly interconnected networks or individual

fragments. In starved cells, mitochondria were highly fused, a

state that is apparently critical for efficient fatty acid import: usually

labeled fatty acids from lipid droplets are homogenously

distributed throughout the mitochondria, but when mitochondria

were fragmented, the label was distributed unevenly [39]. As a

result, fatty acids could not be metabolized as efficiently;

although cells with either fused or fragmented mitochondria upregulate

b-oxidation upon starvation, only those with fused mitochondria

were able to maintain these levels of b-oxidation. For

cells with fragmented mitochondria, levels of b oxidation and,

as a result, total mitochondrial respiration, dropped off with

time, presumably because not all mitochondria had a sufficient

supply of fatty acids (Figure 3B). The likely reason is that there

are many fewer lipid droplets than mitochondrial fragments,

and that only the mitochondria in direct physical contact with

lipid droplets can take up fatty acids efficiently. In fused mitochondria,

those fatty acids can diffuse through the entire

network. In support of this interpretation, when glutamine was

used as an alternative fuel, the fusion state of the mitochondria

did not matter; as glutamine diffuses through the cytoplasm,

its import into mitochondria is not restricted to a limited number

of sites, unlike the supply of fatty acids from lipid droplets. Presumably

because an oversupply of unmetabolized fatty acids is

dangerous, fatty acids were re-exported from the mitochondria,

and either accumulated back in lipid droplets or were released

from the cells into the extracellular space [39].

Efficiency of lipid exchange between mitochondria, ER, and

lipid droplets may also underlie a recent observation that

promotion of mitochondrial fusion is important for lipid-droplet

formation and steroid signaling in Drosophila. Mitochondrial

associated regulatory factor (Marf), the fly ortholog of mammalian

mitofusins, is a small GTPase that promotes fusion of

the outer mitochondrial membrane; thus, loss of Marf leads to

small, round mitochondria [44]. Marf-deficient flies show a

particularly dramatic phenotype in the ring gland, an endocrine

tissue responsible for hormone secretion [45]: mitochondrial

morphology is altered, the ER is fragmented, and lipid-droplet

number is dramatically reduced [44]. Ring gland lipid droplets

receive sterols from the ER and store them as steryl esters;

these, in turn, are the precursors for the production of the steroid

hormone ecdysone in the mitochondrial matrix. Efficient storage

and turnover of steryl esters therefore presumably requires intimate

contacts between the three organelles; in Marf mutants

the contacts between all three organelles were severely reduced

[44]. Lack of Marf in the ring gland also greatly impairs ecdysone

production, with dramatic organism-wide consequences [44].

Mitochondria may not be the only organelle for which close

contacts with lipid droplets promote efficient transfer of fatty

acids. Breakdown of fatty acids is not restricted to mitochondria,

but can also occur in peroxisomes. In the yeast Saccharomyces

cerevisiae, b-oxidation is even entirely restricted to peroxisomes.

Here, lipid droplets and peroxisomes display intimate physical

connections, which have been proposed to promote efficient

coupling of triglyceride breakdown with peroxisomal fatty acid

oxidation [46].

Lipid-Droplet Specialization across a Cell Population

The role of lipid droplets as buffers for fatty acid availability may

even extend to lipid exchange between cells in the same tissue.

A recent study identified a surprising heterogeneity in lipiddroplet

content in hepatocytes [47]: in mouse liver, some cells

have substantially larger numbers of lipid droplets than neighboring

cells (Figure 3C); this variability is especially apparent

under conditions of high overall lipid storage in the liver. Similar

variability was observed in primary hepatocytes in culture and

with a cultured cell line of liver origin (AML12), suggesting that

it is due to cell-intrinsic properties, rather than a reflection of

overall tissue structure.

Such heterogeneity might arise if some cells have acquiredmutations in lipid metabolism. However, a cell sorting strategy

demonstrated that heterogeneity is reversible and appears to

be a population property. After growth on fatty-acid-rich media,

cells were separated by flow cytometry into a low-lipid and a

high-lipid subpopulation. After culture in standard media to promote

breakdown of the stored lipids, the two populations were

again grown under fatty-acid-rich conditions. Remarkably,

both cultures showed the same broad distribution in lipid content.

Inhibitor studies indicate that heterogeneity arises from

fluctuations in biochemical networks controlling lipolysis, fatty

acid oxidation, and protein synthesis.

At the level of a whole organism, heterogeneity of lipid-droplet

content is very common. Many animals have adipose tissues

specialized for storing lipids. It was proposed that heterogeneity

within a single cell population similarly sets aside a subpopulation

of cells that collects lipids particularly well, stores them,

and releases them to their neighbors when needed [47]. To

test this idea, high-lipid cells were isolated in which lipid droplets

had accumulated fluorescently labeled fatty acids. After co-culture

with low-lipid cells (marked with a different fluorescent dye

to distinguish the two original populations), the high-lipid grouphad lost — and the low-lipid group had gained — some of the

labeled fatty acids. Thus, the high-lipid cells can indeed supply

lipids to their neighbors (Figure 3D).

But what is the point of setting aside a subpopulation of cells

with especially high lipid stores if — in the long run — the lipids

are presumably needed equally across cells? One possibility

has to do with the fact that overaccumulation of free fatty acids

is dangerous, both because of disruption of membranes and

because of toxic metabolites generated by fatty acid breakdown.

The high-lipid subpopulation indeed showed higher levels

of oxidative damage, as seen by levels of reactive oxygen species

(ROS; Figure 3C). Importantly, when fluorescently marked

low-lipid cells were co-cultured with either low-lipid or high-lipid

cells (unmarked) and challenged with fatty-acid-rich media, the

marked cells displayed lower ROS levels in the presence of

high-lipid cells. Thus, the presence of high-lipid cells protected

their low-lipid neighbors. High-lipid cells may remove fatty acids

more efficiently from the media, and thus the flux of free fatty

acids into the low-lipid cells is reduced. Remarkably, in the coculture

experiment with high-lipid cells, ROS levels were not

only reduced for the marked low-lipid cells, but also for the population

as a whole.

Although the detailed mechanisms underlying these protective

effects remain to be worked out, the reported experiments

nicely demonstrate a novel strategy, namely heterogeneity in

lipid-droplet accumulation, to alleviate risks from overabundance

of lipids. By accumulating more lipid droplets and more

ROS, the high-lipid subpopulation reduces the overall risk of lipotoxicity.

It is not yet clear how the high-lipid population is able to

handle its increased risk: these cells may induce specific protective

mechanisms, or they might repair their damage during the

time when they find themselves in the low-lipid state (which

will occur sooner or later, due to the stochastic nature of the

heterogeneity). Since heterogeneity has been observed in

cultured cells of various origins [47], this protective strategy

may be employed not just by hepatocytes, but more generally

in other cell types.

Lipid Droplets and the Fight against Pathogens

It has been long known that lipid droplets play important roles in

the immune system. They are sites of synthesis of eicosanoids,

signaling lipids important for inflammation, host defense against

pathogens, and cancer [48]. Various pathogens have, in turn,

evolved strategies to tap into the lipid droplets of the host

to ensure a sufficient lipid supply [49,50]. Recent years have

uncovered how certain viruses appropriate lipid droplets as

assembly platforms and how cells use lipid droplets in novel

ways to fight back.

Lipid Droplets and Viral Assembly

Infection with hepatitis C virus (HCV) is a global public health

threat and can lead to liver cirrhosis and liver cancer [51]. For

part of its life cycle, HCV crucially depends on lipid droplets

(see [52,53] for recent reviews): after infection, two newly expressed

viral proteins transiently accumulate on lipid droplets:

core protein, which is the major structural protein of the virus,

and NS5A, a regulator of viral replication. Droplet-bound viral

proteins then interact with the sites of viral RNA replication, a

process facilitated by the droplet-localized Rab18 [54] and

by dynein-mediated intracellular relocalization of lipid droplets

[55]. For the final maturation, the virus hijacks the pathway

responsible for secretion of the very-low-density lipoprotein

(VLDL) particles, and virions are released into the extracellular

space as low-density lipoviroparticles [56], whose lipids may

ultimately be derived from lipid droplets. HCV is not unique in

its use of lipid droplets; several other viruses assemble with

the help of lipid droplets [57,58].

Droplet accumulation is a necessary step for virus maturation;

if the interaction between core protein and lipid droplets is disrupted,

either with mutations in core protein or via pharmacological

approaches, core protein stability is greatly reduced and

virion assembly is impaired [59,60]. Thus, preventing the recruitment

of viral proteins to droplets is an attractive target for disrupting

the viral life cycle. Droplet targeting requires cis-acting

sequence motifs in both core protein and NS5A [61,62], but

also trans-acting host factors. In particular, DGAT1, one of the

two enzymes mediating triglyceride synthesis (Figure 1A), plays

a crucial role: DGAT1 binds to both NS5A and core protein,

and this interaction mediates recruitment of both proteins to

droplets and is required for efficient virion assembly [63,64].

Interestingly, inhibiting the enzymatic activity of DGAT1 is sufficient

to prevent droplet targeting of these proteins. Since

DGAT1 generates only a subset of lipid droplets (the others

depend on DGAT2; Figure 1A), it was proposed that DGAT1 concentrates

the viral proteins at the sites where it promotes lipiddroplet

formation and thus guides the viral proteins onto just

these lipid droplets [64] (Figure 4A).

Lipid Droplets as Stores for Antiviral and Antibacterial

Proteins

Viperin (which stands for virus inhibitory protein, ER associated,

interferon inducible) is an interferon-induced protein with broad

antiviral activity [65,66]. Viperin is targeted to the cytoplasmic

face of the ER and is also enriched around lipid droplets [62];

targeting to both locations is mediated by an amino-terminal

amphipathic a-helix [62]. Intriguingly, two of the viruses combatted

by viperin, HCV and Dengue virus, employ droplets for

their assembly. Using confocal microscopy and fluorescence

resonance energy transfer (FRET), viperin was shown to interact

with the HCV nonstructural protein NS5A at the droplet surface,

via its carboxy-terminal region [67]. This interaction as well as theamino-terminal droplet-targeting helix are required for viperin’s

antiviral activity against HCV [67]. For Dengue virus, in contrast,

while a physical interaction with the viral protein NS3 was important,

droplet binding was dispensable for the anti-viral effect [68].

The amino-terminal a-helix in viperin was also important to

restrict the replication of chikungunya virus, though presumably

through localization at the ER, rather than at lipid droplets [69].

Thus, at least in some cases, viperin apparently targets a

droplet-dependent step of viral replication and its enrichment

on the droplet surface is necessary for its activity (Figure 4A).

As discussed earlier, lipid droplets can be associated with histones.

This observation potentially has implications for immunity

since histones are increasingly recognized as antibacterial

agents [70,71]: in vitro, histones have broad antibacterial activity

[72], and histones present in extracellular secretions have been

reported to contribute to protection against bacterial pathogens

[73,74]. Analysis in Drosophila suggests that histones bound to

lipid droplets can similarly provide a defense against intracellular

bacterial invaders [75]. Droplets biochemically purified fromDrosophila embryos are associated with high levels of certain

histones [25] and are highly bactericidal in vitro [75]. A number

of independent approaches, including using histone antibodies

and mutations in the histone anchor Jabba [26], showed that

this killing activity of droplets was due to histones.

To test whether droplet-bound histones are protective in vivo,

wild-type and Jabba mutant embryos were injected with GFPlabeled

Escherichia coli; while bacterial numbers diminished in

the wild type, they dramatically increased in the mutants [75]

(Figure 4B). In the same injection assay, wild-type embryos

also showed significantly higher levels of survival when challenged

with a number of Gram-positive and Gram-negative bacteria.

This new immune mechanism may also operate at other

developmental stages: when adult flies were infected with the

intracellular pathogen Listeria monocytogenes, Jabba mutants

were impaired in restricting bacterial titers and were killed

much more readily than wild-type flies [75]. Loading up lipid

droplets with histones to kill bacterial invaders may be a

conserved innate immunity mechanism, since when mice were

challenged with lipopolysaccharide — to mimic bacterial infections

— the levels of droplet-bound histone H1 increased in the

liver [75].

Lipid Droplets and the Nervous System

Lipid metabolism plays crucial roles in the nervous system, for

many membrane functions and signaling events [76–78]. Yet until

recently, there has been only sparse and unconnected information

on the role of lipid droplets in neurons and other cells

of the nervous system. For example, lipid droplets have been detected

in the axons of Aplysia neurons cultured in vitro [79] and in

cultured neurons and brain sections of Huntington’s disease

models [80]. There are also links between a-synuclein, a protein

whose dysfunction or overexpression can cause Parkinson’s

disease, and lipid droplets: a-synuclein has been reported to

bind to lipid droplets in vitro [81] and in cultured cells [82], and

overexpression in yeast promotes droplet accumulation [83],

but the relevance of these observations for a-synuclein’s in vivo

function and for neurodegeneration has yet to be explored. However,

recent papers have identified the presence of lipid droplets

in neurons and in glia under certain disease conditions and suggest

that disrupted lipid-droplet function can contribute to neurodegeneration.

Hereditary Spastic Paraplegias and Lipid Droplets

Hereditary spastic paraplegias (HSPs) are inherited disorders

characterized by motor-sensory axon degeneration, weakness

in lower extremities, and spasticity [84]. Mutations in over 50

loci can cause HSP, and the cellular functions of the encoded

proteins show a surprising heterogeneity. Recently, a number

of HSP candidate genes have been shown to have crucial roles

in lipid-droplet biology: atlastin, REEP1, seipin, spartin, spastin,

and kinesin-1. Atlastin mediates fusion of ER tubules and also

controls the size of lipid droplets [85]. REEP1 maintains the

high curvature of ER tubules and, when overexpressed together

with atlastin, increases lipid-droplet size [85,86]. Seipin, an integral

membrane protein at the ER–droplet junction, is important

for lipid-droplet formation and maintenance [87,88]. Spartin

localizes to lipid droplets, interacts with E3 ubiquitin ligases,

and modulates the turnover of lipid-droplet proteins [89–91];

spartin knockout mice have increased lipid-droplet numbers in

their adipose tissue [92]. Spastin is a microtubule-severing protein

that mediates remodeling of the cytoskeleton; in mammalian

cells, a particular spastin isoform harbors a lipid-droplet targeting

sequence, and depletion of spastin in Drosophila or

C. elegans alters lipid-droplet number and cellular triglyceride

content [93]. KIF5A encodes the microtubule motor kinesin-1;

the same motor powers the motion of lipid droplets in Drosophila

[94]. Finally, several of the HSP candidate genes encode enzymes

implicated in phospholipid or fatty acid metabolism

[95,96]; their dysfunction might therefore alter the supply or

composition of the lipids stored in lipid droplets. These observations

raise the intriguing possibility that aberrant lipid-droplet

biogenesis or function might contribute to axonal degeneration.

However, since all of these proteins also have functions unrelated

to lipid droplets (such as controlling ER structure or promoting

vesicle trafficking), the link between lipid droplets and

HSPs remains tentative.

A much more direct connection to lipid droplets has recently

emerged from the analysis of the HSP gene DDHD2 [97]. Patients

with mutations in DDHD2 exhibit very early onset of the disease

(<2 years) and are often intellectually disabled [98]. The DDHD2

gene is highly expressed in the brain and encodes a serine

hydrolase that displays phospholipase activity in vitro. To determine

its function in vivo, DDHD2 activity was abrogated genetically,

using knockout mice, as well as pharmacologically, withselective inhibitors [97]. In both cases, adults accumulated large

amounts of triglycerides in the brain and the spinal cord, but

there was little to no effect in other tissues; brain phospholipid

content was unchanged. These observations suggest that

DDHD2 has a specific function in triglyceride metabolism of

the central nervous system. It likely acts as a triglyceride lipase

since recombinant DDHD2 expressed in cultured cells displays

triglyceride hydrolase activity and, compared with wild type,

total triglyceride hydrolase activity is significantly reduced in

brain lysates of DDHD2 mutant mice [97]. Finally, the main triglyceride

hydrolase in the fat body of the moth Manduca sexta

shares extensive sequence homology with DDHD2 [99].

The brains of DDHD2 knockout mice displayed abundant lipid

droplets, while lipid droplets were only rarely detected in wildtype

brains [97]. They accumulated predominantly in neurons

and were present in cytoplasm, dendrites, and axons. The

DDHD2 knockout mice also exhibited deficits in motor coordination

and cognition [97], reminiscent of the defects in the human

patients [98]. Intriguingly, in the patients, cerebral magnetic resonance

spectroscopy revealed an abnormal spectrum, with a

peak characteristic of lipid accumulation [98], though it is not

yet known whether this peak represents triglycerides. While

the mechanisms that link droplet accumulation and neuronal

impairment remain obscure, one intriguing observation is that

in the DDHD2 knockout mice some of the large droplets

observed were associated with noticeable swellings of the

neuronal processes and thus might present obstacles to intracellular

trafficking in the relatively thin axons and dendrites.

Glial Lipid Droplets

Glial cells are non-neuronal cells that surround neurons and play

important supportive roles in the central and peripheral nervous

system. Lipid droplets have been observed in culture in primary

glia as well as in glia-derived cell lines [100,101]. When carnitine

palmitoyltransferase 2 (CPT2), a mitochondrial enzyme necessary

for b-oxidation of long-chain fatty acids, is abolished in flies,

massive amounts of triglycerides accumulate specifically in the

brain of adults; glial cells, but not neurons, accumulated abundant

lipid droplets [102]. This seems to be a cell-autonomouseffect because CPT2 is expressed predominantly

in glia and CPT2 expression

solely in glia is sufficient to reverse triglyceride

accumulation in the brain. Flies lacking

CPT2 have a dramatically reduced

lifespan, and glial-specific CPT2 expression

was able to partially rescue this

defect, indicating that triglyceride metabolism

in glia may make an important contribution to overall

organismal energy metabolism.

Lipid droplets can accumulate in glia also non-cell-autonomously,

in response to mitochondrial dysfunction in neighboring

neurons [103]. For a subset of Drosophila mutants known to

cause neurodegeneration in adult photoreceptors [104], abundant

lipid droplets transiently accumulate in the glial cells next

to photoreceptors, prior to or concomitant with the onset of

neurodegeneration. No droplets were observed in the wild type

or in the neurons of mutant animals [103] (Figure 5A,B). The

mutants that showed accumulation of droplets in glial cells all

affect mitochondrial function and, in particular, cause increased

levels of ROS. Elevated ROS are indeed critical for droplet formation

in glia because pharmacological or genetic reduction of

ROS prevented droplet accumulation. Lipid droplets were also

detected in glial cells in a mouse model of neurodegeneration

caused by mitochondrial dysfunction, suggesting an evolutionarily

conserved pathway.

How do ROS promote the accumulation of glial lipid droplets?

The full pathway has yet to be worked out, but activation of

c-Jun-N-terminal kinase (JNK) and sterol regulatory element

binding protein (SREBP) pathways are critical; JNK mediates

stress responses [105] and SREBP controls transcription of

many metabolic genes and, in particular, promotes lipogenesis

[106,107]. Although droplets accumulate in glia, the trigger for

accumulation originates in neurons: when the mitochondrial

genes identified were knocked down in glia, there was no effect;

knockdown only in neurons was sufficient to promote glial lipid

droplets. In addition, expression of an antioxidant enzyme or

knockdown of JNK solely in neurons was able to reduce glial

droplet accumulation. Thus, mitochondrial dysfunction and

elevated ROS in photoreceptors cause accumulation of lipid

droplets in glia in a non-cell-autonomous manner.

Damage to neurons resulting from mitochondrial dysfunction

therefore leads both to transient formation of lipid droplets in

glia and to neurodegeneration. Are these lipid droplets an ultimately

futile protective response, do they promote neurodegeneration,

or are they innocent bystanders? Activation of JNK orSREBP in neurons in the absence of ROS still leads to glial lipid

droplets, but not neurodegeneration [103]. Thus, glial lipid droplets

per se are not detrimental for neurons. The culprit might

be lipids damaged by ROS, given that the mutants leading to

neurodegeneration displayed dramatically elevated levels of

peroxidated lipids. Furthermore, expression of two different

lipases, the ATGL homolog Brummer or the LAL homolog Lip4

(Figure 1B,C), dramatically reduced both lipid-droplet accumulation

and the levels of peroxidated lipids and also delayed neurodegeneration

(Figure 5C) [103]. These observations strongly

suggest that neurodegeneration is driven by altered lipid metabolism,

although the exact role of lipid droplets remains to be

elucidated.

The fatal neurodegenerative disease amyotrophic lateral sclerosis

(ALS) has recently also been linked to lipid droplets.

A particular subtype of ALS is caused by mutations in the human

VAMP (vesicle-associated membrane protein)-associated protein

B (hVAPB). Equivalent mutations in the fly ortholog DVAP,

when ectopically expressed, lead to degeneration of fly photoreceptors.

In genetic screens for enhancers and suppressors of this

phenotype, one of the most represented functional categories

was proteins linked to lipid droplets, including proteins involved

in droplet biogenesis and droplet motility [108]. The proteins

such identified will provide a rich source for follow-up studies to

dissect how lipid droplets might impact neurodegeneration.

Perspective

The crucial roles of lipid droplets in energy homeostasis and lipid

metabolism have focused a lot of recent attention on these still

relatively understudied organelles. Yet the examples discussed

above show that lipid droplets play even broader roles and touch

on biological processes only loosely connected to their traditionally

studied functions.

In particular, lipid droplets contribute to protein trafficking and

protein maturation in the cell. They exchange proteins with the

nucleus, modulate protein stability, and allow concentrated

accumulation of antiviral and antibacterial proteins. We do not

know enough to judge whether these processes have independently

evolved and all just happen to take advantage of lipid

droplets or whether they are indicative of a general cellular

pathway of protein trafficking. Lipid droplets have been proposed

to act as general protein sequestration sites [109]; such

sequestration might modulate the ability of these proteins to

interact with binding partners, promote assembly of protein

complexes, store damaged proteins safely before degradation,

or allow moving droplets to deliver proteins [109,110]. As many

published lipid-droplet proteomes contain proteins from other

compartments, there are ample candidates for testing how widespread

protein sequestration on droplets is. For the verified examples,

much work needs to be carried out to understand how

the sequestered proteins are targeted to lipid droplets, whether

they are bound stably or dynamically, and how release from

droplets is controlled. And why are these proteins sequestered

on lipid droplets and not elsewhere in the cell? Is droplet localization,

say, of histones, just an accident of evolution, or do lipid

droplets provide a unique cellular niche?

The emerging roles of lipid droplets as hubs for fatty acid trafficking

(Figure 3) suggest that the pathways that fatty acids take

from and to lipid droplets are highly regulated. But, apart from

some insights into the importance of direct contacts between

lipid droplets and mitochondria [42], little is known about the

molecular mechanisms controlling this trafficking. For fatty

acid trafficking modulated by droplet heterogeneity between

cells (Figure 3C,D), there are intriguing hints that heterogeneity

is a regulatable property since the extent of heterogeneity is

different between cells of different origin [47], but the control

pathways remain to be worked out.

For lipid droplets in the nervous system, it is now established

that both neurons and glia can accumulate lipid droplets under

certain disease conditions. But what role they play under these

conditions and whether droplets are normally present in the nervous

system is far from clear. For example, in the fly models of

neurodegeneration (Figure 5), it was proposed that accumulation

of lipid droplets in glia promotes neurodegeneration, as long as

high ROS levels provide a second insult [103]. However, lipase

overexpression in glia only mildly delayed neurodegeneration,

whereas lipase overexpression in neurons, where no droplets

were detected, had a much stronger protective effect. It will be

very interesting, in these examples and in the mouse models of

HSP, to examine whether ablation of droplet biogenesis in specific

cell types modulates the disease phenotypes, for better or

for worse, and how these effects compare to disruption or upregulation

of turnover pathways (Figure 1B,C). Real-time imaging

of the trafficking of labeled fatty acids (as in [39]) and characterization

of the lipidomes and proteomes of these droplets will provide

complementary information to characterize exactly how

lipid metabolism is derailed in the disease conditions.

Given the diverse novel roles proposed for lipid droplets, droplets

should be on the radar screen of many a biologist trying to

uncover the mechanistic basis of an ill-characterized process.

With the recent insights into biogenesis and turnover of lipid

droplets [14], one can now systematically determine how a process

is affected if droplets are entirely absent, are structurally

abnormal, or cannot be degraded. Because lipid droplets are

ubiquitous organelles but have been carefully studied in only a

few cell types, it seems likely that, as our understanding of these unique and dynamic organelles deepens, their cellular and physiological

roles will keep expanding.

Answer 1

Functions of lipid droplets:

Lipid droplets are the intracellular sites for neutral lipid storage. They are critical for lipid metabolism and energy homeostasis, and their dysfunction has been linked to many diseases.Lipid droplets are the source of molecules important in the nucleus: they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors.Lipid droplets are the sites where cells store neutral lipids, such as triglycerides, steryl esters, and retinyl esters

• Lipid droplets are important nodes for fatty acid trafficking, both inside the cell and between cells.
• They are also linked to the new roles in immunity but they are not directly linked to lipid metabolism.
• They are directly linked to the neurological system also where the lipid droplets are connected with the neurodegeneration.
• Lipid droplets also allow cells to safely sequester otherwise toxic lipids. For example, as amphipathic molecules, overabundant fatty acids can severely compromise membrane integrity. Once turned into triglycerides and incorporated into lipid droplets,they are relatively inert, stable, and harmless.