Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes

Tomàs, Mariona; Durán, Juan M.; Lázaro‐Diéguez, Francisco; Babià, T; Renau‐Piqueras, Jaime; Egea, Gustavo

doi:10.1016/s0014-5793(04)00245-5

Cited by 20 publications

(22 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In CHO-K1 cells, NBD-sphingomyelin internalized from the plasma membrane to vesicles and other internal sites, and then transported back to the plasma membrane [33, 44]. In a similar fashion, NBD-sphingomyelin in primary cultures of rat astrocytes [45] and in L-cells (present work) were endocytosed and sorted to several intracellular sites, including lipid droplets (results herein). Furthermore, with other NBD-labeled probes such as NBD-cholesterol, there is substantial evidence to suggest that NBD-cholesterol undergoes metabolism and utilizes the same intracellular trafficking pathways as the endogenous lipid [20, 29].…”

Section: Discussionmentioning

confidence: 64%

Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

McIntosh¹,

Storey²,

Atshaves

2010

Lipids

View full text Add to dashboard Cite

During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDphosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.

show abstract

Section: Discussionmentioning

confidence: 64%

Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

McIntosh¹,

Storey²,

Atshaves

2010

Lipids

View full text Add to dashboard Cite

show abstract

“…Ethanol has been shown to induce perturbation in endocytic processes of sphingolipids (Ravasi et al. 2002; Tomás et al. 2004), which may be related to the observed GM2 accumulation in a late endosome/lysosome/phagolysosome fraction of the brain.…”

Section: Discussionmentioning

confidence: 99%

“…Also, it is possible that GM2 accumulates as degradative products in endosomal systems due to perturbed endocytic and sorting processes of sphingolipids by ethanol (Ravasi et al. 2002; Tomas et al. 2004).…”

Section: Discussionmentioning

confidence: 99%

Elevation of GM2 ganglioside during ethanol‐induced apoptotic neurodegeneration in the developing mouse brain

Shimada

Chakraborty

Shah

et al. 2012

Journal of Neurochemistry

View full text Add to dashboard Cite

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase-3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase-3 activation in the 7-day–old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.

show abstract

“…Membrane fluidity was assessed by measuring the fluorescence polarizations of NBD-cholesterol, NBD-sphingomyelin, and DPH-TMA to measure the motional freedom of lipids in the phospholipid monolayer of each LD pool. The use of NBD-labeled lipids was based on numerous studies in living cells (L cells, intestinal enterocytes, CaCo2 cells) that showed that the intracellular distribution, trafficking, binding, and metabolism of NBD-labeled lipids was similar to natural analogs (5,9,19,26,33,38,102). With NBD-sphingomyelin, the probe did not undergo transbilayer movement (58,59) but instead localized to the outer leaflet of the lipid bilayer.…”

Section: Discussionmentioning

confidence: 99%

The phospholipid monolayer associated with perilipin-enriched lipid droplets is a highly organized rigid membrane structure

Storey¹,

McIntosh²,

Senthivinayagam

et al. 2011

American Journal of Physiology-Endocrinology and Metabolism

View full text Add to dashboard Cite

(LD) in lipid metabolism, cell signaling, and membrane trafficking is increasingly recognized, yet the role of the LD phospholipid monolayer in LD protein targeting and function remains unknown. To begin to address this issue, two populations of LD were isolated by ConA sepharose affinity chromatography: 1) functionally active LD enriched in perilipin, caveolin-1, and several lipolytic proteins, including ATGL and HSL; and 2) LD enriched in ADRP and TIP47 that contained little to no lipase activity. Coimmunoprecipitation experiments confirmed the close association of caveolin and perilipin and lack of interaction between caveolin and ADRP, in keeping with the separation observed with the ConA procedure. The phospholipid monolayer structure was evaluated to reveal that the perilipin-enriched LD exhibited increased rigidity (less fluidity), as shown by increased cholesterol/phospholipid, Sat/Unsat, and Sat/ MUFA ratios. These results were confirmed by DPH-TMA, NBDcholesterol, and NBD-sphingomyelin fluorescence polarization studies. By structure and organization, the perilipin-enriched LD most closely resembled the adipocyte PM. In contrast, the ADRP/TIP47-enriched LD contained a more fluid monolayer membrane, reflecting decreased polarizations and lipid order based on phospholipid fatty acid analysis. Taken together, results indicate that perilipin and associated lipolytic enzymes target areas in the phospholipid monolayer that are highly organized and rigid, similar in structure to localized areas of the PM where cholesterol and fatty acid uptake and efflux occur. lipolysis; caveolae; caveolin; concanavalin-A; adipose differentiationrelated protein IT IS INCREASINGLY CLEAR THAT LIPID DROPLETS (LD) are dynamic organelles with regulatory roles in many cellular processes besides lipid metabolism, including cell signaling, immune function, membrane trafficking, and regulation of longevity (42,70). Whereas the mechanism of these processes is only partially understood, much less is known about how the structure of the LD phospholipid monolayer may affect LD protein targeting and function. Embedded in the monolayer that surrounds the LD neutral lipid (NL) core are proteins, such as perilipins (65) and adipose differentiation-related protein (ADRP) (16,41), that coat the LD surface and lipids such as cholesterol (4, 80) that help to define the LD structure. Perilipin and ADRP are two members of the PAT [perilipin, ADRP, tail-interacting protein 47 (TIP47)] family from the Plin group of genes (56) that were initially thought to act simply as barriers to protect the NL core against lipolytic action (63, 65). Current evidence suggests a more complex mechanism, one that requires coordination of perilipin with several lipases, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the activator molecule comparative gene identification-58 (CGI-58), a protein with no lipolytic activity that increases ATGL activity to promote triacylglyerol hydrolysis (11,14,44). Perilipin and HSL upon hormonal stimu...

show abstract

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes

Cited by 20 publications

References 36 publications

Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

Elevation of GM2 ganglioside during ethanol‐induced apoptotic neurodegeneration in the developing mouse brain

The phospholipid monolayer associated with perilipin-enriched lipid droplets is a highly organized rigid membrane structure

Contact Info

Product

Resources

About