Perilipin Family Members Preferentially Sequester to Either Triacylglycerol-

or Cholesteryl Ester-Specific Intracellular Lipid Storage Droplets

Kai Hsieh*, Yun Kyung Lee*, Constantine Londos*, Bruce M. Raaka+, Knut Tomas Dalen*#, and Alan R. Kimmel*^

* Laboratory of Cellular and Developmental Biology, + Clinical Endocrinology Branch,

National Institute of Diabetes and Digestive and Kidney Diseases The National Institutes of Health, Bethesda, MD 20892

and

# Department of Nutrition, Institute of Basic Medical Sciences, Medical Faculty,

University of Oslo, P.O. Box 1046 Blindern, N-0316 Oslo, Norway

^ Telephone: (301) 496-3016 Facsimile: (301) 496-5239 e-mail: ark1@helix.nih.gov

Running Title: Perilipin Lipid Specificities

Key Words: PLIN, ADRP, TIP47, LSDP5, S3-12, triacylglyceride, cholesterol, fatty acids,

lipolysis

© 2012. Published by The Company of Biologists Ltd.Jo

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JCS online publication date 8 June 2012

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SUMMARY

Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid

storage droplets in various cell types. Studies across diverse species

demonstrate that Plins regulate lipid storage metabolism through recruitment of

lipases and other regulatory proteins to lipid droplet surfaces. Mammalian

genomes encode 5 distinct Plin gene members and additional protein forms

derive from specific mRNA splice variants. However, it is not known if the

different Plins have distinct functional properties. Using biochemical, cellular

imaging, and flow cytometric analyses, we now show that within individual cells

of various types, the different Plin proteins preferentially sequester to separate

pools of lipid storage droplets. By examining ectopically expressed GFP fusions

and all endogenous Plin protein forms, we demonstrate that different Plins

sequester to lipid droplets, comprised distinctly of either triacylcerides or of

cholesterol esters. Further, Plins with strong association preferences to TAG (or

CE) droplets can re-direct the relative intracellular TAG/CE balance toward the

targeted lipid. Our data suggest diversity of Plin function, alter previous

assumptions about shared collective actions of the Plins, and indicate that each

Plin can have separate and unique functions.

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INTRODUCTION

Intracellular neutral lipid storage droplets (LSDs) are unique organelles

that store metabolic precursors of cellular energy, membrane biosynthesis,

steroid hormone synthesis, and signaling (Farese and Walther, 2009; Kimmel et

al., 2010; Londos et al., 2005). LSDs contain different lipids (e.g. triacylglycerides

(TAG) or cholesteryl esters (CE) at their core, surrounded by a phospholipid

monolayer. LSD surfaces in organisms as diverged as mammals, Drosophila,

and Dictyostelium are targeted by an evolutionarily related family of proteins

(Kimmel et al., 2010; Lu et al., 2001; Miura et al., 2002), the Perilipins (Plins).

Mammalian genomes encode 5 distinct Plin gene members and additional

protein forms derived from specific mRNA splice variants (Kimmel et al., 2010).

Plin1 is the major LSD coat protein in adipocytes and steroidogenic cells

(Greenberg et al., 1993; Servetnick et al., 1995). Other Plins exhibit different

expression patterns. Plin2 is the predominant, but not exclusive, form in liver

(Dalen et al., 2006), whereas Plin5 is primarily expressed in oxidative tissues,

including heart, soleus muscle, and brown fat (Dalen et al., 2007; Wolins et al.,

2006; Yamaguchi et al., 2006). Based on Plin1 function (Martinez-Botas et al.,

2000; Sztalryd et al., 2003; Tansey et al., 2001; Wang et al., 2009), the Plins are

viewed as fundamental regulators of lipolytic activity. Loss of Plin1 (Martinez-

Botas et al., 2000; Tansey et al., 2001) or Plin2 (Chang et al., 2006) in mice

significantly reduces intracellular lipid levels in adipocytes and hepatocytes,

respectively. Furthermore, heterozygous loss-of-function mutations in human

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PLIN1 leads to a familial partial lipodystrophy, supporting a required role for

Perilipin in TAG storage within human adipocyte LSDs (Gandotra et al., 2011).

Regardless, little is known of lipid interaction specificity of the various Plins. Here,

we show that distinct Plins differentially sequester to either TAG- or CE-specific

LSDs and can alter relative intracellular TAG or CE levels toward the

preferentially targeted lipid. These data demonstrate and emphasize diverse

functions for the different Plins.

RESULTS

Exogenous fatty acids and cholesterol differentially stabilize accumulation

of Plin protein family members.

Intracellular LSDs accumulate substantially when cells are cultured

overnight in the presence of high concentrations of various exogenous lipids (Xu

et al., 2005). Since Plins primarily sequester to LSD surfaces (Miura et al., 2002),

we determined if different Plins exhibited differential regulation in response to

either fatty acids or cholesterol, lipids that mobilize separate pathways.

Y1 mouse adrenocortical cells have robust capacity for steroid hormone

synthesis and accumulate TAG and CE LSDs as energy and metabolic precursor

stores. Further, steroidogenic cells are able to synthesize all 4 Plin1 mRNA splice

variants (Servetnick et al., 1995; Xu et al., 2005) and express all other Plin genes.

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Y1 cells were cultured under standard conditions or in medium

supplemented with oleic acid and/or cholesterol. Endogenous Plin proteins were

quantified in whole cell lysates by specific immunoblotting (Figure 1A). In general,

none of the Plins exhibited significant accumulation in unsupplemented medium.

However, significant Plin accumulation differences were observed in the

presence of oleic acid or cholesterol. The two major Plin1 variants of

steroidogenic cells, Plin1a and Plin1c, exhibited reciprocal patterns. Plin1a was

enhanced by oleic acid, but not by cholesterol, whereas the Plin1c response was

exactly opposite (Figure 1A). The effects were largely activating, since the

expressions of Plin1a and Plin1c were not diminished in cells cultured

simultaneously with oleic acid and cholesterol. Plin1b and Plin1d proteins are not

easily detected in Y1 cells (Servetnick et al., 1995), although Plin1b appears to

be regulated similarly to Plin1a (Figure 1A).

Plin2 and Plin3 accumulate similarly regardless of the exogenous lipid

moiety, although Plin2 may be slightly more responsive to oleic acid. Conversely,

Plin4 and Plin5 show extreme lipid specificity, largely mimicking that of Plin1c

and Plin1a, respectively (Figure 1A).

Since exogenous lipids may have differential regulatory effects on the

transcription or translation of endogenous Plin mRNAs and, thus, indirectly

impact Plin protein accumulation, we also examined the effects of oleic acid and

cholesterol using GFP-Plin protein fusions expressed from identical constitutively

active promoter vectors. McARH7777 rat hepatoma cells were transiently

transfected with vectors to separately express each GFP-Plin protein fusion and

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cultured under standard conditions or in medium supplemented with oleic acid

and/or cholesterol.

The GFP-Plin proteins in McARH7777 cells showed identical responses to

oleic acid and cholesterol as their endogenous counterparts in Y1 cells (Figures

1A and 1B). GFP-Plin1a and GFP-Plin5 were specifically responsive to the

positive effects of oleic acid, whereas GFP-Plin1c and GFP-Plin4 were only

detected in the presence of cholesterol (Figure 1B). GFP-Plin2 and GFP-Plin3

did not show a significant preference to either lipid. The responses of Plin1b and

Plin1d were more clear in this heterologous expression system. Plin1b is

structurally most similar to Plin1a (Lu et al., 2001) and GFP-Plin1b behaves

identically to both Plin1a and GFP-Plin1a. Plin1d is the smallest variant (Lu et al.,

2001) and GFP-Plin1d shows limited lipid preference, acting more like Plin2 and

Plin3.

Differential sub-cellular localizations of FA- or Chl- tagged markers.

Data (Figure 1) suggest that the sequestration and stabilization of

individual Plins to different classes of LSDs may influence the specific

accumulation of particular Plin proteins. To examine this further, we established

conditions to preferentially tag and purify TAG- and CE-specific LSDs.

Y1, McARH7777, and AML12 cells were cultured overnight in the

presence of both oleic acid and cholesterol, plus BODIPY 558/568 C12 [as a

fluorescent fatty acid (FA) dye marker] and cholesteryl BODIPY 500/510 FL C12

[as a fluorescent cholesteryl (Chl) dye marker] and imaged (Figure 2A). All three

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types of cells showed definitive separation FA (red) and Chl (green) markers into

distinct LSDs clusters. Few sectors show any co-localization, a pattern similarly

observed in 4T1 mouse mammary tumor, primary mouse liver, C2C12 mouse

myoblast, 3T3-L1 mouse fibroblast, J774A.1 mouse monocyte-macrophage, and

CHOK1 Chinese hampster ovary cells (Supplemental Figure S1A). In general,

the different markers, though distinctly separate, were largely intermingled.

However, the McARH7777 cells (Figure 2A) were most distinctive. FA- and Chl-

tagged droplets segregated to entirely unique sub-cellular regions. FA-tagged

droplets were polarized to the cell periphery, while Chl-tagged droplets were

centrally localized. Less distinctive labeling and polarized separation of the

droplets is seen in the McARH7777 cells cultured for shorter incubation periods

(Supplemental Figure S1B).

The polarized localizations of FA- and Chl-tagged droplets suggested that

it might be possible to distinguish separate associations of these distinct droplets.

McARH7777 cells were cultured with oleic acid, cholesterol, and FA- or Chl-

tagged fluorescent markers, and co-stained using antibodies against several

organelle-specific proteins (Figure 2B) or organelle-specific dye trackers (Figure

2C). In McARH7777 cells, the FA-tagged droplets localized entirely separate

from lysosomes, mitochondria, and early endosomes (Figures 2B,C). The Chl-

tagged droplets and other organelles are more centrally localized and perinuclear.

Nonetheless, there was nominal overlap of Chl-tagged droplets with these other

structures (Figures 2B,C). Lysosomes and mitochondria remained largely

separate, and there was minimal intermingling with early endosomes. The data

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indicate that the FA- or Chl- fluorescent tags do not broadly label these other

organelles.

FACS purification of TAG- and CE-LSDs.

To determine if FA- and Chl-tagged LSDs had distinct lipid compositions,

we first developed conditions for their separate purifications. Labeled Y1,

McARH7777, and AML12 cells were lysed, LSDs floated by gradient

centrifugation, and the FA- and Chl-fluorescently tagged LSDs separated by

FACS (Figure 3A and Supplemental Figures S2A,B,C). The FA- and Chl- specific

labelings were relatively similar and reproducible. Generally >70% of the particles

were dye-tagged in dual label experiments. FACS separations were distinct with

<5% of unsorted particles carrying both FA and Chl dye markers (Figures 3A,B).

The particles also showed reasonable size homogeneity; ~80% of all particles

had diameters of 2-6 μm. Mixing experiments of labeled and unlabeled lysates

and particles showed <2% dye marker transfer and/or non-specificity

(Supplemental Figures S2A,B,C).

Lipids were extracted from sorted FA- and Chl-tagged LSDs and analyzed

and quantified by thin layer chromatography (Figure 3C, Table I, and

Supplemental Figure S3A). Total LSD content was similar within and between

cell types. The Chl-tagged droplets were primarily comprised of cholesteryl ester

and a smaller amount of cholesterol; we were unable to detect triacylglycerols,

fatty acids, and related metabolites in the sorted Chl-tagged droplets. Conversely,

the isolated FA-tagged droplets were comprised primarily of triacylglycerol and

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minor quantities of metabolites (di- and mono-acylglycerides and fatty acids);

small amounts of cholesterol were present, but cholesteryl ester was not

detected. Thus, we have established conditions to fluorescently tag, image, and

purify LSDs specific to either CE or TAG.

We also traced the cell fate of the Bodipy precursor markers

(Supplemental Figure S3B). The native Bodipy-Chl dye marker migrates at a

unique position that differs from both cholesterol and choleterol ester and retains

its identical motility even after integration within the CE-droplets Thus, Bodipy-

Chl appears to be incorporated directly into CE droplets and is not significantly

metabolized into other droplet-specific lipids during the time frame studied.

Bodipy-FA migrates less far than does untagged FA, but changes mobility when

integrated into TAG droplets. We suggest that this represents the incorporation of

the Bodipy-FA into TAG, which migrates less far than untagged TAG and

distinctly from phospholipids. Regardless, the FA- and Chl-markers represent

specific tracer tags that allow us to separately purify the distinct populations of

LSDs that are biochemically enriched in either triacylglcerol (TAG) or cholesterol

ester (CE), hereafter referred to as TAG- or CE-LSDs, respectively.

Plin associations with TAG- or CE- LSDs.

We next examined Plin protein associations with isolated TAG- or CE-

LSDs. Y1 cells were cultured with oleic acid and cholesterol, plus FA- and Chl-

fluorescent markers. LSDs were floated, TAG- or CE-LSDs purified by FACS,

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and associations of Y1 endogenous Plins within unsorted LSDs or to TAG- or

CE-specific populations then assayed by specific immunoblotting.

All of the Plin proteins were detected in the unsorted LSD population

(Figure 3D), consistent with previous data (Figures 1A,B). The relative increase

in endogenous Plin1b and Plin1d signals probably reflects the enrichment of

these forms in floating droplets compared to whole cell lysates (see Figures 1A,

3D). In this assay, the individual Plins show distinct sequestration to TAG- or CE-

specific LSDs. Plin1a, Plin1b, and Plin5 are restricted to TAG-populations,

whereas Plin1c and Plin4 localize with the CE-LSDs (Figure 3D). Plin2, Plin3,

and Plin1d show limited association preference (Figure 3D).

Sub-cellular localization of GFP-Plins to TAG- or CE-LSDs.

The whole cell accumulations of specific Plin proteins in response to either

oleic acid or cholesterol correlate with the associations of the same Plins, to

either TAG- or CE-LSDs (Figures 1,3D). Still, we wished to investigate specific

Plin/droplet associations by alternative methods. Since McARH7777 cells

accumulate TAG- and CE-LSDs that are completely separated (Figure 2A), we

could visually observe if GFP-Plins exhibited parallel localization differences. In

addition we could quantify the relative distributions of GFP-Plins among CE- or

TAG-LSDs by FACS segregation.

McARH7777 cells were transiently transfected individually with vectors

that specifically encode different eGFP-Plin fusions and cultured with oleic acid

and cholesterol plus either BODIPY 558/568 C12 (TAG marker) or cholesteryl

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BODIPY 576/589 C11 (CE marker). Both dye markers have excitation/emission

spectra that differ from eGFP (488/509). GFP/TAG and GFP/CE experiments

were always performed in pairs and data analyzed in parallel to assess reciprocal

responses (Figures 4, 5, and 6 and Supplemental Figure S4).

GFP-Plin1a localizes to the polarized periphery of McARH7777 cells and

primarily with TAG-LSDs (Figure 4A and 6). In contrast, while CE-LSDs are

sequestered to the cell interior, none of the GFP-Plin1a signal co-localizes with

the CE marker (Figure 4A). FACS profiles of cell populations support these

conclusions; ~95% of expressed GFP-Plin1a co-sorts with the TAG marker, while

only ~5% GFP-Plin1a co-sorts with CE-LSDs (Figures 4A and 6).

GFP-Plin1b also exhibits a localization preference for TAG-LSDs

compared to CE-LSDs, although the FACS profiles show slightly less specificity

(Figures 4B and 6).

GFP-Plin1c shows the expected reciprocal pattern to GFP-Plin1a (see

Figures 1 and 3D). GFP-Plin1c localizes to the cell interior, surrounding CE-LSDs,

and distinctly separate from the polarized TAG-LSDs (Figure 4C). The FACS

data are consistent, where >90% of GFP-Plin1c sorts with CE and separate from

TAG (Figures 4C and 6).

The GFP fusions of Plin1d, Plin2, and Plin3 show minimal preference for

TAG- or CE-LSDs and segregate with both (Figures 4D, 5A, and 5B). All 3

protein fusions are found in both interior and peripheral cell regions, co-localize

with both TAG- and CE-LSDs within the cell, and co-segregate with both by

FACS, but to varying degrees (Figures 4D, 5A, 5B, and 6).

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GFP-Plin4 and GFP-Plin5 exhibit reciprocal LSD preferences and are,

thus, respectively, similar to Plin1c and Plin1a (see Figures 1 and 3). While GFP-

Plin4 sequesters with CE-LSDs in the cell interior (Figure 5C), GFP-Plin5

polarizes with TAG-LSDs to the cell periphery (Figure 5D). FACS data

substantiate these preferences; ~85% of GFP-Plin4 co-sorts with a CE marker,

while ~85% of GFP-Plin5 segregates with TAG (Figures 5C, 5D, and 6).

Plins with TAG- or CE-binding preferences can alter cellular TAG/CE

distributions.

Loss of Plin1 and Plin2 in mice alters the targeted accumulation of lipid

levels in defined cell types (Martinez-Botas et al., 2000; Tansey et al., 2001;

Chang et al., 2006). We were, thus, interested to determine if there were a

preferential relationship among individual Plin proteins, their lipid targeting

preference, and the cellular accumulation of either TAG or CE. We first

attempted siRNA approaches to deplete Plins targeted to either TAG- or CE-

LSDs. Data from other systems clearly show that depletion of one Plin protein

type results in compensation by other Plins (Martinez-Botas et al., 2000; Tansey

et al., 2001; Chang et al., 2006; Sztalryd et al., 2006). Further, Plin2 and Plin3

are expressed in most cells and exhibit no TAG or CE targeting preference. Thus,

experiments directed toward defining Plin effects on TAG or CE levels required

us to simultaneously target multiple Plins in any individual cell. Several cultured

cells lines were selected but we were unsuccessful in depleting any of the TAG-

or CE-LSD specific Plins in combination with Plin2 and Plin3. As we were unable

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to deplete all Plins associated with either TAG- or CE-LSDs, we sought an

alternative approach.

When we compared FACS analyses of GFP-Plin1a- and GFP-Plin1c-

expressing cells (Figure 4), we noticed a conspicuous difference in the relative

distributions of TAG- and CE-LSDs. >60% of total LSD particles in Plin1a-

expressing cells were tagged by the TAG-dye marker (Figure 4A), whereas the

Plin1c-expressing cells were predominantly (>60%) populated with CE-containing

LSDs (Figure 4C). These data suggested that the ectopic expression of Plins

with specific lipid targeting preferences might quantitatively alter the balance of

TAG/CE levels in individual cells. We thus analyzed the relative TAG/CE

distributions in cells expressing various Plin proteins.

AML12 cells were transiently transfected individually with vectors that

specifically encode different Plin proteins and cultured with oleic acid and

cholesterol. Cells cultured without exogenous lipids accumulated only limited

levels of TAG or CE regardless of Plin expression (data not shown), whereas

untransfected control cells accumulate large quantities of both TAG and CE.

LSDs were isolated from the various Plin-expressing and control cells and their

TAG/CE levels quantified by TLC. Cells expressing Plin1a or Plin1c were

analyzed in pairs, and normalized in parallel experiments of lipid loaded and

unloaded control cells (Figure 7). Similar paired and normalized experiments

were used to analyze cells expressing Plin4 or Plin5.

Plin1a and Plin1c exhibited largely reciprocal effects on TAG/CE levels,

with an ~60% TAG lipid bias observed in Plin1a-expressing cells and an ~60%

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CE bias observed for Plin1c-expressing cells (Figure 7). Plin5/Plin4 differences in

relative TAG/CE accumulations were similarly polarized toward either TAG or CE,

respectively. Thus, ectopic expression of Plins that exhibit lipid targeting

preferences can polarize relative cellular lipid-type distributions. These effects

are seen for both the TAG-specific (i.e. Plin1a and Plin5) and the CE-specific (i.e.

Plin1c and Plin4) Plin proteins, but not for the non-preferential Plins 2, 3, and 1d

(data not shown).

DISCUSSION

We have demonstrated that distinct Plins differentially sequester to either

TAG- or CE-specific LSDs, emphasizing diversity of function for the different

Plins. These significant functional differences towards TAG- or CE-LSDs impact

previous assumptions about commonality of Plin action. The large unilocular

TAG-LSDs that are targeted by Plin1a in adipocytes are proposed to derive from

nascent droplets marked by Plin2, Plin3, and Plin4 (Wolins et al., 2005). While

Plin2 and Plin3 interact with TAG-LSDs, this association, unlike that of Plin1a, is

not exclusive, as Plin2 and Plin3 also co-segregate with CE-LSDs. Since Plin4

primarily targets CE-LSDs, conclusions regarding linear development of Plin-

specific LSD populations, based upon static global imaging of cells cultured with

exogneous fatty acids (Wolins et al., 2005), must be tempered.

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Plin structural domains that direct LSD targeting are still poorly defined. All

Plins share an N-terminal, ~100 amino acid PAT domain and a distal 11-mer

amphipathic helical repeat (Bussell and Eliezer, 2003; Lu et al., 2001; Miura et al.,

2002). C-terminal to both, Plins are increasingly diverged. Several groups have

probed for LSD-interacting motifs through domain-specific expressions (Garcia et

al., 2003; Hickenbottom et al., 2004; McManaman et al., 2003; Nakamura and

Fujimoto, 2003; Ohsaki et al., 2006; Subramanian et al., 2004a; Subramanian et

al., 2004b; Yamaguchi et al., 2006), but these studies have focused on TAG-

enriched cells and ignored effects of CE-LSDs. In addition, some constructs have

exposed amphipathic helices that are usually masked in endogenous Plins and,

thus, may target inappropriately (Hickenbottom et al., 2004).

No simple and consistent structural model for preferential Plin

associations with TG- or CE-LSDs can be easily deduced from sequence or

structural motif scanning and interrogation. While the PAT and associated 11-

mer domains may be involved in LSD interaction, these regions cannot be

sufficient determinants for specific targeting. Although all Plin1 variants have

identical N-terminal 198 residues, which include the PAT and 11-mer regions

(Kimmel et al., 2009; Lu et al., 2001; Miura et al., 2002), they have dissimilar LSD

targeting. Further, the PAT and 11-mer domains differ greatly among the other

Plin proteins in both length and sequence. Lipid binding discrimination can also

not be simply deduced by analyses of C-termini, which differ highly among Plin4,

Plin5, and each of the unique Plin1 forms. Signaling or targeting motifs may

instead reside in non-common segments, as hydrophobic segments are

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suggested to facilitate Plin1 targeting to LSDs (Garcia et al., 2003). While Plin-

specific interacting proteins possibly help direct TAG- or CE-LSD recognition,

one must consider that the extremely diverged single Plin species in

Dictyostelium specifically targets LSDs when expressed in mammalian CHO cells

(Lu et al., 2001; Miura et al., 2002). Furthermore, although Plin interactions that

are unique to TAG- or CE-specific LSDs may involve the surrounding

phospholipid monolayer, these would also require commonality in both diverse

tissues (e.g. adrenal and liver cells) and species.

Pathogenesis associated with abnormal lipid storage has serious health

consequences. Thus, understanding the mechanisms that direct lipid storage and

lipolytic breakdown is paramount. The Plins regulate access of lipases to lipids

stored within the LSD core (Granneman et al., 2009; Granneman et al., 2010;

Martinez-Botas et al., 2000; Miyoshi et al., 2007; Sztalryd et al., 2003; Tansey et

al., 2001; Wang et al., 2010; Wang et al., 2009; Yamaguchi et al., 2004), and the

cellular content of Plins and accumulated LSDs seem intimately coordinated. In

addition, various Plins may have significantly different regulatory effects on

cellular lipolytic activity depending upon tissue context (Dalen et al., 2007;

Tansey et al., 2003). Accordingly, recent attention has been directed towards

possible causal linkages of aberrant Plin function with human disease. In

particular, heterozygous loss-of-function mutations of PLIN1 causes a familial

partial lipodystrophy in humans (Gandotra et al., 2011) and polymorphisms in

human genes for Plin1 (Qi et al., 2004) and Plin4 (Richardson et al., 2011) have

been associated with obesity.

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Our data add novel conceptual parameters for LSD and Perilipin function

and analyses. The various Plins target different classes of LSDs even within a

single cell population and Plins with LSD lipid specificities can preferentially

affect the accumulation of the targeted LSD class. Intriguingly, tissues such as

adipose, heart, and oxidative muscle, that predominantly accumulate TAG, have

highest expression of Plin 1a, 1b, or 5, which specifically target TAG-LSDs.

Conversely, steroidogenic cells, which accumulate CE, express these Plins more

poorly in comparison to Plin1c. We suggest that Plin expression may not impact

all cellular lipids and LSDs equivalently, but that the actions of individual Plin-

types may be differentially targeted to distinct LSD classes within a given cell. It

will, thus, be of interest to evaluate the full spectrum of Plin variants in broad

tissue panels that exhibit differential TAG/CE biases. Our new findings

underscore the view that each Plin is likely to have very separate and perhaps

unique functions associated with their LSD-specific targeting.

MATERIALS AND METHODS

Materials.

Fatty acid free bovine serum albumin (BSA) was purchased from Fisher

Scientific (Pittsburgh, PA). Phosphate buffered saline (PBS), glutamine, fetal

bovine serum (FBS), horse serum, cholesteryl BODIPY® 500/510 FL C12

[cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-

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dodecanoate; cholesteryl dye], BODIPY® 558/568 C12 [4,4-difluoro-5-(2-thienyl)-

4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; fatty acid dye], cholesteryl

BODIPY® 576/589 C11 [cholesteryl 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-

s-indacene-3-undecanoate; cholesteryl dye], and BODIPY® 500/510 FL C12 [4,4-

difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate; fatty acid dye]

were from Invitrogen (Carlsbad, CA). MitoTracker (579/599) and LysoTracker

(577/590) were also from Invitrogen (Carlsbad, CA). Antibodies to LAMP1 and

EEa1 were from Abcam (Cambridge, MA) and to TOM2 from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA). The 250 microns silica gel H TLC plates

were from Analtech (Newark, DE). Complete protease inhibitor cocktail tablets

were from Roche Diagnostics (Indianapolis, IN). Paraformaldehyde was from

Electron Microscopy Sciences (Hatfield, PA). Cell media and all other chemicals

were from Sigma-Aldrich (St. Louis, MO).

Cell culture.

Y1 mouse adrenalcortical, AML12 mouse liver, and McARH7777 rat

hepatoma cells were obtained from the American Tissue Culture Collection

(Manassas, VA). Cells were grown to subconfluence in medium supplemented

with 100 μg/mL penicillin and 100 μg/mL streptomycin and incubated in

humidified air containing 5% CO2 at 37°C. Y1 adrenal cells were grown in

Dulbecco's modified Eagle medium (DMEM)/Ham’s nutrient mixture F-12

supplemented with 15% horse serum, 2.5% FBS, and 2 mM glutamine.

McARH7777 cells were grown in DMEM supplemented with 10% FBS. AML12

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cells were grown in DMEM/Ham’s nutrient mixture F-12 supplemented with 5

mg/ml insulin, 5 mg/ml tranferrin, 5 ng/ml selenium, 10% FBS, and 2 mM

glutamine

Plin-GFP fusion constructs and transfection.

The perilipin expression vectors were generated using Multi-Site Gate-

Way (Invitrogen, Carlsbad, California). Mouse Plins 1a, 2, 3, and 5 cDNAs were

amplified from previously described pSG5 vectors (Dalen et al., 2007). Plins 1b,

1c, 1d, and 4 cDNAs were amplified from Y-1 cells or adipose tissue mRNA

using PfuTurbo® DNA Polymerase (Stratagene). Primers used contained

overhangs for insertion into the pDONR-221 P4r-P3r vector, Kozak sequence

(ACCTAG) and stop codon (CTA), and were designed using Vector NTI 10.0

(Invitrogen) with Tm set to 65ºC.

PCR products were recombined into the pDONR-221 P4r-P3r vector using

BP Clonase II (Invitrogen) to produce pENTR-vectors. GFP was amplified from

pEGFP-C1 (Clontech) and cloned into the pDONR-221 P1-P4. The V5-6x-His-

Gly epitope was cloned into the pDONR-221 P3-P2 vector.

The destination vector was generated by replacing the multi-cloning site of

pcDNA3 (Invitrogen) with the attR1-ccdB-chloramphenicol-attR2-cassette (R1-R2)

from pLenti6/v5-DEST (Invitrogen). The R1-R2 cassette was amplified with

PfuTurbo, and digested with HindIII/ApaI prior to ligation into HindIII/ApaI

digested pcDNA3 vector. The ligation mixture was transformed into ccdB Survival

TR cells (Invitrogen) and clones selected on Ampicilin (100 µg/ml) and

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Chloramphenicol (25 µg/ml) plates. The novel pcDNA3-R1- R2 vector (pcDNA3-

KTD2-DEST) was subsequently recombined with the above pENTR-vectors

using LR clonase II (Invitrogen) to generate the pKTD2-G-Perilipin-VH

expression vectors. Due to the stop codon inserted into the pENTR-perilipin

vectors, the 3-end V5-6xHis-G tag will not be translated. Correctly amplified

sequences were confirmed by sequencing (Macrogen, Korea).

Transient transfection was carried out following the manufacturer's

instructions (Invitrogen) using lipofectamine LTX reagent. McARH7777 cells (cell

density 6.25x104/cm2) were incubated in standard growth conditions in medium

lacking antibiotic for one day and then incubated with fresh media containing

plasmid DNA (250 ng/cm2), lipofectamine LTX reagent (1 µl/cm2), and Opti-MEM

(50 µl/cm2) for one day.

Immunoblot analyses.

Proteins were separated by electrophoresis in 10% NuPAGE gels

(Invitrogen, Carlsbad, CA) using MOPS running buffer and then subjected to

immunoblot analyses as described (Kim et al., 2002). For immunoblot analyses,

we used rabbit polyclonal antisera (1:3000) to mouse Plin1 (Servetnick et al.,

1995), Plin2 (Xu et al., 2005), or Plin3 (Sztalryd et al., 2006), and guinea pig

polyclonal antisera (1:3000) to human Plin4 (American Research Product,

03-GP31) and human Plin5 (Progen Biotechnik, GmbH GP34). Rabbit antibody

(1:5000) to β-actin and GFP were, respectively, from Abcam (Cambridge, MA)

and Invitrogen (Carlsbad, CA). Secondary antibodies to rabbit or guinea pig

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IgG were from Jackson ImmunoResearch (West Grove, PA) and used

at 1:5000 dilution.

Lipid loading.

Cells were grown overnight to <50% confluence in media supplemented

with 100-200 μM oleic acid bound to fatty acid free BSA (2.5:1 mol oleic acid: mol

BSA) (Dalen et al., 2006) and/or 50 μM cholesterol complexed with β-methyl

cyclodextrin (8:1 mol β-methyl cyclodextrin: mol cholesterol) (Christian et al.,

1997). For fatty acid and cholesteryl dye labeling, cells were grown overnight to

<50% confluence in media supplemented with 100 μM oleic acid and 50 μM

cholesterol, plus 1.0 μM BODIPY® 558/568 C12 (fatty acid dye) and 0.5 μM

cholesteryl BODIPY® 500/510 FL C12 (cholesteryl dye). GFP-Plin expressing

cells were grown overnight to <50% confluence in media supplemented with 100

μM oleic acid and 50 μM cholesterol, plus either 1.0 μM BODIPY® 558/568 C12

(fatty acid dye) or 0.5 μM cholesteryl BODIPY® 576/589 C11 (cholesteryl dye).

Lipid droplet preparation.

Cells were grown overnight, washed with phosphate-buffered saline (PBS),

scraped into PBS, and pelleted by centrifugation (300xg for 5 min). The cell pellet

was resuspended in 4 ml hypotonic lysis solution (50 mM HEPES pH 7.3, 0.1 M

KCl, 2 mM MgCl2; 4 °C) containing protease inhibitors (20 μg/ml leupeptin, 1 mM

benzamidine and 100 μM 4-(2-aminoethyl)-benzenesulfonylfluoride) and lysed by

incubation on ice for 30 min. 1 ml of 50% (w/v) sucrose in lysis solution was

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added to the cell lysate for a total volume of 5 ml. This 10% w/v sucrose solution

was layered at the bottom of an ultracentrifuge tube and a step gradient of 5%,

2.5%, and 0% sucrose was carefully layered above; centrifugation (Beckman

Coulter Optima XL100K Ultracentrifuge) was at 154,000xg for 1 h at 4 °C. The

floating lipid layer was collected using a Beckman tube slicer (Brea, CA) for

immunoblotting or flow cytometry (Brasaemle et al., 2004; Hsieh and Huang,

2005)

FACS separation of fluorescently labeled lipid droplets.

Lipid droplets isolated from cells labeled with fluorescent fatty acid dye,

fluorescent cholesteryl dye, and/or expressing GFP were sorted using a

FACSAria II cytometer (BD Biosciences). All fluorochromes were excited using a

488 nm laser. Fluorescent emissions from lipid droplets labeled with cholesteryl

BODIPY® 500/510 FL C12 or with GFP-tagged perilipin proteins were collected

through a 502 nm longpass filter followed by a 530/30 nm bandpass filter.

Emissions from lipid droplets labeled with BODIPY® 558/568 C12 or with

cholesteryl BODIPY® 576/589 C11 were collected through a 556 nm longpass

filter followed by a 575/26 bandpass filter. Compensation settings for

experiments with multiple fluorescent dyes were established using lipid droplets

isolated from cells labeled with a single fluorescent dye (see Figures S1 and S3).

The forward scatter threshold was set at the lowest possible linear signal height

of 200 (within an allowed range of 200 to 262,143), permitting detection of lipid

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droplets with diameters >1 μm. For sorting, ~5,000 events/second were

processed and sort precision was set on the default purity mode.

Confocal laser scanning microscopy (CLSM).

CLSM was carried out using a Zeiss LSM 510 (Jena, Germany) inverted

confocal microscope with a 100x (Plan-Apochromat, NA1.40) oil objective lens.

GFP and cholesteryl BODIPY® FL 500/510 FL C12 were imaged using argon

488-nm laser and a 505-530-nm BP emission filter. BODIPY® 558/568 C12 and

cholesteryl BODIPY® 576/589 C11 were imaged using a He/Ne 543-nm laser

excitation and a 580-nm LP emission filter. The software for confocal microscopic

image generation was LSM510 software 3.2. The cells were seeded and

maniplulated in 35 mm glass bottom culture dishes (MatTek, Ashland, MA), fixed

with 4% paraformaldehyde in PBS for 30 minutes, and washed twice for 5

minutes before observation. To visualize isolated lipid droplets, the unsorted or

sorted lipid droplet populations were first mixed with an equal volume of glycerol

before observation.

Lipid extraction and analyses.

Lipid droplets were extracted twice with 2 volumes of

chloroform:heptane:methanol (4:3:2; v/v/v) (Hsieh and Huang, 2007). The lipids

were applied to TLC plates and separated in hexane:diethyl ether:acetic acid

(70:30:1; v/v/v); the plates were stained overnight in an iodine chamber to

visualize the lipids. Extracted lipids were separated in parallel to a dilution series

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of lipid standards applied to the same plate. The standards were cholesteryl

oleate (cholesteryl ester; CE), glyceryl trioleate (triacylglycerol; TAG), oleic acid

(fatty acid; FA), cholesterol (Chol), dioleoylglycerol (diacyclglycerol; DAG), DL-α-

Monoolein (monoacyclglycerol; MAG), and L-α-phosphatidylcholine (phospholipid;

PL).

TIFF images of the iodine stained TLC plates were analyzed by

ImageQuant TL with module 1D gel analyses (GE Health Life Sciences,

Piscataway, NJ). Stored 8 bit grayscale images were created manually, since

single lane images had various shapes and spot distances. Spot intensities were

corrected for background and data exported to generate curve standards for the

various lipid marker controls. Masses of the unknown lipids were extrapolated in

comparison to standard curves (see Figure S3A).

Statistical analyses.

Confocal data (Figures 4,5,6) are from at least 3 independent, paired

experiments (see Figure 3), where the TAG/CE sum for each pair was 98-102%

(see Figure 3). McARH7777 cells were transiently transfected with the indicated

GFP-Plin expressing constructs and cultured overnight in the presence of 100

μM oleic acid and 50 μM cholesterol, plus either 1 μM BODIPY 558/568 C12 (FA)

or 0.5 μM cholesteryl BODIPY 576/589 C11 (Chl) dye markers. TAG or CE

distributions represent the % of the total GFP+ signal that co-sorts, respectively;

with either FA+ or Chl+ tags. The numbers are shown ± standard deviation

(Figure 6).

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ACKNOWLEDGEMENTS

This research was supported by the Intramural Research Program of the

National Institutes of Health, the National Institute of Diabetes and Digestive and

Kidney Diseases. K.T.D. was supported by a travel grant from the Henning and

Johan Throne-Holst's Foundation. There are no conflicts or competing interests.

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perilipin gene haplotype with obesity risk in a white population. Obes Res 12, 1758-1765.\

Richardson. K., Louie-Gao, Q., Arnett, D.K., Parnell, L.D., Lai, C.Q., Davalos, A., Fox, C.S., Demissie, S., Cupples, L.A., Fernandez-Hernando, C. and Ordovas, J.M. (2011). The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site. PLoS One 6, e17944. Servetnick, D. A., Brasaemle, D. L., Gruia-Gray, J., Kimmel, A. R., Wolff, J. and Londos, C. (1995). Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. J Biol Chem 270, 16970-3. Subramanian, V., Garcia, A., Sekowski, A. and Brasaemle, D. L. (2004a). Hydrophobic sequences target and anchor perilipin A to lipid droplets. J Lipid Res 45, 1983-91. Subramanian, V., Rothenberg, A., Gomez, C., Cohen, A. W., Garcia, A., Bhattacharyya, S., Shapiro, L., Dolios, G., Wang, R., Lisanti, M. P. et al. (2004b). Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes. J Biol Chem 279, 42062-71. Sztalryd, C., Bell, M., Lu, X., Mertz, P., Hickenbottom, S., Chang, B. H., Chan, L., Kimmel, A. R. and Londos, C. (2006). Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line. J Biol Chem 281, 34341-8. Sztalryd, C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J. A., Kimmel, A. R. and Londos, C. (2003). Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol 161, 1093-103. Tansey, J. T., Huml, A. M., Vogt, R., Davis, K. E., Jones, J. M., Fraser, K. A., Brasaemle, D. L., Kimmel, A. R. and Londos, C. (2003). Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols. J Biol Chem 278, 8401-6. Tansey, J. T., Sztalryd, C., Gruia-Gray, J., Roush, D. L., Zee, J. V., Gavrilova, O., Reitman, M. L., Deng, C. X., Li, C., Kimmel, A. R. et al. (2001). Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity. Proc Natl Acad Sci U S A 98, 6494-9. Wang, H., Bell, M., Sreenevasan, U., Hu, H., Liu, J., Dalen, K., Londos, C., Yamaguchi, T., Rizzo, M. A., Coleman, R. et al. (2010). Unique Regulation of Adipose Triglyceride Lipase (ATGL) by Perilipin 5, a Lipid Droplet-associated Protein. J Biol Chem 286, 15707-15. Wang, H., Hu, L., Dalen, K., Dorward, H., Marcinkiewicz, A., Russell, D., Gong, D., Londos, C., Yamaguchi, T., Holm, C. et al. (2009). Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins. J Biol Chem 284, 32116-25. Wolins, N. E., Quaynor, B. K., Skinner, J. R., Schoenfish, M. J., Tzekov, A. and Bickel, P. E. (2005). S3-12, Adipophilin, and TIP47 package lipid in adipocytes. J Biol Chem 280, 19146-55.

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Wolins, N. E., Quaynor, B. K., Skinner, J. R., Tzekov, A., Croce, M. A., Gropler, M. C., Varma, V., Yao-Borengasser, A., Rasouli, N., Kern, P. A. et al. (2006). OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization. Diabetes 55, 3418-28. Xu, G., Sztalryd, C., Lu, X., Tansey, J. T., Gan, J., Dorward, H., Kimmel, A. R. and Londos, C. (2005). Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem 280, 42841-7. Yamaguchi, T., Matsushita, S., Motojima, K., Hirose, F. and Osumi, T. (2006). MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor alpha. J Biol Chem 281, 14232-40. Yamaguchi, T., Omatsu, N., Matsushita, S. and Osumi, T. (2004). CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome. J Biol Chem 279, 30490-7.

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FIGURE LEGENDS

Figure 1. Differential accumulation of Plins in cells cultured in the absence

or presence of fatty acid and/or cholesterol.

A. Y1 adrenal cells were cultured overnight in the absence or presence of oleic

acid and/or cholesterol. Whole cell lysates were prepared and endogenous Plins

assayed by immunoblot. Data are representative of 3 experiments.

B. McARH7777 rat liver cells were transiently transfected with the indicated GFP-

Plin expressing constructs and cultured overnight in the absence or presence of

oleic acid and/or cholesterol. Whole cell lysates were prepared and GFP-Plin

assayed by immunoblot. Data are representative of 3 experiments.

Figure 2. Spatially distinct intracellular accumulations of fatty acid- and

cholesteryl-dye markers.

A. Y1 adrenal, McARH7777 rat liver, and AML12 mouse liver cells were cultured

overnight in the presence of oleic acid and cholesterol, plus BODIPY 558/568 C12

[fatty acids (FA)] and cholesteryl BODIPY 500/510 FL C12 (Chl). Representative

confocal images are shown with red and green indicating the localization of FA

and Chl, respectively.

B. McARH7777 cells were cultured overnight in the presence of oleic acid and

cholesterol, plus BODIPY 558/568 C12 [fatty acids (FA)] and cholesteryl BODIPY

500/510 FL C12 (Chl) and probed for organelle localization by immunoflorescence

detection of LAMP1 (Lysosomal-associated membrane protein 1), TOM2

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(translocase of outer mitochondrial membranes 20 kDa), and EEA1 (Early

Endosome Antigen 1.

C. McARH7777 cells were cultured overnight in the presence of oleic acid and

cholesterol, plus BODIPY 500/510 C12 [fatty acids (FA)] or cholesteryl BODIPY

500/510 FL C12 (Chl) and probed for organelle localization by staining with dye

specific markers.

Figure 3. Differential Localization of Plins to TAG or CE droplets.

Y1 adrenal, McARH7777 rat liver, and AML12 mouse liver cells were

cultured overnight in the presence of oleic acid and cholesterol, plus BODIPY

558/568 C12 [fatty acids (FA)] and cholesteryl BODIPY 500/510 FL C12 (Chl).

A. Representative FACS profiles of purified FA- and Chl-tagged lipid droplets are

shown, with relative distributions of total lipid droplet numbers indicated in the

different quadrants (FA-/Chl-, FA+/Chl-, FA-/Chl+, and FA+/Chl+); blue represents

dual signals of FA and Chl probes. Y1 cells (25,000 total droplets); McARH7777

cells (20,000 total droplets); AML12 cells (25,000 total droplets).

B. Confocal images of unsorted and FACS sorted FA- and Chl-tagged lipid

droplets from Y1, McARH7777, and AML12 cells.

C. Lipids isolated from FA- and Chl-sorted droplets were separated by TLC in

parallel with lipid markers and detected by staining with iodine vapor. 1x indicates

2x105 LSD particles; 5x indicates 1x106 LSD particles. Marker lanes have 50μg

of CE: Cholesteryl Ester* and 10μg each of TAG: Triacylglycerols; FA: Fatty

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Acids; DAG: Diacylglycerides; Chol: Cholesterol; MAG: Monoacylglyceride; PL:

Phospholipids. PLs do not migrate from the origin in this system.

D. Proteins were prepared from unsorted and FACS sorted TAG- or CE-LSD

isolated from Y1 cells. Endogenous Plin proteins were assayed by immunoblot.

Identical lipid droplet particle numbers were loaded in each lane. Data are

representative of 3 experiments.

Figure 4. Differential localization of Plin1 proteins to TAG or CE lipid

droplets.

McARH7777 cells were transiently transfected with the indicated GFP-

Plin1 expressing constructs and cultured overnight in the presence of oleic acid

and cholesterol, plus either BODIPY 558/568 C12 (TAG) or cholesteryl BODIPY

576/589 C11 (CE).

Representative confocal images are shown for each with red indicating the

localization of either TAG or CE droplets, green indicating localization of GFP-

Plin1 proteins, and yellow (or rings) indicating co-localization.

Representative FACS profiles of TAG- and GFP-labeled lipid droplets or of

CE- and GFP-labeled lipid droplets are shown, with relative distributions of total

particle numbers indicated in the different quadrants, and the relative Plin1

variant localizations indicated as % of total GFP+ signal that co-sorts (blue) with

either TAG+ or CE+ tags. Each experiment was performed at least 3 times (see

Figure 6).

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A. Plin1a (25,000 total droplets for each population); B. Plin1b (20,000

total droplets for each population); C. Plin1c (25,000 total droplets for each

population); D. Plin1d (10,000 total droplets for each population).

Figure 5. Differential localization of Plin2-5 proteins to TAG or CE lipid

droplets.

McARH7777 cells were transiently transfected with the indicated GFP-Plin

expressing constructs and cultured overnight in the presence of oleic acid and

cholesterol, plus either BODIPY 558/568 C12 (TAG) or cholesteryl BODIPY

576/589 C11 (CE).

Representative confocal images are shown for each with red indicating the

localization of either TAG or CE droplets, green indicating localization of GFP-

Plin proteins, and yellow (or rings) indicating co-localization.

Representative FACS profiles of TAG- and GFP-labeled lipid droplets or of

CE- and GFP-labeled lipid droplets are shown, with relative distributions of total

particle numbers indicated in the different quadrants, and the relative Plin2-5

variant localizations indicated as % of total GFP+ signal that co-sorts (blue) with

either TAG+ or CE+ tags. Each experiment was performed at least 3 times (see

Figure 6).

A. Plin2 (15,000 total droplets for each population); B. Plin3 (20,000 total

droplets for each population); C. Plin4 (25,000 total droplets for each population);

D. Plin5 (10,000 total droplets for each population).

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Figure 6. Relative distributions of Plin family proteins to TAG- or CE-

specific intracellular lipid storage droplets.

McARH7777 cells were transiently transfected with the indicated GFP-Plin

expressing constructs and cultured overnight in the presence of oleic acid and

cholesterol, plus either BODIPY 558/568 C12 (FA) or cholesteryl BODIPY

576/589 C11 (Chl) dye markers. TAG or CE distributions represent the % of the

total GFP+ signal that co-sorts, respectively; with either FA+ or Chl+ tags (see

Figures 4,5). Data are from at least 3 independent, paired experiments, where

the TAG/CE sum for each pair was 98-102%. The numbers are shown as the

mean ± standard deviation.

Plin1a, Plin1b, and Plin5 show strong preference for localization to TAG-

LSDs, with Plin1a consistently exhibiting a stronger TAG signal than did Plin1b or

Plin5. Plin1c and Plin4 show strong preference for localization to CE-LSDs, with

Plin1c consistently exhibiting a stronger CE signal than did Plin4. Plin1d, Plin2,

and Plin3 exhibit minor localization preferences.

Figure 7. Relative change in TAG- or CE- specific intracellular lipid storage

upon expression of various Plin proteins.

AML12 cells were transiently transfected with the indicated GFP-Plin

expressing constructs and cultured overnight in the presence of oleic acid and

cholesterol. Transfection efficiencies were confirmed by visualizing GFP

fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and

cholesterol, were grown in parallel. LSDs were isolated by centrifugation and

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lipids were extracted, separated by TLC in parallel with lipid markers, and TAG

and CE detected after staining with iodine vapor. Relative TAG/CE ratios were

quantified in cells transfected with each specific Plin-expressing construct and

analyzed in parallel with identically grown untransfected cells for normalization

and TLC background correction.

The TAG/CE-ratio for untransfected lipid loaded cells (controls) was set to

0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c- expressing cells were

always analyzed in parallel and normalized to those of control cells, and then

secondarily compared to results determined for its Plin-expressing counterpart.

Numbers >0.5 indicate a proportional increase in TAG lipid bias, whereas

numbers <0.5 indicate a proportional increase in CE lipid bias. Relative

distributions of TAG/CE levels are shown as the mean ± standard deviation for

each paired comparison. Data for each pair are based on three independent

experiments. Plin4 and Plin5 expressing cells were similarly analyzed as pairs

and internally compared as described for the Plin1a/Plin1c pair.

Plin1a and Plin5 show a relative bias for cellular TAG accumulation and a

strong preference for binding specificity to TAG-LSDs (see Figure 6). Conversely,

Plin1c and Plin4 show a relative increase in cellular CE levels and a strong

preference for binding specificity to CE-LSDs.

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SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Spatially distinct intracellular accumulations of fatty acid- and

cholesteryl-dye markers.

A. 4T1 mouse mammary tumor, primary mouse liver, C2C12 mouse myoblast,

3T3-L1 mouse fibroblast, J774A.1 mouse monocyte-macrophage, and CHOK1

Chinese hampster ovary cells were cultured overnight in the presence of oleic

acid and cholesterol, plus BODIPY 558/568 C12 [fatty acids (FA)] and cholesteryl

BODIPY 500/510 FL C12 (Chl). Representative confocal images are shown with

red and green indicating the localization of FA and Chl, respectively.

B. McARH7777 cells were cultured for the times indicated in the presence of

oleic acid and cholesterol, plus BODIPY 558/568 C12 [fatty acids (FA)] and

cholesteryl BODIPY 500/510 FL C12 (Chl). Representative confocal images are

shown with red and green indicating the localization of FA and Chl, respectively.

Figure S2. Control FACS profiles of lipid dye-marked Y1 cells.

Cells were cultured overnight in the presence of 100 μM oleic acid and 50

μM cholesterol, and in the absence or presence of 1 μM BODIPY 558/568 C12

(FA) or 0.5 μM cholesteryl BODIPY 500/510 FL C12 (Chl). Cells were

hypotonically lysed and lipid droplets purified by sucrose gradient centrifugation.

Representative FACS profiles of unlabeled lipid droplets and of FA- or Chl-

labeled lipid droplets are shown, with relative distributions of total particle

numbers indicated in the different quadrants (FA-/Chl-, FA+/Chl-, FA-/Chl+, and

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FA+/Chl+). In one FACS panel (FA lysate + Chl lysate), lysates were prepared

from cells that had been separately labeled for either FA or Chl and then mixed

and sorted simultaneously. A. Y1 cells; B. McARH7777 cells; C. AML12 cells.

Figure S3. Lipid quantification.

A. Lipid markers were separated by TLC and detected by staining with iodine

vapor (see Figure 3C). TIFF images of the iodine stained TLC plates were

analyzed to generate standard curves for each lipid. CE: Cholesteryl Ester; TAG:

Triacylglycerols; FA: Fatty Acids; DAG: Diacylglycerides; Chol: Cholesterol; MAG:

Monoacylglyceride.

B. Lipids isolated from FA- and Chl-sorted droplets were separated by TLC in

parallel with lipid markers and detected by fluorescence under UV or by staining

with iodine vapor. 1x indicates 2x105 LSD particles; 5x indicates 1x106 LSD

particles. Marker lanes had various lipid concentrations. Bodipy-FA is the input

BODIPY 558/568 C12 marker and Bodipy-Chl is the input cholesteryl BODIPY

500/510 marker. CE: Cholesteryl Ester; TAG: Triacylglycerols; FA: Fatty Acids;

DAG: Diacylglycerides; Chol: Cholesterol; MAG: Monoacylglyceride; PL:

Phospholipids. PLs do not migrate from the origin in this system.

Figure S4. Control FACS profiles of lipid dye-marked or GFP-Plin

expressing McARH7777 cells cells.

McARH7777 cells were cultured overnight in the presence of 100 μM oleic

acid and 50 μM cholesterol. In one cell population, 1 μM BODIPY 558/568 C12

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(FA) was added to the culture media. Another population of McARH7777 cells

was transiently transfected with a GFP-Plin1a expressing construct before

culturing. Cells were then hypotonically lysed and lipid droplets purified by

flotation following centrifugation. Representative FACS profiles of unlabeled lipid

droplets and of FA- or GFP-labeled lipid droplets are shown, with relative

distributions of total particle numbers indicated in the different quadrants (FA-

/GFP-, FA+/GFP-, FA-/GFP+, and FA+/GFP+). In one FACS panel (FA lysate +

GFP lysate), lysates were prepared from cells that had been separately labeled

for either FA or GFP and then mixed and sorted simultaneously.

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Table I

Lipid Specificities in FACS Purified Lipid Droplets

LIPID Chl-Tagged Droplets FA-Tagged Droplets s

...... Y1 McA AML12 Y1 McA AML12

CE 90μg 65μg 70μg ND (3<μg) ND (3<μg) ND (3<μg)

TAG ND (0.5<μg) ND (0.5<μg) ND (0.5<μg) 70μg 100μg 100μg

FA ND (0.5<μg) ND (0.5<μg) ND (0.5<μg 10μg 15μg 20μg

DAG ND (0.5<μg) ND (0.5<μg) ND (0.5<μg 20μg 10μg 10μg

Chol 50μg 15μg 15μg 3-5μg 3-5μg 3-5μg

MAG ND (2<μg) ND (2<μg) ND (2<μg) 15μg 10μg 10μg

Chl- and FA-tagged lipid droplets from Y1, McARH7777, and AML12 cells were separated by FACS

and lipid profiles analyzed by TLC (see Figure 3C). Values/2x106 droplets were extrapolated (± 30%)

from relative staining intensity data in comparison to standards (see Figure S3B) for CE: Cholesteryl

Ester, TAG: Triacylglycerol, FA: Fatty Acid, DAG: Diacylglycerol, Chol: Cholesterol, and MAG:

Monoacylglycerol. Phospholipid (PL) spot staining was similar for each, but did not resolve from the

origin. ND: not detected.

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- - + + Oleic Acid

- + - + Cholesterol

Plin1a

Plin1b

Plin1c

Plin1d

Plin2

Plin3

Plin4

Plin5

actin

Endogenous

Y1 Plins

Figure 1A

A.

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- - + + Oleic Acid

- + - + Cholesterol

Plin1a

actin

Plin1b

actin

Plin1c

actin

Plin1d

actin

Plin2

actin

Plin3

actin

Plin4

actin

Plin5

actin

GFP-Plin B.

Figure 1B

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Y1 Mouse Adrenalcortical

FA Chl

A.

Figure 2A

McARH7777 Rat Liver

FA Chl

AML12 Mouse Liver

FA Chl

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LAMP1 (lysosomes)

FA

Chl

EEA1 (early endsomes) Tom20 (mitochondria)

B.

Figure 2B

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MitoTracker LysoTracker

C.

Figure 2C

FA

Chl

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Fatt

y A

cid

s

Cholesteryl

Figure 3A

A.

Y1

Cholesteryl Cholesteryl

McARH7777 AML12

0.381

0.194

0.400

0.025

0.356

0.042 0.330

0.272 0.515

0.294

0.149

0.042

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McARH7777

Unsorted

Droplets

FA-

Sorted

Droplets

Chl-

Sorted

Droplets

Y1 AML12

10mm

B.

Figure 3B

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AML12 Sorted

Lipid Droplets

DAG Chol

MAG

McA Sorted

Lipid Droplets Lipid Markers

(10 mg each)

4x105 Chl-Droplets

4x105 FA-Droplets

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

5x 5x

CE

TAG

FA

PL

DAG Chol

MAG

Y1 Sorted

Lipid Droplets

CE

FA

PL

Chol

MAG

TAG

DAG

+*

-

-

-

-

-

-

-

-

5x 1x 1x 1x

1x 1x 1x

* (50mg) CE

TAG

FA

(origin) PL

C.

Figure 3C

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+ - + TAG Lipid Droplets

+ + - CE Lipid Droplets

Plin1a

Plin1b

Plin1c

Plin1d

Plin2

Plin3

Plin4

Plin5

Endogenous

Y1 Plins

FACS

Purified

D.

Figure 3D

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TA

G

GFP-Plin1a GFP-Plin1a

CE

95% of

GFP+

5% of

GFP+

0.032

0.574

0.305

0.0

89

0.505

0.027

0.080

0.3

87

Plin1a

TAG

Plin1a

CE

A.

Figure 4A

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GFP-Plin1b GFP-Plin1b

80% of

GFP+

0.092

0.358

0.361

0.1

89

19% of

GFP+

0.328

0.079

0.133

0.4

60

Plin1b

TAG Plin1b

CE

TA

G

CE

B.

Figure 4B

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GFP-Plin1c GFP-Plin1c

8% of

GFP+

0.562

0.048

0.063

0.3

28

94% of

GFP+

0.041

0.639

0.274

0.0

46

Plin1c

TAG

Plin1c

CE

TA

G

CE

C.

Figure 4C

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GFP-Plin1d GFP-Plin1d

47% of

GFP+

0.207

0.191

0.299

0.3

03

57% of

GFP+

0.173

0.225 0

.27

3

Plin1d

TAG

Plin1d

CE

0.329

TA

G

CE

D.

Figure 4D

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GFP-Plin2 GFP-Plin2

65% of

GFP+

0.112

0.210

0.480

0.1

98

32% of

GFP+

0.225

0.108 0

.36

5

Plin2

TAG

Plin2

CE

0.302

TA

G

CE

A.

Figure 5A

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GFP-Plin3 GFP-Plin3

55% of

GFP+

0.141

0.171

0.383

0.3

05

43% of

GFP+

0.154

0.118 0

.31

8

Plin3

TAG Plin3

CE

0.410

TA

G

CE

B.

Figure 5B

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GFP-Plin4 GFP-Plin4

19% of

GFP+

0.301

0.072

0.4

39

84% of

GFP+

0.072

0.1

41

0.384

Plin4

TAG

Plin4

CE

0.403 0.189

TA

G

CE

C.

Figure 5C

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GFP-Plin5 GFP-Plin5

88% of

GFP+

0.051

0.356

0.441

0.1

52

13% of

GFP+

0.339

0.050

0.205

0.4

06

Plin5

TAG Plin5

CE

TA

G

CE

D.

Figure 5D

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Plin1a

Plin1b

Plin1c

Plin1d

Plin2

Plin3

Plin4

Plin5

TAG CE

Plin1a 93.0 2.4% 6.0 1.2%

Plin1b 82.6 6.7% 16.1 6.2%

Plin1c 9.5 2.3% 91.4 3.7%

Plin1d 46.2 3.9% 57.2 3.1%

Plin2 67.8 3.6% 34.5 3.0%

Plin3 57.2 3.3% 46.0 3.6%

Plin4 17.7 2.0% 81.9 2.4%

Plin5 87.9 1.5% 12.5 0.4%

GFP-Plin % TAG Distribution % CE Distribution

Figure 6

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No

rma

lize

d T

AG

/CE

0

0.5

1.0

1a 1c 5 4

Lipid Quantity

Figure 7

Plin

p<0.05 p<0.05

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