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The endoplasmic reticulum (ER) and Golgi apparatus are primarily responsible for the proper sorting of lipids and proteins in cells. Consequently, most of the cell-permeant probes for these organelles are either lipids or chemicals that affect protein movement. Several of the most effective probes for the Golgi apparatus are fluorescent ceramides and sphingolipids, which are discussed below and in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3. Certain aspects of lipid trafficking through the ER and Golgi apparatus related to signal transduction are described in Probes for Lipid Metabolism and Signaling—Section 17.4. In both live and fixed cells, the flattened membranous sacs of the ER and the Golgi apparatus can be stained with a variety of lipophilic probes and then distinguished by their morphology.
In addition to these fluorescent organelle stains, we offer several CellLight reagents that comprise BacMam expression vectors encoding targeted autofluorescent proteins for visualizing the endoplasmic reticulum and Golgi apparatus in live mammalian cells. For labeling fixed-cell preparations, we offer the SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit (S34200), which contains an antibody directed against the ER-associated protein disulfide isomerase (PDI). Enzymes in the ER are also involved in synthesis of cholesterol and in the detoxification of hydrophobic drugs through the cytochrome P450 system (Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes—Section 10.6). Furthermore, some fluorescent lectins are useful markers for the Golgi apparatus because several enzymes in this organelle function to glycosylate lipids and proteins (). Nissl bodies principally comprise ordered structures of alternate lamellae of rough endoplasmic reticulum and polyribosome arrays; our NeuroTrace fluorescent Nissl stains are described in Probes for the Nucleus—Section 12.5. An excellent compendium of human diseases that affect intracellular transport processes through lysosomes, Golgi and endoplasmic reticulum (ER) has been published.
CellLight reagents combine the utlity and selectivity of targeted fluorescent proteins with the efficiency of the BacMam gene delivery and expression technology. These reagents incorporate all the customary advantages of BacMam technology, including high efficiency transduction of mammalian cells and long-lasting, titratable expression ( BacMam Gene Delivery and Expression Technology—Note 11.1). CellLight reagents are provided in a ready-to-use format—simply add, incubate and image—with highly efficient expression in cell lines, primary cells, stem cells and neurons. A complete list of CellLight reagents and their targeting sequences can be found in CellLight reagents and their targeting sequences—Table 11.1.
The CellLight ER and Golgi markers are generally useful for identification and demarcation of their respective target organelles in live-cell imaging investigations of protein trafficking. CellLight ER-GFP (C10590, Figure 12.4.1) and CellLight ER-RFP (C10591) are BacMam expression vectors encoding fusions of Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP) with the calreticulin ER insertion sequence and the KDEL tetrapeptide retention sequence. Because the localization of CellLight ER-GFP and CellLight ER-RFP is directed by cellular protein trafficking infrastructure, it is more specific than that of dyes such as DiOC6(3), which are largely driven by simple hydrophobic partition. CellLight Golgi-GFP (C10592, Figure 12.4.2) and CellLight Golgi-RFP (C10593) are BacMam expression vectors encoding fusions of GFP or RFP with the human Golgi-resident enzyme N-acetylgalactosaminyltransferase 2.
ER-Tracker dyes are cell-permeant, live-cell stains that are highly selective for the ER. These dyes rarely stain mitochondria, unlike the conventional ER stain DiOC6(3) (D273), and staining at low concentrations does not appear to be toxic to cells. When cells are stained using the optimized protocol provided, staining patterns are retained after treatment with formaldehyde, although at reduced intensities.
ER-Tracker Blue-White DPX Dye
ER-Tracker Blue-White DPX (E12353, ) is a highly selective and photostable stain for the ER in live cells (, ). ER-Tracker Blue-White DPX is a member of our Dapoxyl dye family and thus exhibits an unusually large Stokes shift and long-wavelength emission with a high extinction coefficient and high quantum yield when in a hydrophobic environment. Its fluorescence is highly environment sensitive—with increasing solvent polarity, the fluorescence maximum shifts to longer wavelengths (Figure 12.4.3) and the quantum yield decreases—and peak fluorescence emission ranges from 430 nm to 640 nm; we recommend visualizing its ER staining with a standard DAPI or UV longpass optical filter set. The ER-Tracker Blue-White DPX dye is also readily visualized by two-photon microscopy.
Figure 12.4.3 Normalized fluorescence emission spectra of Dapoxyl (2-aminoethyl)sulfonamide in 1) hexane, 2) chloroform, 3) acetone, 4) acetonitrile and 5) 1:1 acetonitrile:water.
ER-Tracker Green and Red Dyes
ER-Tracker Green (E34251) and ER-Tracker Red endoplasmic reticulum stains (E34250) are fluorescent sulfonylureas—BODIPY FL glibenclamide () and BODIPY TR glibenclamide—which exhibit excitation/emission maxima of ~504/511 nm and 587/615 nm, respectively. Glibenclamide binds to sulfonylurea (SUR) receptors of ATP-sensitive K+ channels, which are prominent on ER but may have more disseminated tissue- and cell type–dependent distributions. BODIPY FL glibenclamide also generates SUR-independent labeling in some cases. Despite these mechanistic nuances, ER-Tracker Green () and ER-Tracker Red are effective and widely used endoplasmic reticulum markers in live-cell imaging applications.
Short-Chain Carbocyanine Dyes
Terasaki and co-workers used the short-chain carbocyanine DiOC6(3) (D273) to visualize the ER in both live and aldehyde-fixed cells. This dye and the similar DiOC5(3) (D272) have since been used extensively to study structural interactions and dynamics of the ER in neurons, yeast and onion epidermis, and to examine the morphological relationships between the ER, mitochondria, intermediate filaments and microtubules in various cell types. DiOC6(3) and DiOC5(3) pass through the plasma membrane and stain intracellular membranes with a fluorescein-like fluorescence; ER membranes can easily be distinguished by their characteristic morphology. Caution must be exercised, however, in using the carbocyanines as probes for the ER. It has been reported that ER staining with DiOC6(3) does not occur until the mitochondria round up and lose the fluorochrome. Rhodamine 6G and the hexyl ester of rhodamine B (R634, R648MP; Probes for Mitochondria—Section 12.2) appear to stain like DiOC6(3), except they are apparently less toxic and they fluoresce orange, providing possibilities for multicolor labeling. When used at very low concentrations, these slightly lipophilic rhodamine dyes tend to stain only mitochondria of live cells.
Long-Chain Carbocyanine Dyes
Terasaki and Jaffe have used the long-chain carbocyanines DiIC16(3) and DiIC18(3) (D384, D282) to label ER membranes. They achieved selective labeling of the ER by microinjecting a saturated solution of DiI in oil into sea urchin eggs. This method has been successful in several other egg types but was not effective in molluscan or arthropod axons. As noted in the discussion of dialkylcarbocyanine and dialkylaminostyryl probes in Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4, DiI diffuses only in continuous membranes.
NBD C6-ceramide (N1154) and BODIPY FL C5-ceramide (D3521), both of which can be used with fluorescein optical filter sets, are selective stains for the Golgi apparatus. With spectral properties similar to those of Texas Red dye, BODIPY TR ceramide (D7540) is especially useful for double-labeling in combination with Green Fluorescent Protein (GFP) fusion proteins ( Using Organic Fluorescent Probes in Combination with GFP—Note 12.1), as well as for staining cells and tissues that have substantial amounts of green autofluorescence. In addition, the BODIPY TR fluorophore is ideal for imaging microscopy with CCD cameras or other red-sensitive detectors. Uptake of fluorescent ceramides, at least in Paramecium cells, appears to be an ATP-dependent process.
NBD Ceramide and NBD Sphingomyelin
NBD C6-ceramide (N1154) and NBD C6-ceramide complexed with defatted BSA (N22651) have been used extensively as a selective stain of the trans-Golgi in both live and fixed cells. Complexing fluorescent ceramides with bovine serum albumin (BSA) facilitates cell labeling without requiring the use of organic solvents to dissolve the probe. Furthermore, the fluorescence of NBD C6-ceramide is apparently sensitive to the cholesterol content of the Golgi apparatus, a phenomenon that is not observed with BODIPY FL C5-ceramide. If NBD C6-ceramide–containing cells are starved for cholesterol, the NBD C6-ceramide that accumulates within the Golgi apparatus appears to be severely photolabile. However, this NBD photobleaching can be reduced by stimulation of cholesterol synthesis. Thus, NBD C6-ceramide may be useful in monitoring the cholesterol content of the Golgi apparatus in live cells.
NBD C6-ceramide's conversion to the NBD C6-glycosyl ceramide and NBD C6-sphingomyelin (N3524) has been observed in vivo. Metabolism of the probe in live Chinese hamster ovary (CHO) fibroblasts has been used to define lipid-transport pathways. Like NBD C6-ceramide, NBD C6-sphingomyelin has been used for the study of lipid trafficking between organelles. Normal fibroblasts hydrolyze NBD C6-sphingomyelin and process it through the Golgi apparatus. However, in human skin fibroblasts from patients with Niemann–Pick disease, which is characterized by a lack of lysosomal sphingomyelinase activity, NBD C6-sphingomyelin accumulates in the lysosomes.
BODIPY Ceramides, BODIPY Sphingomyelin and Related Derivatives
The green-fluorescent BODIPY FL C5-ceramide (D3521) is more fade-resistant and brighter than the NBD derivative and can likely be substituted for the NBD C6-ceramide in many of its applications. The red-fluorescent BODIPY TR ceramide (D7540) has proven useful for two-color immunofluorescence using a fluorescein-labeled antibody. As with NBD C6-ceramide, we also offer BODIPY FL C5-ceramide and BODIPY TR ceramide complexed with defatted BSA (B22650, B34400) to facilitate cell labeling without the use of organic solvents to dissolve the probe.
During normal resting intracellular transport, the kinetics of dye loading and transport may differ somewhat between the BODIPY and NBD analogs. BODIPY FL C5-ceramide has proven to be an excellent structural marker for the Golgi apparatus, visualized either by fluorescence microscopy or, following diaminobenzidine (DAB) conversion, electron microscopy. BODIPY FL C5-ceramide has also been used to:
- Delineate the Golgi apparatus in the cytoarchitecture of size-excluding compartments in live cells
- Investigate both the inhibition of glycoprotein transport by ceramides and the possible link between protein secretory pathways and sphingolipid biosynthesis
- Isolate mammalian secretion mutants
- Study sphingolipid distribution during human keratinocyte differentiation
- Visualize tubovesicular membranes induced by Plasmodium falciparum
BODIPY FL C5-ceramide exhibits concentration-dependent fluorescence properties that provide additional benefits for imaging the Golgi apparatus. At high concentrations, the nonpolar BODIPY FL fluorophore forms excimers, resulting in a shift of the fluorophore's emission maximum from 515 nm (green) to ~620 nm (red). BODIPY FL C5-ceramide accumulation is sufficient for excimer formation in the trans-Golgi but not in the surrounding cytoplasm. Longpass optical filters that isolate the red emission can thus be used to selectively visualize the Golgi apparatus (, ). Moreover, this two-color property can be used to quantitate BODIPY FL C5-ceramide accumulation by ratio imaging. Like BODIPY FL C5-ceramide, the red-fluorescent BODIPY TR ceramide appears to form long-wavelength excimers when concentrated in the Golgi apparatus; in this case, however, the excimers exhibit infrared fluorescence. In an unexpected application, it has been shown that cells infected with some intracellular bacteria, including Chlamydia psittaci, accumulate BODIPY FL C5-ceramide (D3521) in their inclusion membranes rather than in the Golgi of the host cells. Certain CellTracker reagents (Membrane-Permeant Reactive Tracers—Section 14.2) that were used in combination with BODIPY FL C5-ceramide were also found to selectively label intracellular bacteria and parasites.
We also offer BODIPY FL C5-sphingomyelin (D3522)—the likely metabolic product of BODIPY FL C5-ceramide —as well as BODIPY FL C12-sphingomyelin (D7711) and BODIPY FL C5-lactosylceramide (D13951, B34402). The concentration-dependent fluorescence shift of BODIPY FL C5-sphingomyelin from green to red has been used to follow the initial steps of lipid uptake and transport by early endosomes through the cytoplasm. BODIPY FL C5-glucocerebroside is reportedly internalized by endocytic and nonendocytic pathways that are quite different from those governing the internalization of BODIPY FL C5-sphingomyelin (D3522). Addition of BODIPY FL C5-lactosylceramide to the culture medium of cells from patients with sphingolipid-storage diseases (sphingolipidosis) results in fluorescent product accumulation in lysosomes, whereas this probe accumulates in the Golgi apparatus of normal cells and cells from patients with other storage diseases. Pagano and collaborators have published reviews of the use of BODIPY ceramides and BODIPY sphingolipids to study the endocytic pathway in mammalian cells.
The SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit (S34200) provides all the reagents required to fix and permeabilize mammalian cells and then specifically label the ER. To achieve ER labeling, this kit employs a primary antibody directed against an ER-associated protein, protein disulfide isomerase (PDI), and an Alexa Fluor 488 dye–labeled secondary antibody. The Alexa Fluor 488 dye exhibits bright green fluorescence that is compatible with filters and instrument settings appropriate for fluorescein.
Each SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit contains:
- Mouse IgG2b anti–protein disulfide isomerase (PDI) antibody
- Highly cross-adsorbed Alexa Fluor 488 goat anti–mouse IgG antibody
- Concentrated fixative solution
- Concentrated phosphate-buffered saline (PBS)
- Concentrated permeabilization solution
- Concentrated blocking solution
- Detailed protocols for mammalian cell preparation and staining (SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit )
Wheat Germ Agglutinin and Concanavalin A
Various proteins and lipids found in the Golgi apparatus are glycosylated; consequently, lectin conjugates (Lectins and Other Carbohydrate-Binding Proteins—Section 7.7) have been found to be particularly useful for staining Golgi structures in fixed-cell preparations (). Wheat germ agglutinin (WGA) conjugates are commonly used as markers of the trans-Golgi. Fluorescent conjugates of concanavalin A (Con A) also stain the Golgi but with reduced specificity. We prepare WGA and Con A conjugates whose fluorescence spans the entire visible and near-infrared spectrum ( Molecular Probes lectin conjugates—Table 7.24). Our Alexa Fluor conjugates of these important lectins are particularly recommended for their enhanced brightness and photostability. We also offer a Wheat Germ Agglutinin Sampler Kit (W7024), which contains 1 mg quantities each of WGA conjugates of the Alexa Fluor 350, Oregon Green 488, tetramethylrhodamine and Texas Red-X dyes.
Griffonia simplicifolia Lectin GS-II
Lectin GS-II from Griffonia simplicifolia is the only known lectin that binds with high selectivity to terminal, nonreducing α- and β-N-acetyl-D-glucosaminyl (GlcNAc) residues of glycoproteins. Because of the affinity of lectin GS-II for GlcNAc, conjugates of this lectin are useful for staining intermediate-to-trans Golgi—the site of N-acetylglucosaminyltransferase activity. The Golgi apparatus of oligodendrocytes and ganglion neurons are readily stained by fluorescent GS-II conjugates. We have prepared the green-fluorescent Alexa Fluor 488 (L21415, ), red-fluorescent Alexa Fluor 594 (L21416) and far-red–fluorescent Alexa Fluor 647 (L32451) conjugates of lectin GS-II for use in Golgi staining.
Helix pomatia (Edible Snail) Agglutinin
Helix pomatia agglutinin (HPA) selectively binds to terminal α-N-acetylgalactosaminyl residues—an intermediate sugar added in O-linkage to serine and threonine residues in cis-Golgi cisternae and then substituted with galactose and sialic acid in the trans-Golgi. HPA conjugates are principally used as markers for the Golgi. Our fluorescent Alexa Fluor 488 and Alexa Fluor 647 conjugates of HPA (L11271, L32454) should be particularly useful for Golgi staining.
Isolated from from Penicillium brefeldianum, brefeldin A (BFA, B7450) has multiple targets in cells. Exposing cells to BFA causes a distortion in intracellular protein traffic from the ER to the Golgi apparatus and the eventual loss of Golgi apparatus morphology; removal of BFA completely reverses these effects. BFA also alters the morphology of endosomes and lysosomes. BFA has been used to prevent retinoic acid potentiation of immunotoxins, to study translocation of proteins in polarized epithelial cells and to investigate the regulation of ADP-ribosylation factor binding to the Golgi apparatus. BFA action can be monitored using fluorescent endosomal markers such as lucifer yellow CH (L453, L682, L1177, L12926; Polar Tracers—Section 14.3) and tetramethylrhodamine-labeled transferrin (T2872, Probes for Following Receptor Binding and Phagocytosis—Section 16.1). Researchers have also used BFA to detect the intracellular expression of cytokines. BFA disrupts Golgi-mediated intracellular transport and allows cytokines to accumulate, producing an enhanced cytokine signal that can be detected by flow cytometry.
|D3522||766.75||FF,D,L||see Notes||505||77,000||512||MeOH||1, 3|
|N3524||740.88||FF,D,L||see Notes||466||22,000||536||MeOH||3, 6|