Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4

The endoplasmic reticulum (ER) and Golgi apparatus (Figure 12.1) 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. The lipophilicity of these probes enables their facile loading into live cells, usually from a dilute solution in dimethylsulfoxide. 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.

An excellent compendium of human diseases that affect intracellular transport processes through lysosomes, Golgi and endoplasmic reticulum (ER) has been published. Enzymes in the ER are also involved in synthesis of cholesterol and membranes and in the detoxification of hydrophobic drugs through the cytochrome P450 system (Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6). Because several enzymes in the Golgi glycosylate lipids and proteins, some fluorescent lectins are useful markers for this organelle (). Nissl bodies principally comprise ordered structures of alternate lamellae of rough endoplasmic reticulum and polyribosome arrays (, , ), and our NeuroTrace fluorescent Nissl stains are described in Polar Tracers - Section 14.3.

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. For labeling fixed-cell preparations, we also offer antibodies specific for the yeast ER and for both mammalian and yeast Golgi proteins, as well as the SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit (S34200), which contains an antibody directed against the ER-associated protein disulfide isomerase (PDI).

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 (Figure 1.111). Its fluorescence is highly environment sensitive — with increasing solvent polarity, the fluorescence maximum shifts to longer wavelengths and the quantum yield decreases — and peak fluorescence emission ranges from 430 nm to 640 nm. The ER-Tracker Blue-White DPX dye is readily visualized by two-photon microscopy. We recommend visualizing its ER staining with a standard DAPI or UV longpass optical filter set (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11).

Figure 1.111 Normalized fluorescence emission spectra of Dapoxyl (2-aminoethyl)sulfonamide (D10460) in 1) hexane, 2) chloroform, 3) acetone, 4) acetonitrile and 5) 1:1 acetonitrile:water.

ER-Tracker Green and Red Dyes

The green-fluorescent ER-Tracker Green and red-fluorescent ER-Tracker Red dyes (E34251, E34250) correspond to the drug conjugates BODIPYFL glibenclamide and BODIPY TR glibenclamide, which exhibit excitation/emission maxima of ~540/511 nm and 587/615 nm, respectively. Glibenclamide (glyburide), a drug taken daily by millions of diabetic patients to correct hyperglycemia, has been used to investigate pancreatic-cell activity and insulin secretion and to study myocardial cell function and heart arrhythmia. Glibenclamide binds to the sulphonylurea receptors of ATP-sensitive K⁺ channels, which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling.

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, 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 Dialkylcarbocyanine and Dialkylaminostyryl Probes - Section 13.4 on dialkylcarbocyanine and dialkylaminostyryl probes, DiI diffuses only in continuous membranes.

The fungal metabolite brefeldin A (BFA) has proven valuable for dissecting the cellular processes, including vesicle formation and kinesin distribution, involved in exporting newly synthesized proteins. In addition to the natural product isolated from Penicillium brefeldianum (B7450), we offer green- and orange-fluorescent BODIPY derivatives of brefeldin A (B7447, B7449).

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.

Fluorescent Brefeldin A

The green-fluorescent BODIPY FL and red-orange–fluorescent BODIPY 558/568 BFA derivatives (B7447, B7449; ) are selectively localized in the ER and Golgi apparatus in four different cell lines. BODIPY 558/568 BFA may be used in conjunction with NBD C₆-ceramide (N1154) to investigate the ER and Golgi apparatus simultaneously. BODIPY 558/568 BFA has also been used in combination with ER Tracker Blue-White DPX (E12353) to label the endoplasmic reticulum of hyphae. However, the biological activity of the fluorescent BFA derivatives may be limited and may be dependent on cleavage of the BODIPY fluorophore from BFA by intracellular esterases. Two isomeric esters are isolated in the synthesis of the fluorescent brefeldins; we offer only "isomer 1" of each product.

NBD C₆-ceramide and BODIPY FL C₅-ceramide (N1154, D3521; (NBD- and BODIPY(R)-Dye–Labeled Sphingolipids)), both of which can be used with fluorescein optical filter sets (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11), 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 experiments, including with chimeras of green-fluorescent proteins, 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 C₆-Ceramide and NBD C₆-Sphingomyelin

NBD C₆-ceramide (N1154) has 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. For this application, we provide NBD C₆-ceramide complexed with defatted BSA (N22651). Researchers have employed NBD C₆-ceramide to investigate:

• Defective trans-Golgi acidification in cystic fibrosis
• Effects of BFA (B7450) on the transport of proteins from the Golgi apparatus to the ER
• Farber's disease, a genetically inherited disease of lipid metabolism
• Inhibition of glycoprotein traffic through secretory pathways
• Intracellular trafficking and targeting of thrombin receptors
• Secretory activity during the isolation of secretion mutants by fluorescence-activated cell sorting
• Subcellular distribution of the verotoxin B subunit in Vero cells

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. NBD C₆-ceramide is reported to be metabolized to NBD C₆-sphingomyelin in Plasmodium falciparum–infected erythrocytes, but not in normal erythrocytes. 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 C₅-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), BODIPY FL C₅- and C₁₂-glucocerebrosides (D7548, D7547; Sphingolipids, Steroids, Lipopolysaccharides and Related Probes - Section 13.3) and BODIPY FL C₅-lactosylceramide (D13951, B34402; NBD- and BODIPY(R)-Dye–Labeled Sphingolipids). 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 kit contains:

• Mouse IgG_2b 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(R) Alexa Fluor(R) 488 Endoplasmic Reticulum Labeling Kit)

The SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit can be used in conjunction with probes for other cell targets to achieve multicolor cell staining.

Anti–Human Golgin-97

Originally isolated from the serum of a patient with the autoimmune disease known as Sjögren's syndrome, anti–human golgin-97 antibodies recognize a 97,000-dalton protein called golgin-97, a member of the granin family of proteins and a peripheral membrane protein localized on the cytoplasmic face of the Golgi apparatus. Because the antibody recognizes a protein unique to the Golgi apparatus of most vertebrate species, it is a useful for immunodetection and identification of the Golgi apparatus in cells, Western blotting and immunoprecipitation; however, we have a report that it may not work for rat cells (, , ). Molecular Probes offers mouse IgG₁ monoclonal anti–human golgin-97 antibody (A21270, Anti-Human Golgin-97, Mouse Monoclonal CDF4).

Monoclonal Antibody Specific for the Yeast Late-Golgi Compartment

For detection of the late-Golgi compartment of yeast, we offer a mouse monoclonal antibody to Vps10p (A21274, Monoclonal Antibodies for Yeast Cell Biology). Yeast Vps10p is an ~180,000-dalton membrane protein that resides in the late-Golgi compartment. This monoclonal antibody is a valuable tool for detecting the late-Golgi compartment and late-Golgi membranes in yeast subcellular fractions by Western blot. However, due to the low abundance of the Vps10p antigen, this monoclonal antibody cannot be used to visualize Golgi by immunocytochemistry unless the signal is amplified, such as with our tyramide signal amplification (TSA) technology (Tyramide Signal Amplification (TSA) Technology - Section 6.2).

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. Molecular Probes prepares WGA (Wheat Germ Agglutinin Conjugates) and Con A (Concanavalin A Conjugates) conjugates whose fluorescence spans the entire visible and near-infrared spectrum (Molecular Probes' selection of 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 (Fluorescent Conjugates of Lectin GS-II from Griffonia simplicifolia).

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, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 conjugates of HPA (L11271, L32452, L32453, L32454; Lectin HPA Conjugates) should be particularly useful for Golgi staining.