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 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.

SummaryNeutral 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 membraneassociated 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.

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.freeze-fracture immunocytochemistry ͉ lipid droplet-associated proteins ͉ mammary epithelial cells ͉ milk secretory granules

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.