A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

Ktistakis, Nicholas T.; Kao, Chia Yi; Wang, R. H.; Roth, Michael G.

doi:10.1091/mbc.6.2.135

Cited by 20 publications

(16 citation statements)

References 41 publications

(34 reference statements)

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“…When vector-and SPP-1-transfected HEK 293 cells were loaded with DMB-Cer at 4°C, the fluorescence was observed in the nuclear envelope and in a diffuse, reticular ER pattern. Upon warming the cells to 37°C for 30 min, the DMB fluorescence exited the ER and accumulated in juxtanuclear regions corresponding to the Golgi, in agreement with many previous studies (5,8,15,24,43,45,(50)(51)(52), as indicated by colocalization with the fluorescently tagged Golgi marker, DsRed-Monomer Golgi (Fig. 4A).…”

Section: Spp-1 Preferentially Increases Long-chain Ceramide Speciessupporting

confidence: 92%

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

Giussani

Maceyka

Stunff

et al. 2006

Molecular and Cellular Biology

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Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4°C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.All sphingolipids contain ceramide, which consists of a sphingoid base, typically sphingosine or dihydrosphingosine, and an amide linked acyl chain. Ceramide, a bioactive molecule (39), can be deacylated to sphingosine, which in turn can be phosphorylated to sphingosine-1-phosphate (S1P), also a potent lipid mediator that regulates vital biological processes (56). S1P is the ligand of a family of specific cell surface G protein-coupled receptors, hereafter referred to as S1PRs. These receptors couple to various G proteins to regulate cell migration, angiogenesis, vascular maturation, heart development, neurite retraction, and lymphocyte trafficking and immune responses (reviewed in references 49, 53, and 56). Dihydrosphingosine-1-phosphate (dihydro-S1P), which is identical to S1P but lacks the trans 4,5 double bond, binds to all of the S1PRs and activates them and yet does not mimic all of the effects of S1P, especially those related to cell survival and protection against apoptosis (56). Hence, S1P might also have intracellular functions independent of S1PRs. Indeed, S1P also regulates important functions in yeast and plant cells that lack S1PRs (reviewed in references 53 and 56).In contrast to S1P, ceramide, a central molecule in sphingolipid metabolism, has been implicated in cell growth arrest, differentiation, and apoptosis (10,22,39). Abundant evidence indicates that the balance between ceramide and S1P is a critical factor that determines cell fate ...

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Section: Spp-1 Preferentially Increases Long-chain Ceramide Speciessupporting

confidence: 92%

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

Giussani

Maceyka

Stunff

et al. 2006

Molecular and Cellular Biology

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“…In transfected COS cells, the wild-type XL␣s was localized to a compact perinuclear area that can be identified as Golgi with the anti-58-kDa protein antibody (Figure 3, a and b). We also found colocalization of endogenous and overexpressed XL␣s with another Golgi marker, BODIPY FL C 5 -ceramide (Ktistakis et al, 1995), in PC12 cells.…”

Section: Intracellular Localization Of Xl␣s Mutantssupporting

confidence: 54%

“…Cells, washed four times with 0.1% BSA in PBS and once with distilled water, were mounted in Prolong antifade reagent (Molecular Probes) and visualized with the use of a Zeiss (Thornwood, NY) Axioskop microscope equipped for fluorescence microscopy with a 63ϫ, 1.4 numerical aperture Plan-Apochromat oil immersion objective or a Leica (Wetzlar, Germany) LSM confocal microscope. For Golgi labeling of live PC12 cells with BODIPY FL C 5 -ceramide (Molecular Probes), the method described by Ktistakis et al (1995) was used. For treatment with brefeldin A, transfected COS cells were incubated with BODIPY TR ceramide (1 M, mixed with equimolar defatted BSA in serum-free DMEM; Molecular Probes) for 30 min at 4°C, washed three times with DMEM with 10% FBS, and incubated at 37°C for 30 min.…”

Section: Immunocytochemistry and Fluorescence Microscopymentioning

confidence: 99%

A Proline-rich Region and Nearby Cysteine Residues Target XLαs to the Golgi Complex Region

Uğur

Jones

2000

MBoC

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XL␣s is a splice variant of the heterotrimeric G protein, G␣ s , found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XL␣s for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XL␣s incorporated [3 H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XL␣s without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a G␣ i2 splice variant, dynamin II, ␤III spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein ␣ subunits and possibly for other peripheral membrane proteins as well.

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“…1) are nearly equipotent to induce trans-Golgi swelling in smooth muscle cells, based on the formation of giant vacuoles that exclude cytosolic proteins such as HcRed and whose membranes are labeled with C5-ceramide (Fig. 3), a recognized marker of the Golgi network and downstream secretory pathway (Pagano et al, 1991;Ktistakis et al, 1995). Both golgin-97 and p230 are peripheral membrane proteins associated with the cytosolic face of the trans-Golgi network (Kjer-Nielsen et al, 1999;Munro and Nichols, 1999); the corresponding cell labeling in smooth muscle cells concerns a minority of contiguous giant vacuoles in amine-treated cells (Figs.…”

Section: Discussionmentioning

confidence: 99%

N-Substituted 4-Aminobenzamides (Procainamide Analogs): An Assessment of Multiple Cellular Effects Concerning Ion Trapping

Morissette¹,

Moreau²,

C.‐Gaudreault³

et al. 2005

Mol Pharmacol

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Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-B inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPasedependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma). Giant vacuole formation, of confirmed trans-Golgi origin (labeled with C5-ceramide, p230, golgin-97), is a cellular response to all tested amines in the series (Ն2.5 mM), including triethylamine. These drugs and the V-ATPase inhibitor bafilomycin A1 inhibited smooth muscle cell proliferation, suggesting that acidification of a cellular compartment is essential to cell division. The cytotoxicity was maximal with metoclopramide, and this effect was minimally influenced by bafilomycin A1; furthermore, metoclopramide (2.5 mM) induced apoptosis in tumor cells as judged by poly(ADP-ribose)polymerase (PARP) cleavage. Triethylamine and procainamide exhibit a low level of cytotoxicity variably reduced by bafilomycin cotreatment. In Morris cells, the secretion of ␣-fetoprotein is inhibited by amines, consistent with the impairment of the secretory pathway. The most highly substituted 4-aminobenzamides are significant NF-B inhibitors in smooth muscle cells. Although some effects of 4-aminobenzamides are independent of VATPase-driven ion trapping (inhibition of NF-B nuclear translocation, agent-specific cytotoxicity, PARP cleavage), other effects are dependent on this phenomenon (vacuolization, a component of the cytotoxicity, inhibition of secretion).We have recently addressed the mechanism of the massive cell vacuolization induced by many organic amines at millimolar concentrations; procainamide, an N-substituted 4-aminobenzamide, was the prototype drug used in most assays (Morissette et al., 2004a), but the effect was shared by structurally simpler tertiary amines, such as triethylamine (Fig.

show abstract

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

Cited by 20 publications

References 41 publications

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

A Proline-rich Region and Nearby Cysteine Residues Target XLαs to the Golgi Complex Region

N-Substituted 4-Aminobenzamides (Procainamide Analogs): An Assessment of Multiple Cellular Effects Concerning Ion Trapping

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