Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes.

Blanchette‐Mackie, E. Joan; Dwyer, Nancy K.; Barber, T.; Coxey, R.A.; Takeda, Tomomi; Rondinone, Cristina M.; Theodorakis, Janice L.; Greenberg, Andrew S.; Londos, Constantine

doi:10.1016/s0022-2275(20)41129-0

Cited by 424 publications

(70 citation statements)

References 37 publications

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“…Medium was removed from the cells, which were then washed twice with PBS. Cells were incubated with 3% paraformaldehyde in PBS for 20 minutes at room temperature (Blanchette-Mackie et al 1995) and then washed four times with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…All incubations were done in antibody diluent (PBS with 0.1 mg/ml saponin and 0.5 mg/ml goat IgG); all washes were done with PBS. Fixed cells were incubated for 45 min at RT in antibody diluent with 0.2 M glycine (Blanchette-Mackie et al 1995) to block nonspecific antibody binding and to quench background fluorescence due to aldehydes. Cells were incubated with primary antibody in antibody diluent overnight at 4C and warmed at RT for 30 min before four 10-min PBS washes.…”

Section: Methodsmentioning

confidence: 99%

“…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; Blanchette-Mackie et al 1995; Servetnick et al 1995). In these cells it restricts the access of hormone-sensitive lipase and other cytosolic lipases to the lipid droplet under basal conditions (Souza et al 1998,2002; Brasaemle et al 2000b; Martinez-Botas et al 2000; Tansey et al 2001), and facilitates lipase access to the droplet under lipolytically stimulating conditions (Tansey et al 2001; Souza et al 2002).…”

mentioning

confidence: 99%

“…These fixatives have been used before staining for adipophilin in cultured adipocytes (Jiang and Serrero 1992), macrophages (Chen et al 2001), fibroblasts (Frolov et al 2000; Atshaves et al 2001), and various cell lines derived from dogs and cattle (Heid et al 1998), and for the core protein of the hepatitis C virus expressed in Chinese hamster ovary (CHO) fibroblasts (Barba et al 1997). Chemical crosslinkers such as paraformaldehyde and glutaraldehyde have also been used as fixatives to study proteins associated with many cell compartments, including lipid droplets (Blanchette-Mackie et al 1995; Brasaemle et al 2000a,b). In this study we have investigated the effects of cold methanol, cold acetone, and paraformaldehyde fixation procedures on the imaging of lipid droplets and their associated proteins by fluorescence microscopy, and on cellular lipid and protein composition.…”

mentioning

confidence: 99%

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Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy

DiDonato

Brasaemle

2003

J Histochem Cytochem.

132

124

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy

DiDonato

Brasaemle

2003

J Histochem Cytochem.

132

124

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“…This architecture at the ER-LD junction constitutes a barrier for ER residential integral membrane proteins and excludes them from the LD monolayer (Khaddaj et al, 2020). Early cryo electron microscopy (EM) studies have revealed that the cytoplasmic leaflet of the ER membrane is continuous with the LD surface, and freeze fracture EM suggested that the ER membrane forms a cup-like structure that holds the LD in place (Blanchette-Mackie et al, 1995;Robenek et al, 2006). Quantitative 3D-EM in mammalian cells indicates that LDs remain connected to the ER and that this connection is mediated by seipin, a protein that specifically localizes to ER-LD junctions (Salo et al, 2016).…”

Section: The Endoplasmic Reticulum-lipid Droplet Junction Forms a Unique Interface Between The Endoplasmic Reticulum Lipid Bilayer And LImentioning

confidence: 99%

A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets

Choudhary

Schneiter

2021

Front. Cell Dev. Biol.

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Lipid droplets (LDs) constitute compartments dedicated to the storage of metabolic energy in the form of neutral lipids. LDs originate from the endoplasmic reticulum (ER) with which they maintain close contact throughout their life cycle. These ER–LD junctions facilitate the exchange of both proteins and lipids between these two compartments. In recent years, proteins that are important for the proper formation of LDs and localize to ER–LD junctions have been identified. This junction is unique as it is generally believed to invoke a transition from the ER bilayer membrane to a lipid monolayer that delineates LDs. Proper formation of this junction requires the ordered assembly of proteins and lipids at specialized ER subdomains. Without such a well-ordered assembly of LD biogenesis factors, neutral lipids are synthesized throughout the ER membrane, resulting in the formation of aberrant LDs. Such ectopically formed LDs impact ER and lipid homeostasis, resulting in different types of lipid storage diseases. In response to starvation, the ER–LD junction recruits factors that tether the vacuole to these junctions to facilitate LD degradation. In addition, LDs maintain close contacts with peroxisomes and mitochondria for metabolic channeling of the released fatty acids toward beta-oxidation. In this review, we discuss the function of different components that ensure proper functioning of LD contact sites, their role in lipogenesis and lipolysis, and their relation to lipid storage diseases.

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Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

1996

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In this report, we provide direct evidence for the presence of a lipid droplet-associated capsule in hamster steroidogenic Leydig cells by using a monoclonal antibody A2. Leydig cells are characterized by containing many lipid droplets and having 3 beta-hydroxysteroid dehydrogenase activity. Immunofluorescence staining with this antibody demonstrated a rim or capsule surrounding the lipid droplets in Leydig cells, a pattern not seen with anti-vimentin antibody. Immunogold labelling confirmed ultrastructurally that antibody binding was distributed on the lipid droplet surface. In order to investigate the possible function of the capsule, we examined the morphological changes induced in the capsule following stimulation with LH or dibutyryl cAMP; the fluorescent intensity of the capsule was seen to gradually decrease, accompanied by a decrease in number and size of lipid droplets, and the response to both reagents was time- and concentration-dependent. We thus conclude that hormonal stimulation resulting in the detachment of certain capsular proteins from the surface of lipid droplets is mediated via the cAMP signaling pathway and may allow cholesterol ester hydrolytic enzyme direct access to its substrate in the lipid droplet.

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Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes.

Cited by 424 publications

References 37 publications

Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy

Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy

A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets

Immunocytochemical demonstration of a lipid droplet-specific capsule in cultured Leydig cells of the golden hamsters

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