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. Author manuscript; available in PMC: 2015 Aug 4.
Published in final edited form as: Traffic. 2013 Jun 23;14(9):974–986. doi: 10.1111/tra.12087

A test of current models for the mechanism of milk-lipid droplet secretion

Jaekwang Jeong 1,*, Ivonne Lisinski 1,*, Anil KG Kadegowda 1, Hyunsu Shin 1, FB Peter Wooding 2, Brian R Daniels 3, Jerome Schaack 4, Ian H Mather 1,+
PMCID: PMC4524534  NIHMSID: NIHMS488394  PMID: 23738536

Abstract

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts, either with itself, or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced-green-fluorescent-protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5–0.7%, (w/w) of the total protein, i.e., over fifty-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets, mark it as a potential mobile signaling molecule in milk.

Keywords: Butyrophilin, Xanthine oxidoreductase, Perilipin-2, Milk-lipid secretion, Mouse, Lactation, Exocrine biology

Lipid droplets are secreted into milk by a unique mechanism. Droplets of triacylglycerol assembled within, or in cytoplasmic regions close to the membrane of the rough endoplasmic reticulum (rER), are transported through the cytoplasm to the cell apex and then progressively coated with cellular membranes by a budding process that terminates with the expulsion of the droplets into milk (Figure 1A) (1, 2). Thus unlike, e.g., the secretion of lipoproteins in liver, the droplets are assembled in the cytoplasm and bypass the classical secretory pathway, entirely. Evolution of the ability to secrete membrane-coated lipid droplets from the cytoplasm is one key event that was required for milk to replace egg yolk as the primary nutrient source for neonatal survival in mammals (3).

Figure 1. Proposed mechanisms of milk-lipid secretion.

Figure 1

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(A) Electron micrograph of a lipid droplet at the point of secretion (lactating guinea-pig mammary gland), Bar 1.0 μm. (B) Postulated topology of BTN, XOR, and PLIN2 in secreted lipid droplets. BTN is a type 1 glycoprotein with two Ig domains (IgI and IgC1, green rectangles) in the exoplasmic (exo) domain. The two N glycosylation sites discussed in the text are indicated by black triangles. The B30.2 domain (black rectangle) in the cytoplasmic (cyto) region binds to XOR (grey ball). PLIN2 (blue rectangle), a member of the PAT protein family, binds to the phospholipid monolayer (light grey) on the lipid droplet surface (orange). (C) Proteins of bovine MLGM separated in one dimension by SDS-polyacrylamide gel electrophoresis. The major protein bands of XOR, BTN and PLIN2 and their apparent Mrs are indicated. Note that most of the 66 kDa protein band comprises BTN, unlike mouse MLGM. (D) Current molecular models for the secretion of milk-lipid droplets. (i) BTN (red bars) in the apical plasma membrane (APM) self associates and binds to XOR (green triangles) to form dimers or aggregates of higher order, which bind to proteins (black dots) on the surface of cytoplasmic lipid droplets (LD), possibly including PLIN2 or XOR (1). Formation of this macromolecular complex drives the expulsion of lipid droplets from the cell. (ii) PLIN2 (blue dots) on the surface of cytoplasmic lipid droplets directly binds to the phospholipid bilayer of the apical plasma membrane through a four-helix bundle motif at the C terminus. Binding induces membrane curvature and BTN and XOR are subsequently recruited into the budding lipid droplet (16). (iii) BTN on the surface of cytoplasmic lipid droplets binds to BTN in the apical plasma membrane to form a network of adhesive molecules encircling the droplet. XOR, PLIN2, or other proteins are not required for this interaction (18).

Most current molecular models for the terminal steps in lipid droplet secretion are based on the presumption that proteins associated with the droplet surface interact with integral membrane proteins, thus driving apposition of the globule surface with the outer membrane bilayer (1, 2). Such interactions are assumed to give rise to a 10–20 nm thick protein coat (4–6) visible in electron micrographs on the cytoplasmic face of isolated membrane stripped from the core lipid [called here the milk-lipid-globule membrane (MLGM)] (1). In many species, major coat constituents include the integral protein butyrophilin 1A1 (BTN) (7, 8), and the peripheral proteins, xanthine oxidoreductase (XOR) (9) and perilipin-2 [PLIN2, also known as adipose differentiation-related protein, or adipophilin (10, 11))] (Figure 1B,C) (12).

Functional evidence that BTN and XOR are required for milk-lipid secretion is provided by analysis of several mutant mouse lines. Thus, lipid secretion in Btn1a1−/− mice (13) and one strain of Xdh+/− mouse (14) is severely compromised, leading to the accumulation of large droplets of triacylglycerols in the cytoplasm, and unstable masses of aggregated fat in luminal spaces. In addition, ablation of the mouse gene Cidea, which encodes a transcriptional coactivator of the Xdh gene, is deleterious to milk-lipid secretion (15). In the case of PLIN2, expression of a mutant form lacking the C-terminus blocks lipid secretion in wild-type mice, presumably by acting as a dominant negative inhibitor (16).

Three distinct schemes have been proposed to explain the potential roles of these three proteins in milk-lipid secretion (Figure 1D). In the model of Mather and Keenan (1), BTN serves as a transmembrane scaffold, which binds to XOR (17) and other proteins on the lipid droplet surface, possibly including PLIN2 (Figure 1Di). In the model of Chong et al. (16), binding between the apical membrane and lipid droplet surface is initiated by PLIN2 and is then stabilized by the formation of a tripartite complex with BTN and XOR (Figure 1Dii). In the model of Robenek et al. (18) lipid secretion is solely driven by adhesive interactions between BTN in the plasma membrane and BTN on the lipid droplet surface (Figure 1Diii). Thus in this latter scheme, BTN, which is a type 1 integral membrane protein (7, 19), is presumed to stably associate with a phospholipid monolayer on the surface of triacylglycerol droplets with both extensive exoplasmic and cytoplasmic domains facing the cytoplasm.

We tested a series of predictions, which arise from all three models using a combination of biochemical and morphological approaches. The results are not compatible with any of these hypotheses and suggest that interactions between MLGM proteins are more dynamic and transient than previously proposed.

Results

Experimental predictions

The models of Mather and Keenan (1), Robenek et al. (18), and Chong et al. (16) all predict that BTN is a major immobile component of an extensive network of adhesive molecules encircling the secreted droplets in all mammals (Figure 1D, Table 1). As a consequence, the abundance of BTN is presumed to give rise to the extensive protein coat identified in transmission electron micrographs as a dense amorphous layer on the cytoplasmic face of the MLGM. In the model of Robenek et al. this coat will arise from both BTN in the apical plasma membrane and BTN on the surface of lipid droplets prior to secretion. This droplet-associated BTN is assumed to derive from sites of synthesis in the rER, and to be carried with the lipid droplets through the cytoplasm to the cell apex, at which point it self associates with BTN in the plasma membrane. If this is the case, a substantial fraction of the BTN molecules associated with the secreted lipid should contain immature, high mannose N-linked glycans, which have not been processed though the Golgi complex and therefore remain sensitive to cleavage by endoglycosidase-H (endo-H) (20). In the models of Mather and Keenan, and Chong et al., all the BTN is assumed to be routed through the Golgi complex and targeted to the apical plasma membrane as a fully processed, type 1 glycoprotein, which should be resistant to endo-H digestion. As noted above, all three models predict that BTN should be immobile in the plane of the outer lipid bilayer of the MLGM and be an abundant membrane constituent that gives rise to an extensive MLGM-associated protein coat. Experiments were designed to test each of these predictions (Table 1).

Table 1.

Predictions from current models for milk-lipid secretion1

Test Prediction
Chong et al.(16, 35) Mather and Keenan (1) Robenek et al. (18)
Distribution of BTN mainly apical PM mainly apical PM intracellular LDs/apical PM
BTN N-glycans endoH resistant endoH resistant endoH sensitive/ resistant (approx. 4:1)
BTN mobility immobile immobile immobile
BTN amount abundant abundant abundant
MLGM coat present present present
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1

Based on the assumption that the mechanism of milk-lipid secretion is the same in all mammals.

Is BTN a bona fide constituent of the surface of intracellular lipid droplets?

We first determined whether BTN associates with the surface of lipid droplets, in either cultured cells, or lactating mammary gland using morphological methods, which as far as possible did not compromise the intracellular localization of proteins, or the integrity of the lipid droplets. Mouse BTN was fused to either enhanced green fluorescent protein (EGFP) or an appropriate analogue at either the N- or C- terminus and expressed in cell lines, which had been induced to assemble cytoplasmic lipid droplets by adding oleic acid to the medium. Lipid droplets were identified by either co- expressing fluorescent forms of PLIN2, or by staining with nile red or BODIPY 665. In all cell lines examined, the fluorescent BTN was targeted to the plasma membrane and accumulated at intracellular sites in punctate structures, which aggregated together (Figures 2,3). We were unable to detect BTN on intracellular lipid droplets, either as a bona fide constituent on the droplet surface, or in association with aggregated forms of BTN (examples for bovine mammary MAC-T cells and human HEK 293T cells, Figure 2). Similar results were obtained with 3T3 cells, which had been converted into 3T3L1 adipocytes (data not shown), and HC 11 cells, a mouse mammary cell line, which had been induced to differentiate with lactogenic hormones (Figure 3). A detailed three-dimensional analysis of such HC 11 cells showed no significant co-localization of BTN-EGFP and BODIPY 665-stained lipid droplets in the interior of the cell (Figure 3A–F). However, larger droplets towards the cell margin were coated with BTN in regions that were in direct contact with the plasma membrane (arrowheads, Figure 3A,B,E,G). Thus, as predicted from the models of Mather and Keenan (1) and Chong et al. (16), BTN only associates with lipid droplets at the cell surface (Figure 1Di,ii).

Figure 2. Distribution of BTN in established cell lines.

Figure 2

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Cells were co-transfected with pECFP-BTN, and pPLIN2-EYFP and the unfixed cells examined by microscopy. (A–C) Epifluorescent images of bovine MAC-T cells. (D–F) Confocal images of HEK 293T cells, which were treated with digitonin to remove excess cytoplasmic PLIN2-EYFP. Note that in each case, ECFP-BTN (green) is targeted to the plasma membrane (arrowheads) and intracellular aggregates (arrows), but does not associate with lipid droplets (red), which were identified by PLIN2-EYFP. Bars 10 μm.

Figure 3. Distribution of BTN in HC 11 cells.

Figure 3

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HC 11 cells were treated with insulin, dexamethasone and prolactin to induce XOR expression, and then transduced with Adv-BTN-EGFP. Cultures were subsequently stained with BODIPY 665 to localize lipid droplets and fixed with 4% (w/v) paraformaldehyde. Three-dimensional images of cells were constructed using Imaris software from serial confocal sections. (A,B) Example from a stack of 25 0.32 μm confocal sections. (A) BTN-EGFP; (B) BODIPY 665; (C–E) Confocal section 1.3 μm from the base of the cell, showing separate, (C) green (BTN-EGFP), and (D) red (BODIPY 665) channels in grey scale, and (E) a colored merged image; (F,G) y/z sections 1 and 2, as indicated in (E) showing separate green (G) and red (R) channels in grey scale, and colored merged images (M). Note BTN associates with large lipid droplets in the periphery of the cell (arrowheads in A,B,E,G) (section 2) but not with smaller droplets towards the center of the cell (section 1). Bars 5 μm.

To confirm that BTN is not a bona fide surface component of intracellular lipid droplets in mammary tissue in vivo, BTN-EYFP was expressed in the mammary glands of CD1 mice using an adenoviral vector. Confocal microscopy of transduced tissue showed that BTN-EYFP was targeted to the apical plasma membrane, but did not associate with intracellular lipid droplets, identified by staining with nile red (Figure 4A–D). In addition, endogenous BTN was localized by immunocytochemistry in paraformaldehyde-fixed frozen sections of lactating mammary tissue (Figure 4E,F). BTN was detected on apical surfaces (Figure 4E) but was undetectable on intracellular lipid droplets, separately identified with an antibody to PLIN2 (Figure 4F). Because of the difficulty of stabilizing lipid droplets in frozen sections, we also analyzed formaldehyde/glutaraldehyde-fixed, Araldite-embedded lactating mammary tissue from several species. Resin was subsequently cleared from the tissue sections with sodium ethoxide. This unconventional method provides morphologically well-preserved tissue, which retains reactivity towards many antibodies and does not affect the expected distribution of organelle-specific antigen markers, e.g., for PLIN2 (lipid droplets) (Figure 4H) and MUC 1 (apical plasma membrane) (21). Using this method, BTN was detected on the apical plasma membrane of mammary epithelial cells in a number species, including cow (Figure 4G), guinea pig (Figure S1A, Supporting Information) and sheep (Figure S1C), but not on intracellular lipid droplets (Figures, 4G,H, S1).

Figure 4. Distribution of BTN in lactating mammary gland.

Figure 4

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(A–D) Mouse mammary gland transduced in vivo with Adv-BTN-EYFP, fixed in 4% paraformaldehyde and frozen in O.C.T. compound. (A) merged image of BTN-EYFP (green), nile-red stained lipid droplets (red), and DAPI-stained nuclei (blue); (B) BTN-EYFP (green), (C) nile-red stained lipid (red); (D) merged image of B and C; (E,F) Frozen sections of lactating mouse mammary gland labeled with (E) anti-peptide antibody to mouse BTN, and (F) mouse PLIN2, followed in both cases with goat anti-(rabbit IgG)-Alexa Fluor™-488. (G,H) Resin-embedded lactating bovine mammary gland sections labeled with anti-peptide antibody to (G) BTN, or (H) PLIN2, followed in each case with goat anti-(rabbit IgG)-colloidal gold (4 nm), and enhanced with silver. The same two alveoli in adjacent sections in G and H are labeled X and Y. Note in E and G that BTN is largely associated with the apical plasma membrane (single arrowheads), compared with PLIN2 in F and H which associates with cytoplasmic lipid droplets (CLD) (arrows). AL, alveolar lumen; N, nucleus; Bars (A–D) 5 μm; (E–H) 25 μm.

In mouse, BTN was concentrated around budding lipid droplets at the apical plasma membrane and on secreted droplets (Figure S2, Supporting Information). Very little BTN was detected in regions of the apical membrane not in contact with lipid droplets, unlike the staining pattern seen with paraformaldehyde-fixed frozen sections (Figure 4E,F). This apparent discrepancy most likely reflects differences in the accessibility and relative abundance of BTN epitopes in frozen versus plastic-embedded tissue, fixed with both paraformaldehyde and glutaraldehyde.

Are the N-linked glycans of BTN cleaved with endo-H?

The dearth of BTN on intracellular lipid droplets compared to budding or secreted milk-lipid droplets was corroborated by an alternative biochemical approach. As indicated above, if a large fraction of BTN is targeted to nascent lipid droplets in the rER (18), then the majority of BTN molecules associated with secreted lipid should not have been processed through the Golgi complex and acquired complex-type oligosaccharide chains (22). Thus, the two N-linked glycans in the exoplasmic domain should remain sensitive to endo-H, which cleaves the β1–4 linkage within the chitobiose sequence of high mannose chains (20). In contrast, glycans associated with BTN that is targeted to the plasma membrane should become resistant to digestion with endo-H, provided they acquire terminal sugars, such as galactose and neuraminic acid in the trans- Golgi and trans- Golgi network. We tested these possibilities by digesting milk-lipid droplets and MLGM with endo-H to determine the relative amounts of complex or hybrid, to high-mannose glycans associated with BTN in secreted lipid droplets. For reference purposes, the same samples were digested with N-glycanase, an enzyme which completely removes both complex and high mannose glycans from N-linked glycoproteins (23).

On digestion with endo-H, close to 40% of BTN in bovine milk-lipid droplets or MLGM was cleaved into fragments with Mrs that were intermediate between untreated BTN and BTN that was fully deglycosylated by treatment with N-glycanase (Table 2, Figure 5). In no case were we able to completely remove the bulk of the sugar from even a fraction of the total BTN (at least five samples each, tested from different animals), strongly suggesting that most BTN molecules in bovine milk-lipid droplets and MLGM contain a mixture of complex- with some hybrid-glycans. The BTN in lipid droplets from either CD1, or C57/Bl6 mouse milk was even more resistant to digestion; the partially digested protein, i.e., with one remaining glycan, comprised about 20% of the total and no completely deglycosylated protein was detected (Table 2, Figure 5). Control experiments verified that the batch of endo-H used for these experiments was fully active as it completely deglycosylated pancreatic ribonuclease B within 4h (data not shown). Thus, from these endo-H digestion experiments (Table 2, Figure 5), the cell expression data and immunocytochemical localizations (Figures 2– 4, S1, S2), we conclude that BTN has little propensity to directly associate with lipid-droplet surfaces in situ, certainly not at the levels suggested by Robenek et al. (18), i.e., 3.7 times higher than in the plasma membrane.

Table 2.

Digestion of proteins in the MLGM, and milk-lipid droplets with endoglycosidase H

Species Sample Digestion with endoglycosidase-H after 4 h (%)1
Intact Partially digested Completely digested
Holstein cow
Droplets 58.34 +/− 1.91 39.14 +/− 0.92 2.51 +/− 1.51
MLGM 60.62 +/− 4.48 37.82 +/− 4.41 1.56 +/− 0.20
Mouse
CD1 Droplets 81.64 +/− 1.94 17.86 +/− 2.04 0.49 +/− 0.51
C57/Bl6 Droplets 77.91 +/− 0.66 21.85 +/− 0.77 0.24 +/− 0.24
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1

Mean and standard deviation (n = 5, in each case)

Figure 5. Digestion of mouse or bovine MLGM proteins with endo-H, or N- glycanase.

Figure 5

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Lipid droplets or MLGM samples were digested with either endo-H, or N-glycanase and the samples analyzed by immunoblotting after SDS-PAGE. (A) bovine lipid droplets; (B) bovine MLGM; (C) CD1 lipid droplets; (D) C57/Bl6 lipid droplets. Individual samples from five animals digested with endo-H are shown for each species or strain to the left, and time courses for the digestion of single samples with N-glycanase analyzed on the same blot are to the right as indicated. Densitometric analysis of the blots in (A–D) is shown in Table 2.

Is BTN mobile on the surface of secreted lipid droplets?

We next tested the prediction from all three models that BTN should be fixed in an immobile complex in the MLGM, if it forms an interlocking network of adhesive molecules, by using fluorescence recovery after photobleaching (FRAP) analysis to measure the mobility of BTN in secreted lipid droplets. BTN-EGFP was expressed in lactating mammary gland by adenoviral transduction and the unfixed lipid droplets in the collected milk examined by confocal microscopy (Figure 6). Distribution of the fluorescent fusion proteins on the droplet surfaces was strikingly heterogeneous, as previously noted for other MFGM constituents using fluorescent-lectin conjugates and lipophilic probes (24, 25). Although some droplets were surrounded by an even layer of BTN-EGFP (Figure 6A, yellow arrowheads), many droplets displayed areas of condensed fluorescent material in confocal optical sections (Figure 6A, yellow arrows).

Figure 6. FRAP analysis of the mobility of BTN-EGFP in milk-lipid droplets and HC 11 cells.

Figure 6

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(A) Unfixed milk-lipid droplets containing BTN-EGFP showing examples of droplets with a “continuous” membrane (yellow arrowheads), and “condensed” membrane (yellow arrows); (B,C) Examples of FRAP analysis of the mobility of BTNEGFP in lipid droplets showing time-dependent recovery of fluorescence in bleached regions in, (B) a “continuous” area of membrane, and, (C) a “condensed” area of membrane; (D) Example of FRAP analysis of the mobility of BTNEGFP in the plasma membrane of HC 11 cells; (E) Summary of FRAP analysis of the mobility of BTN-EGFP in lipid droplets and HC 11 cell plasma membrane, as indicated. Numbers to the right of the graph indicate the number of estimates in each case. Bleached areas in B–D are indicated by dotted circles before photobleaching and arrowheads after photobleaching; Bars, (A) 50 μm, (B–D) 5 μm.

These condensed regions have previously been identified by electron microscopy and arise in the alveolar lumen after secretion (5). Areas of the droplets were divided into condensed regions (Figure 6A, yellow arrows), or continuous areas (Figure 6A, yellow arrowheads) and the mobility of BTN-EGFP in each area determined by FRAP analysis (Figure 6B,C,E). Diffusion coefficients for BTN-EGFP in the “continuous” areas were in the range, 0.012 +/− 0.002 μm2/sec, and essentially all of the molecules were mobile (mobile fraction R of 101 +/− 4%) (Table 3, Figure 6E). In contrast, only about 20% of the BTN-EGFP molecules in the condensed regions were mobile and the diffusion coefficients were approx. half (0.005 +/− 0.002 μm2/sec) (Table 3, Figure 6E).

Table 3.

Mobility of BTN in different cellular contexts determined by fluorescence recovery after photobleaching (FRAP) analysis

Location Mobile fraction (R) (%)1 Diffusion coefficient (D) (μm2/s)1
MLGM:
“Continuous” 101 +/− 4a 0.012 +/− 0.002c
“Condensed” 19 +/− 7b 0.005 +/− 0.002d
Plasma membrane:
HC 11 cells 108 +/− 7a 0.006 +/− 0.002d
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1

Mean and standard deviation in each case.

Data in columns with different superscript letters are significantly different;

a and b

p < 0.001;

c and d

p < 0.05.

For reference purposes, the mobility of BTN-EGFP was measured in the plasma membrane of mouse HC 11 cells. Essentially all of the molecules were mobile (R of 108 +/− 7 %) with diffusion coefficients about half the values for BTN-EGFP in the continuous regions of lipid droplets (0.006 +/− 0.002 μm2/sec) (Table 3, Figure 6D,E). In toto, these data and previous ultrastructural studies (5) show that formation of the MLGM at the time of secretion is followed by global changes in the structure of the membrane and that BTN is mobile in the MLGM until the membrane rearranges and thickens on the droplet surface. With some caveats (discussed below), these results are difficult to reconcile with any of the current models for milk-lipid secretion.

Is BTN an abundant protein in mouse MLGM?

All three models predict that BTN should be an abundant constituent of the MLGM in all mammals. Therefore, we measured the amount of BTN in mouse MLGM by quantitative immunoblot of one-dimensional SDS gels, to compare with other species, e.g., cow, in which BTN is a major constituent, viz, 20–40% of total membrane protein (Figure 1C) (26, 27). In sharp contrast, the amount of BTN in the milk-lipid droplet fraction of C57/Bl6 and CD1 mice was only 0.5–0.7% of total protein (5.65 +/− 1.31 and 6.63 +/− 0.81 ng/μg protein, respectively, for duplicate determinations from four mice, each). Qualitative comparison of the MLGM protein compositions of C57/Bl6 Btn1a1+/+ and Btn1a1−/− mice by two-dimensional gel electrophoresis and immunoblot (Figure 7A,B), confirmed that the isoelectric variants of mouse BTN constitute a minor fraction of the total membrane protein and that a substantial amount of the one-dimensional band of 66-kDa protein, previously assumed to be BTN comprises glycosylated variants of milk-fat-globule epidermal-growth-factor 8 (Mfg-e8; lactadherin). These data cannot be explained by contamination of the cream samples with skim milk proteins and cytoplasmic material entrained with the lipid droplets, as the samples contained very little casein (determined by inspection of two-dimensional gels, data not shown) and only 4.65 +/− 2.24% of the droplets in C57/Bl6 mouse milk contained extraneous material, which was identified by staining the droplets with acridine orange (7,028 droplets counted; total of 6 mice).

Figure 7. Comparison of the protein composition of milk-lipid droplets from Btn+/+ and Btn−/− mice by oneand two-dimensional gel electrophoresis and immunoblot.

Figure 7

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Separation of the proteins associated with unfractionated lipid droplets from, A (a–c) Btn+/+, and B (d–f) Btn−/− mice. Gels were stained with Coomassie blue. The isoelectric focusing (IEF) pH gradients increase from left to right, and Mrs of proteins separated by SDS-PAGE decrease from top to bottom. One– dimensional SDS gels are shown to the left of each figure and the 66 kDa protein band containing BTN is indicated with an asterisk (*). Detailed analyses of the 66 kDa band by two-dimensional gel electrophoresis and immunoblot are shown in (a–c) for Btn+/+ mice and (d–f) for Btn−/− mice. (a,d) The 66 kDa region stained with Coomassie blue (CB). (b,e) Immunoblots of the same regions in a,d stained with anti-peptide antibody to mouse BTN. (c,f) Immunoblots of the same regions in a and d stained with a commercial antibody to Mfg-e8. Note that the BTN variants, indicated by a “fence” in a,b, are minor components of this Mr region of the gel and that variants of Mfg-e8, indicated by a bracket in a,c,d,f are major components.

Does mouse MLGM have an extensive protein coat?

Since BTN is assumed to be one of the major constituents of the membrane- associated protein coat located on the cytoplasmic face of the membrane (8, 28, 29), we examined isolated mouse MLGM by transmission electron microscopy for possible differences in the quantity, or morphology of the coat. Typically, preparations of isolated bovine MLGM form homogeneous sheets of membrane, which are unable to form vesicles because of the large amount of coat protein, which can be removed by digestion with proteinase [see refs (5, 30, 31) for examples from goat, cow and human]. In contrast, mouse MLGM was strikingly heterogeneous, comprising a mixture of membrane-bounded vesicles and sheets, which generally maintain a similar morphology after digestion with papain (Figure 8A–E). MLGM from Btn1a1−/− mice was similar (Figure 8F). Thus, mouse MLGM lacks sufficient BTN and its binding partner, XOR (17, 32) to form a substantial protein coat.

Figure 8. Analysis of the morphology of mouse MLGM by electron microscopy.

Figure 8

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Mouse MLGM samples were prepared for standard transmission electron microscopy. (A) Survey micrograph at low magnification; (B–D) Examples of the heterogeneous nature of mouse MLGM at higher magnification. Note that the mouse membrane lacks the extensive protein coat seen on the cytoplasmic face of bovine MLGM (6), and that the membrane forms sheets in B, and numerous vesicles in C,D, some of which contain electron dense material (example in D); (E) MLGM after treatment with papain. Note that the morphology is similar to untreated membrane (compare C and E), unlike bovine MLGM, which is depleted of the associated protein coat after treatment with protease [see Figure 2 of ref (30) for an example]; (F) MLGM of Btn−/− mice is similar to that of wild-type mice. Bars, (A) 1 μm; (B–F) 200 nm.

Discussion

The data described provide novel insights and challenge current molecular models for the process of milk-lipid secretion. Regarding the model of Robenek et al. (18), fluorescently tagged forms of BTN were not detectable on the surface of intracellular lipid droplets in cultured cells (Figures 2,3), or in secretory epithelial cells in the lactating mouse mammary gland (Figure 4A–D), whether the fluorescent reporter was placed at either the N-, or C- terminus. Across species, endogenous BTN in lactating mammary gland was not detected on intracellular lipid droplets, which were separately identified by immunoreactivity to PLIN2 (Figures 4E–H, S1, S2). BTN was only associated with lipid droplets at the plasma membrane, either in cultured cells (Figure 3), or on the apical surfaces of mammary epithelial cells and secreted lipid droplets (Figures 4E–H, S1, S2). At least one of the two N-linked glycans associated with either mouse or bovine BTN appear to be of the hybrid-, or complex- type and to thus have been processed through the secretory pathway, a conclusion in agreement with the chemical analysis of glycans associated with bovine BTN, many of which contain Gal and GlcNAc in the outer termini (33) Thus, BTN is not a bona fide component of the phospholipid monolayer on the surface of cytoplasmic lipid droplets, certainly not at the levels required to form extensive networks with BTN in the outer membrane bilayer, or at the levels suggested by Robenek et al. (18), i.e., approx. 3.7 times higher than in the plasma membrane. These data do not exclude the possibility that small vesicles containing BTN as an integral protein may transiently associate with intracellular droplets, as has been observed for MUC1-containing vesicles close to the apical surface (21).

The possibility that BTN is locked in an extensive macromolecular complex (a posit of all three models), is difficult to reconcile with the FRAP data. BTN-EGFP was freely mobile over extensive regions of the droplet surface with diffusion coefficients (0.012 +/− 0.002 μm2/s at 37 °C; Table 3), in the same range as fluorescently labeled “non-raft” transmembrane proteins in the apical surfaces of MDCK or Caco-2 cells (0.007 to 0.021 μm2/s for two constructs at 25 °C, or 37 °C; (34)). The rate of diffusion of BTN-EGFP in HC11 cells (0.006 +/− 0.002; Table 3) was about half the MLGM value, possibly because of interactions between BTN-EGFP and the underlying cytoskeleton, or with endogenous plasma membrane proteins.

There is no reason to suspect that the fluorescently-tagged proteins behaved differently from native BTN, as they were correctly targeted to the apical surface and incorporated into secreted lipid droplets. Immobile BTN-EGFP molecules were confined to the thickened regions of membrane, which form on the surface of the droplets after secretion in the alveolar lumina (4, 5). Thus, these thickened regions cannot be the sites for either interactions between BTN, XOR, and PLIN2 (1, 16, 35), or BTN molecules alone (18), that are proposed to occur at the time of secretion.

The most compelling functional evidence that BTN and its binding partner XOR, play a role in milk-lipid secretion is provided by analysis of the Btn1a1−/− knock-out mouse and one of two Xdh+/− mouse strains, in which lipid secretion is disrupted (13, 14), and the fact that the two proteins interact with high affinity and in a pH- and salt-sensitive manner (17). Furthermore, when soluble forms of the cytoplasmic domain of BTN are expressed in wild-type mice using adenoviral vectors, the interaction between BTN and XOR is disrupted in a dominant negative manner and large lipid droplets accumulate in the cytoplasm, i.e., lipid droplet secretion is inhibited (Jeong, J. and Mather, I.H., unpublished observations). The potential importance of BTN in lipid secretion is also highlighted by its accumulation around budding lipid droplets at the apical surface even in species such as mouse (Figure S2), which express relatively low amounts of BTN (Figure 7).

These disparate data can be reconciled, if it is assumed that BTN, processed through the secretory pathway and inserted into the apical plasma membrane, binds to XOR to form a transient secretion complex, which drives formation of the MLGM at the physiological pH of the cytoplasm. As the droplets emerge from the cell, BTN may partially disengage from XOR because the pH of milk is acidic [mean of 6.32 in the case of goat's milk (36)] and BTN binds less tightly to XOR below pH 7.0 (17). This would explain the presence of soluble and membrane-bound forms of XOR previously characterized in milk-lipid droplets and the MLGM (9, 37), and the separate distributions of BTN and XOR in immunolabeled freeze-fracture micrographs (18). Following secretion, the external oxidizing environment of luminal spaces and the presence of sulfhydryl oxidase in milk (38) may allow a fraction of BTN to form the disulfide-bonded complexes previously described between itself and other proteins in the MLGM (35) and thus generate the condensed regions identified on the droplet surface by both live cell imaging (Figure 6) and electron microscopy (5). An underlying assumption of this more dynamic model is that there is sufficient BTN, even in mouse, to initiate interactions between the lipid droplet and outer membrane bilayer; interactions, which could be augmented by the direct binding of the C-terminus of PLIN2 to phospholipids on the cytoplasmic face of the apical plasma membrane as proposed by Chong et al. (16). An alternative possibility is that BTN and XOR are constituents of a signaling complex, in which case BTN would not be required in large quantities to be physiologically functional (discussed in ref (17)).

Outside the context of milk secretion, members of the BTN family, including BTN1A1 (39) function as co-repressors of T-cell activation (40–42) and intracellular lipid droplets play important roles in cellular immunity (43). Our observations that a significant fraction of BTN molecules are mobile on the surface of milk-lipid droplets and that the MLGM is a dynamic structure, suggest an additional function for lipid droplets as regulators of gland immunity by engaging with immune cells in milk (44), together with milk exosomes (45) or vesicles shed from secreted milk-lipid droplets (5).

Materials and Methods

Materials

Electrophoresis and immunoblotting supplies were from Bio-Rad (Hercules, CA). Goat anti-mouse Mfg-e8 antibody was from R&D Systems (Minneapolis, MN) and goat anti-human PLIN2 (C-terminus) antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-peptide antibodies to mouse and bovine BTN have been described previously (7, 13). Endo-H was from New England Biolabs (Ipswich, MA) and N-glycanase from Glyko/ProZyme (San Leandro, CA). Oleic acid was from CalBiochem (San Diego, CA) and bovine insulin, 3-isobutyl-1-methylxanthine and dexamethasone from Sigma (St. Louis, MI). MAC-T and HC 11 cells were gifts from Drs. Juan Loor (University of Illinois) and Bernd Groner (Georg-Speyer-Haus, Frankfurt, F.R.G.), respectively. Cell culture media were from Gibco/Invitrogen and fetal bovine serum (FBS) from Atlanta Biologicals (Lawrenceville, GA). All other reagents were of AR grade from standard supply houses.

Animal Care

Mice were maintained on Formulab Diet 5008 (PMI Nutrition International, Richmond, IN) and water ad libitum. Maintenance and experimental procedures with animals had the prior approval of the Institute's Animal Care and Use Committee, University of Maryland, College Park.

Collection and Fractionation of Milk

Milk was collected from C57/Bl6 and CD1 mice and separated into cream and skim milk fractions as described (13). MLGM samples were prepared by subjecting the cream fractions to three alternating freeze-thaw cycles at −80°C and room temperature in PBS (10 mM NaH2PO4/NaOH and 150 mM NaCl, pH 7.2) and the membrane collected by centrifugation at 100,000 × g for 1h. The membrane was washed once by re-suspending the pellet in 50-mM Na citrate, pH 7.0 and re-centrifuging at 100,000 × g for 1h. Bovine milk samples (10 l) were collected during the regular morning milking from individual cows at peak lactation in the dairy herd at the U.S.D.A. Beltsville Agricultural Research Center (Beltsville, MD) and washed cream and MLGM fractions prepared by standard procedures (46).

Production of Expression Plasmids and Adenoviral Vectors

Construction of expression plasmids is described in Jeong et al. (17). Recombinant adenovirus was generated from pShuttle-CMV vectors by homologous recombination in E. coli using the AdEasy™ system (Quantum Biotechnologies) (47), as modified by Orlicky and Schaack (48).

Cell Culture and Protein Expression

MAC-T cells (49) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% (wt/vol) FBS, 4.5% (wt/vol) D-glucose, 5.0 μg/ml bovine insulin, and L-glutamine, penicillin and streptomycin [1% (wt/vol) each]. HEK 293T cells were grown under similar conditions, without insulin. HC 11 cells were maintained in 10% bovine calf serum in RPMI 1640 with 10 ng/ml insulin, 10 ng/ml epidermal growth factor and 2 mM L-glutamine and the medium replaced once every 2 days. Cells were split 1:6 when at or near confluency. After 2 days of growth in EGF-containing medium (10 ng/ml), cells were treated with 5 μg/ml insulin, 1 μM dexamethasone, and 5 μg/ml prolactin to induce differentiation and the expression of XOR.

For protein expression, MAC-T cells were cotransfected with 0.15 μg each, pECFP-BTN and pPLIN2-EYFP. Lipid droplet formation was induced by adding 25 μg/ml oleic acid and the unfixed cells examined by epifluorescence microscopy 24 h later. HEK 293 cells were treated similarly, except that 50 μg/ml oleic acid was used. HC 11 cells were treated with insulin, dexamethasone and prolactin, as above, together with 14 μg/ml oleic acid. Cells were transduced with Adv-BTN-EGFP (106 pfu) 4–6 h later. After 24–28 h, cells were fixed with 4%(w/v) paraformaldehyde for 15 min and stained with BODIPY 665 (10 μM).

Transduction of Mammary Tissue with Adenoviral Vectors

To express fluorescent fusion proteins in vivo, recombinant adenoviral vectors were infused via the nipple into the mammary glands of C57/Bl6 mice on the 18th day of pregnancy using glass micropipettes, essentially as described by Russell et al. (50). The mice were milked on days 3 to 5 of lactation and then asphyxiated by immersion in CO2. Samples of milk were spotted directly onto microscope slides, sealed under coverslips and used directly for FRAP analysis. Pieces of lactating mammary gland were fixed overnight in 4% (wt/vol) paraformaldehyde and then flash frozen in isopentane held in liquid nitrogen. Frozen sections (10 μm) were placed on microscope slides, stained with nile red (1 μg/ml) and mounted in Vectashield mounting medium containing DAPI (1.5 μg/ml) (Vector Laboratories, Burlingame, CA) and examined by confocal microscopy.

Fluorescence Microscopy

Cells, tissues or milk-lipid droplets were examined by epifluorescence or confocal microscopy. Epifluorescence micrographs in Figure 2 A–C were obtained as described by Jeong et al. (17). For the confocal micrographs in Figures 2 D–F and 4 A–F, DAPI was excited at 405 nm and the emission recorded at 411–431 nm, ECFP was excited at 458 nm and the emission recorded at 470–480 nm, Alexa Fluor™-488 was excited at 488 nm and the emission recorded at 502–521 nm, EYFP was excited at 512 nm and the emission recorded at 525–540 nm, and nile red was excited at 540 nm and the emission recorded at 590–600 nm, using a Leica TCS SP5-X Laser scanning confocal microscope. For the confocal micrographs in Figure 3, excitation wavelengths of 488 nm and 633 nm, and emission wavelengths of 505–605 nm, and 655–755 nm were used for EGFP and BODIPY 665, respectively. Optical sections (0.32 μm) through a depth of 8 μm were recorded with an Olympus Fluoview 1000 confocal microscope (Olympus America, Center Valley, PA) and three-dimensional images reconstructed and analyzed with Imaris software (Bitplane AG, Zurich, Switzerland).

For FRAP analysis, milk-lipid droplets containing expressed BTN-EGFP were placed on coverslips, and kept at 37 °C. A circular area (2 μm diameter) on the surface of the droplet was photobleached at 488 nm using the maximum power of an Argon laser in a Leica TCS SP5-X Laser scanning confocal microscope. Three images were acquired at 1 sec intervals before photobleaching. Fluorescence recovery was monitored between 510–525 nm at low laser intensity for 160 sec. FRAP experiments were performed on at least 25 milk-lipid droplets from 3 mice, and data were averaged to generate a single FRAP curve, following the procedures of Soumpasis (51) [see Daniels et al. (52) for examples]. For FRAP of HC 11 cells, a total of 12 determinations were made on cells from four separate cultures.

Immunohistology

Tissues were fixed by perfusion with formaldehyde and/or glutaraldehyde and embedded in Araldite. Semi-thin sections were deresinated before immunocytochemistry with immunogold, which was enhanced with silver as detailed in Wooding et al. (53). Immunocytochemical controls, in which the primary antibodies were omitted were consistently negative.

Electron Microscopy

Samples of mouse MLGM were fixed simultaneously with 2.5% (wt/vol) glutaraldehyde and 2% (wt/vol) OsO4 (54), dehydrated in acetone and embedded in either Epon or EMbed 812. Sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM10CA electron microscope.

Electrophoresis and Immunoblotting Techniques

Proteins were separated by one- and two-dimensional electrophoresis, essentially as described (55–57). For two-dimensional gel electrophoresis, milk-lipid droplet fractions were dissolved in a lysis buffer containing 9.8M urea, 2% (w/v) ampholines (BioRad), 4% NP-40, and 100 mM dithiothreitol, and the solutions clarified by centrifugation at 15,000 × g for 15 min. Western blots were developed by enhanced chemiluminescence (GE Healthcare) using appropriate secondary antibodies conjugated to horse-radish peroxidase as detecting agents.

Digestion with endo-H and N-glycanase

Milk-lipid droplets and MLGM samples (30 μg protein, each) were digested with 10 mU of endo-H in 30 μl of 50 mM sodium acetate buffer, pH 5.5 containing 0.1% (wt/vol) SDS, 1% (vol/vol) NP-40, 25 mM EDTA, 100 mM β-mercaptoethanol and 0.10% (wt/vol) NaN3 at 37°C. For N-glycanase digestions, samples (30 μg protein, each) were digested with 10 mU of enzyme in 30 μl of 100 mM NaH2PO4/NaOH buffer, pH 7.5 containing 0.1% (wt/vol) SDS, 0.75% (vol/vol) NP-40, 50 mM β-mercaptoethanol and 0.1% (wt/vol) NaN3 at 37 °C.

Assays

The amount of mouse BTN in milk-lipid droplets was determined by quantitative densitometry of immunoblots using a fusion protein of GST and the cytoplasmic domain of mouse BTN (GST-BTNcyto) (17) as a standard. GST-BTNcyto served as an appropriate standard because the entire C-terminal epitope used to generate the anti-peptide antibody (13) was contained within the fusion protein. The 10,000-dalton difference between native mouse BTN and GST-BTNcyto was taken into account when calculating the absolute amounts of protein. Milk-lipid droplet samples were separated by SDS-PAGE in duplicate alongside the standards and blotted with antibody to mouse BTN (13). Chemiluminescent images were captured using the Bio-Rad ChemiDoc XRS system and immunoreactive bands quantified using Quantity One v4.5.2 software (Bio-Rad). Total protein was assayed with the bicinchoninic acid reagent (58) using bovine serum albumin as a standard.

Supplementary Material

Supp Fig S1-S2

Synopsis.

Molecular models for the secretion of milk- lipid droplets are based on the assumption that the integral protein, butyrophilin (BTN), interacts with proteins on the lipid droplet surface to form an immobile secretion complex. Using a combination of morphological and biochemical approaches, we show that in mouse milk-lipid droplets, BTN is a minor and highly mobile component on the droplet surface. A more dynamic model, in which BTN transiently interacts with xanthine oxidoreductase and other lipid-associated proteins is proposed.

Acknowledgements

We thank Tim Reardon and Liane West for animal care and maintenance and Dr. Dale Hailey (University of Washington, Seattle, WA) for Clontech vectors. Dr. Roberto Weigert (IMTU, NIDCR, National Institutes of Health, Bethesda MD) provided access to the Olympus confocal microscope and Imaris software. This work was funded by grants from the USDA NRI Program (2005-04637), NICHD-NIH (1R01 HD048588-01A1) and the Maryland Agricultural Experiment Station to IHM.

Footnotes

None of the authors have conflicts of interest to declare.

Supporting Information Additional Supporting Information may be found in the online version of this article.

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