Our existing understanding of the structure, protein organization and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. The electron microscopic technique of freeze-fracture replica immunogold labeling (FRIL) overcomes these problems, and is currently providing new perspectives in the field. Because of the property of frozen lipids to deflect the fracture plane, en face views of the lipid droplet and its component layers are revealed for high resolution visualization. By means of immunogold labeling, proteins involved in the accretion and mobilization of lipids, notably the PAT family proteins, can be localized at and in the droplet. Application of this approach demonstrates that, contrary to prevailing wisdom, the PAT family proteins are not invariably restricted to the surface of the lipid droplet but can occur throughout the core. The notion that lipid droplet biogenesis involves neutral lipid accumulation within the ER membrane bilayer followed by budding off, enclosed by a protein-containing phospholipid monolayer, is not substantiated. Instead, lipid droplets appear to develop externally to both ER membranes at specialized sites in which the ER enwraps the droplet, and the facing leaflets of the ER membrane and droplet surface are enriched in adipophilin. PAT family proteins are not, as often stated, specific to the lipid droplet, but are widely present in the plasma membrane where, under conditions of lipid loading, they adopt a similar configuration to that of specialized sites in the ER. FRIL has further provided new insights into the mechanism of secretion of a special type of lipid droplet, the milk fat globule. These examples highlight the contribution of the FRIL technique to critical appraisal and development of concepts in the lipid droplet field. (C) 2008 Elsevier B.V. All rights reserved.

Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.

Low density lipoprotein (LDL) is retained in the extracellular matrix of the arterial wall where it is considered to be atherogenic, but little is known about how cell adhesion to the matrix is affected by collagen-bound LDL. We tested the effect of native, oxidized and acetylated LDL reacted with adsorbed monomeric type I, III and V collagen on endothelial cell adhesion to collagen using a colorimetric adhesion assay. We found that none of the LDL species affected adhesion to type I and III collagen, but that collagen-bound native and acetylated LDL enhanced attachment to type V collagen, whereas bound oxidized LDL inhibited adhesion to this collagen. We therefore suggest that oxidized LDL associated with type V collagen in the arterial wall would favor de-endothelialization and contribute to atherogenesis and thrombosis.

Of all the techniques in freeze-fracture cytochemistry attempted and tested over the last three decades, the freeze-fracture replica immunolabeling technique (FRIL) [1], has proven to be the most successful, and is currently having a substantial impact in solving questions in cell biology that have hitherto been impossible to address with other ultrastructural, cell biological or molecular approaches. For FRIL the specimen is rapidly frozen, freeze-fractured and platinum/carbon replicas made following the standard protocol used for freeze-fracture electron microscopy. However, instead of removing the biological material from the replicas with bleach as in the standard technique, the replica is treated with sodium dodecylsulphate (SDS). The aim of SDS treatment is to dissociate most of the biological material from the replica so that structure is visible by electron microscopy, leaving just sufficient material adherent to the replica (ideally a single molecular layer) to retain epitopes for labeling. By applying immunogold techniques, the spatial distribution of the targeted epitopes can be viewed superimposed upon standard planar freeze-fracture views of the membrane interior.

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.

The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.

The prevailing hypothesis of lipid droplet biogenesis proposes that neutral lipids accumulate within the lipid bilayer of the ER membrane from where they are budded off, enclosed by a protein-bearing phospholipid monolayer originating from the cytoplasmic leaflet of the ER membrane. We have used a variety of methods to investigate the nature of the sites of ER-lipid-droplet association in order to gain new insights into the mechanism of lipid droplet formation and growth. The three-dimensional perspectives provided by freeze-fracture electron microscopy demonstrate unequivocally that at sites of close association, the lipid droplet is not situated within the ER membrane; rather, both ER membranes lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg cup (the ER) holding an egg (the lipid droplet). Freeze-fracture cytochemistry demonstrates that the PAT family protein adipophilin is concentrated in prominent clusters in the cytoplasmic leaflet of the ER membrane closely apposed to the lipid droplet envelope. We identify these structures as sites at which lipids and adipophilin are transferred from ER membranes to lipid droplets. These findings call for a re-evaluation of the prevailing hypothesis of lipid droplet biogenesis.

We studied the distribution of caveolin-1 in cell membranes and how caveolin-1 accesses lipid droplets from the endoplasmic reticulum (ER) in vascular smooth muscle cells (SMC) using freeze-fracture immunocytochemistry. We found caveolin-1 in the cytoplasmic leaflet of the plasma membrane, but surprisingly exclusively in the endoplasmic leaflet of all intracellular membranes. Furthermore, we localized caveolin-1 in notable amounts both on the surface and within the cores of lipid droplets.

The PAT family proteins, named after perilipin, adipophilin, and the tail-interacting protein of 47 kDa (TIP47), are implicated in intracellular lipid metabolism. They associate with lipid droplets, but how is completely unclear. From immunofluorescence studies, they are reported to be restricted to the outer membrane monolayer enveloping the lipid droplet and not to enter the core. Recently, we found another kind of lipid droplet-associated protein, caveolin-1, inside lipid droplets. Using freeze-fracture immunocytochemistry and electron microscopy, we now describe the distributions of perilipin and caveolin-1 and of adipophilin and TIP47 in lipid droplets of adipocytes and macrophages. All of these lipid droplet-associated proteins pervade the lipid droplet core and hence are not restricted to the droplet surface. Moreover, lipid droplets are surprisingly heterogeneous with respect to their complements and their distribution of lipid droplet-associated proteins. Whereas caveolin-1 is synthesized in the endoplasmic reticulum and is transferred to the lipid droplet core by inundating lipids during droplet budding, the PAT proteins, which are synthesized on free ribosomes in the cytoplasm, evidently target to the lipid droplet after it has formed. How the polar lipid droplet-associated proteins are accommodated among the essentially hydrophobic neutral lipids of the lipid droplet core remains to be determined.

Neutral lipid accumulation is frequently observed in some Gram-negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester- and triacylglycerol (TAG)-bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid-body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid-bodies starts with the docking of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid-body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane-associated enzyme. SLDs conglomerated subsequently to membrane-bound lipid-prebodies which are then released into the cytoplasm. The formation of matured lipid-bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half-unit membrane of phospholipids.

Proteins of the PAT family, named after perilipin, adipophilin, and TIP47 (tail-interacting protein of 47 kDa), are associated with lipid droplets and have previously been localized by immunofluorescence microscopy exclusively to the droplet surface. These proteins are considered not to be present in any other subcellular compartment. By applying the high resolution technique of freeze-fracture electron microscopy combined with immunogold labeling, we now demonstrate that in macrophages and adipocytes PAT family proteins are, first, distributed not only in the surface but also throughout the lipid droplet core and, second, are integral components of the plasma membrane. Under normal culture conditions these proteins are dispersed in the cytoplasmic leaflet of the plasma membrane. Stimulation of lipid droplet formation by incubation of the cells with acetylated low density lipoprotein leads to clustering of the PAT family proteins in raised plasma membrane domains. Fractures penetrating beneath the plasma membrane demonstrate that lipid droplets are closely apposed to these domains. A similar distribution pattern of labeling in the form of linear aggregates within the clusters is apparent in the cytoplasmic monolayer of the plasma membrane and the immediately adjacent outer monolayer of the lipid droplet. The aggregation of the PAT family proteins into such assemblies may facilitate carrier-mediated lipid influx from the extracellular environment into the lipid droplet. Lipid droplets appear to acquire their PAT proteins by interaction with plasma membrane domains enriched in these proteins.

We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call “lipid sails.” TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.

The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.

Caveolin-1, a putative mediator of intracellular cholesterol transport, is generally assumed to be integrated into the cytoplasmic leaflets of all cellular membranes. Lipid droplets form by budding at the endoplasmic reticulum (ER), and caveolin-1 is thought to be transferred to the droplet surface along with the cytoplasmic leaflet of ER membranes and not to enter the droplet core. We explored how caveolin-1 accesses lipid droplets from the ER by localizing caveolin-1 in ER membranes and in lipid droplets in cultured smooth muscle cells using freeze-fracture immunocytochemistry. We detected caveolin-1 in endoplasmic leaflets of ER membranes but never in cytoplasmic leaflets. Caveolin-1 was also present in lipid droplet cores. These findings are incompatible with the current hypothesis of lipid droplet biogenesis. We suggest that the inherent high affinity of caveolin-1 for neutral lipids causes caveolin-1 molecules to be extracted from the endoplasmic leaflets of ER membranes and to be transferred into the droplet core by inundating lipids during droplet formation.

Caveolin-1, a ubiquitous integral protein of cell surface caveolae, binds directly to cholesterol and is believed to participate facultatively in the bidirectional shuttling of free cholesterol between the plasma membrane and membranes of the endoplasmic reticulum (ER), Golgi and lipid droplets via the endocytotic pathway [1]. At the plasma membrane caveolin-1 does not entirely span the lipid bilayer, its lipophilic moiety being inserted primarily into the cytoplasmic leaflet of the membrane and its amino and carboxy terminals both projecting into the cytosol. Consequently, caveolin-1 molecules are extensively oligomerized and react strongly with proteins in the cytosol.

Caveolin-1, a major protein of cell surface invaginations called caveolae, is currently believed to cycle between the plasma membrane and intracellular compartments via the endocytotic pathway, at least for part of its itinerary. We studied the distribution of caveolin-1 in cell membranes, using ultrathin cryosections and freeze-fracture immunolabeling and found this protein not only in the cytoplasmic leaflet of the plasma membrane, but also in the exoplasmic leaflet of all intracellular membranes. This sidedness implies that caveolin-1 switches from one membrane leaflet to the other somewhere on its way through the cell and rules out the classic mechanism of endocytotic membrane budding and fusion for caveolin-1 intracellular trafficking. Underlying the sidedness of caveolin-1 may be a fundamental, hitherto unrecognized, mechanism by which proteins transit membranes.