David Troyer

Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis

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.

Mechanism of lipid-body formation in prokaryotes: how bacteria fatten up.

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.

Production of Type VI Collagen by Human Macrophages: A New Dimension in Macrophage Functional Heterogeneity

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.

Butyrophilin controls milk fat globule secretion

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.

Lipids partition caveolin-1 from ER membranes into lipid droplets: updating the model of lipid droplet biogenesis

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.

Compartmentalization of proteins in lipid droplet biogenesis

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.

Differences in the effects of HMG-CoA reductase inhibitors on proliferation and viability of smooth muscle cells in culture

We investigated the influence of lovastatin, simvastatin and pravastatin on proliferation and viability of vascular smooth muscle cells (SMC) in vitro and studied the effects of lovastatin on a mouse SMC line transgenic for a temperature-sensitive mutant of SV40 large T antigen (TAg), known to inhibit the function of p53 and pRb family members. We found that lovastatin and simvastatin inhibited cell proliferation by provoking G0/G1 phase arrest with concomitant depression of the proliferation antigen Ki-67/MIB-1. Lovastatin at high concentrations of 20 micromol/l caused cell death in the presence of serum but not under serum starved conditions, which was verified on the basis of increased DNA strand breaks, decreased DNA content and morphological alterations seen by electron microscopy. Cell death was also found for simvastatin, whereas pravastatin did not exhibit antiproliferative or cytotoxic effects. Mouse SMC transgenic for TAg did not show any impaired sensitivity to the antiproliferative and cell death inducing effect of lovastatin, but both effects could be antagonized by the supplementation of mevalonate. The data indicate that antiproliferative and cytotoxic effects of lovastatin are caused by the using up of products of mevalonate metabolism and do not require the presence of p53 or pRb.

Cholesterol transporter caveolin-1 transits the lipid bilayer during intracellular cycling

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.

Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

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.

Collagen-bound LDL modifies endothelial cell adhesion to type V collagen: Implications for atherosclerosis

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.