S U M M A R YThe study of proteins associated with lipid droplets in adipocytes and many other cells is a rapidly developing area of inquiry. Although lipid droplets are easily visible by light microscopy, few standardized microscopy methods have been developed. Several methods of chemical fixation have recently been used to preserve cell structure before visualization of lipid droplets by light microscopy. We tested the most commonly used methods to compare the effects of the fixatives on cellular lipid content and lipid droplet structure. Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. We found this method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are discovered and studied, it is critical to use appropriate methods to avoid studying artifacts. L ipid droplets are found in many different organisms and across many different tissues and cell types. Although the lipid droplets within adipocytes in the adipose tissue of animals are the largest and most easily observed, these specialized structures have been found across the biological kingdom and are now characterized as ubiquitous components of most types of cells (Murphy 2001). Lipid droplets play an important role in lipid storage and trafficking in mammalian cells. These structures provide energy and substrates for synthesis and repair of cell membranes in most types of cells, for synthesis of mediators of the inflammatory response, for steroid hormone biosynthesis in adrenal cortical, testis, and ovarian cells, for surfactant synthesis in lung, and for secretion of the lipid component of milk from mammary epithelial cells (Murphy 2001). In addition, the accumulation of cholesterol esters in lipid droplets in macrophages leads to the formation of foam cells, which play a major role in the development of atherosclerotic lesions. In each cell type, some of the proteins that are associated with the surfaces of lipid droplets regulate the accumulation and mobilization of the lipids that fulfill these functions.To date, only a few lipid droplet-associated proteins have been identified. Adipophilin is a ubiquitously expressed protein in all mammalian cell types and is found only in lipid droplets and in no other subcellular compartment (Brasaemle et al. 1997b;Heid et al. 1998). Perilipin, a structurally related protein, associates with lipid droplets in adipocytes and steriodogenic cells (Greenberg et al. 1991; BlanchetteMackie et al. 1995;Servetnick et al. 1995). In these cells it restricts the access of hormone-sensitive lipase and other cytosolic lipases to the lip...
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