DiOC<sub>6</sub>(3): a Useful Dye for Staining the Endoplasmic Reticulum

Sabnis, R. W.; Deligeorgiev, Todor; Jachak, Madhukar N.; Dalvi, Tukaram S.

doi:10.3109/10520299709082249

Cited by 78 publications

(43 citation statements)

References 41 publications

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“…Other portions of the ER, however, contained relatively low amounts of myc-tagged DGAT1 or DGAT2 protein fluorescence compared with the ConA fluorescence in these same regions ( Figure 5, black arrowheads). Similar results were observed when the ER in myc-DGAT1 or myc-DGAT2 cells was labeled with 3,39-dihexyloxacarbocyanine iodide, another commonly used stain for the general ER (Sabnis et al, 1997) (see Supplemental Figure 4 online).…”

Section: Dgat1 and Dgat2 Proteins Are Enriched In Subdomains Of The Ersupporting

confidence: 70%

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

et al. 2006

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Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing ;80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

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Section: Dgat1 and Dgat2 Proteins Are Enriched In Subdomains Of The Ersupporting

confidence: 70%

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

et al. 2006

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“…For experiments in uninfected cells, tunicamycin (2.5 g/ml) was added 24 h after subculturing and the monolayer was fixed after another 24 h. Dicarbocyanine dye localization. DiOC 6 (3-3-dihexyloxacarbocyanine iodide) was obtained in powder form from Molecular Probes (D-273) and dissolved (0.7 mg/ml) in ethanol (41). An aliquot of the DiOC 6 stock (2.8 l/ml yielded a final concentration of DiOC 6 of 2 g/ml) was added to warm cell culture medium; this medium was applied to live cells for 30 min and then rinsed twice with phosphate-buffered saline (PBS) and processed for fluorescence microscopy as described above.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…To quantify the increase in the size of the ER due to VZV infection, we measured the relative size of the ER in VZVinfected MRC-5 cells by staining the cells with DiOC 6 , a polar dye that preferentially localizes to polarized membranes, such as those found in mitochondria and the ER (41,50). A series of confocal fluorescent images at 630ϫ were then taken of both uninfected and VZV-infected MRC-5 cells.…”

Section: Enumeration Of the Cellular Constituents Within A Vesiclementioning

confidence: 99%

Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response

Carpenter

Jackson

Benetti

et al. 2011

J Virol

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Autophagy is a recently recognized component of the life cycle of varicella-zoster virus (VZV).We have documented abundant autophagosome formation in skin vesicles (final site of virion assembly) from randomly selected cases of varicella and zoster. The fact that autophagy was an early event in the VZV replication cycle was documented by finding infected vesicle cells with the VZV IE62 protein confined to the nucleus. Next, we pursued studies in VZV-infected cultured cells to define whether autophagy was preceded by endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). First, we demonstrated that autophagosome formation in infected cells closely resembled that seen after treatment of cells with tunicamycin, a potent initiator of ER stress. Second, we demonstrated a marked expansion of ER size in both VZV-infected cells and cells transfected with the predominant VZV glycoprotein complex gE/gI. An enlarged ER is critical evidence of ER stress, which in turn is relieved by the UPR. To this end, we documented the UPR by detecting the alternatively spliced form of the XBP1 protein as well as CHOP (C/EBP homologous protein), both transcriptional activators of other UPR genes in an ER stress-dependent manner. Because VZV does not encode inhibitors of autophagy, the above results suggested that autophagy was a common event in VZV-infected cells and that it was provoked at least in part by ER stress secondary to overly abundant VZV glycoprotein biosynthesis, which led to UPR activation in an attempt to maintain cellular homeostasis.Varicella-zoster virus (VZV) is a human pathogen that causes chicken pox (varicella) and shingles (zoster) (55). Zoster is the disease associated with reactivation of latent VZV in the elderly. The virus exists as a spherical particle approximately 200 nm in diameter, including a 125-kb DNA genome enclosed in an icosahedral capsid which is itself surrounded by an amorphous shell of proteins called the tegument and an outer lipid envelope containing viral glycoproteins (9). The most prominent viral glycoprotein is called gE and is part of the gE/gI complex (20,22). Within a few days after infection, viral replication leads to the assembly of nascent viral particles in the head and neck region. A viremia ensues within T lymphocytes, after which viral particles exit the capillaries and replicate within the epidermis to cause the characteristic vesicular rash (28). The skin vesicle is considered to be the final site of assembly and envelopment of the mature VZ virion (52). Relatively little is known about the innate immune response within the cutaneous microenvironment (2).How a cell responds to viral infection and in turn how the virus attempts to moderate that response have been a topic of renewed research. One such response of the host cell is to increase macroautophagy (29). Macroautophagy is a catabolic process by which whole or parts of organelles are sequestrated into double-membraned autophagosomes in the cytoplasm and then degraded when the autophagosomes fuse with ly...

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“…The ER was labeled with the lipophilic, cationic DiOC 6 dye applied for 15 min at a final concentration of 2 mg ml À1 (Terasaki et al, 1984). This marker, used in the concentration range 1 -10 mg ml À1 , was reported to be highly specific for ER and, compared to other possible candidates, is brightest with the slowest bleaching rate (Sabnis et al, 1997). At the end of the double staining, the labelling solution was removed by gentle rinsing with RPMI 1640 containing 25 mm Hepes, and microspectrofluorometry measurements or observations by confocal fluorescence microscopy were performed.…”

Section: Treatment Of Cells Prior To Microspectrofluorometry and Confmentioning

confidence: 99%

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan® localisation in cultured tumour cells

et al. 2003

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Intracellular photosensitiser localisation significantly influences the mechanism of response to photodynamic therapy (PDT), since the primary sites of damage are closely related to the specific sensitiser distribution. Foscan s subcellular localisation in the MCF-7 human adenocarcinoma cell line has been studied by means of confocal microscopy and microspectrofluorometry. The fluorescence topographic profiles recorded after cells costained with Foscan s and organelle-specific fluorescent probes revealed that Foscan s presents low localisation in lysosomes and a weak accumulation in mitochondria. Alternatively, the Foscan s fluorescence topographic profile turned out to colocalise perfectly with that obtained for the endoplasmic reticulum (ER) and the Golgi apparatus. The patterns of fluorescence derived from confocal microscopy studies were consistent with predominant localisation of Foscan s in these organelles. Furthermore, evaluation of enzymatic activity of selected organelles immediately after laser light irradiation (650 nm) indicated the Golgi apparatus and ER as the primary damaged sites resulting from Foscan s -mediated PDT in the MCF-7 cell line. To our knowledge, this is the first study to demonstrate unambiguously that the ER and the Golgi apparatus are preferential sites of Foscan s accumulation.

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DiOC₆(3): a Useful Dye for Staining the Endoplasmic Reticulum

Cited by 78 publications

References 41 publications

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan® localisation in cultured tumour cells

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DiOC6(3): a Useful Dye for Staining the Endoplasmic Reticulum

Cited by 78 publications

References 41 publications

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Tung Tree DGAT1 and DGAT2 Have Nonredundant Functions in Triacylglycerol Biosynthesis and Are Localized to Different Subdomains of the Endoplasmic Reticulum

Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan® localisation in cultured tumour cells

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DiOC₆(3): a Useful Dye for Staining the Endoplasmic Reticulum