Molecular Probes

Section 12.3 — Probes for Lysosomes, Peroxisomes and Yeast Vacuoles

Molecular Probes' acidotropic reagents can be used to stain lysosomes and yeast vacuoles, as well as several other types of acidic compartments such as trans-Golgi vesicles, endosomes and subpopulations of coated vesicles in fibroblasts, secretory vesicles in insulin-secreting pancreatic β-cells, acrosomes of spermatozoa and plant vacuoles. Lysosomes contain glycosidases, acid phosphatases, elastase, cathepsins, carboxypeptidases and a variety of other proteases. Chapter 10 describes a number of substrates for detecting the activity of these hydrolytic enzymes. An excellent compendium of human diseases that affect intracellular transport processes through lysosomes, Golgi and endoplasmic reticulum (ER) has been published.

Like lysosomes, peroxisomes are single membrane–bound vesicles that contain digestive enzymes. The chief function of these basic organelles is to enzymatically oxidize fatty acids and to subsequently catalyze the breakdown of H₂O₂, a by-product of fatty acid degradation. Recently, interest in peroxisomes has increased, especially studies related to peroxisomal origin and maintenance. Morphological abnormalities in peroxisomes related to disease states and diet have also been the subject of current research. The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit (S34201), described below, provides an antibody-based method for labeling peroxisomes in fixed cells.

LysoTracker Probes: Acidic Organelle–Selective Cell-Permeant Probes

Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes. One frequently used probe for acidic organelles, DAMP (D1552), is not fluorescent and therefore must be used in conjunction with anti-DNP antibodies (Section 7.4) directly or indirectly conjugated to a fluorophore or enzyme in order to visualize the staining pattern. The fluorescent probes neutral red (N3246) and acridine orange (A1301, A3568) are also commonly used for staining acidic organelles, though they lack specificity.

These limitations have motivated us to search for alternative acidic organelle–selective probes, both for short-term and long-term tracking studies. The LysoTracker probes (LysoTracker(R) and LysoSensor Probes) are fluorescent acidotropic probes for labeling and tracing acidic organelles in live cells. These probes have several important features, including high selectivity for acidic organelles and effective labeling of live cells at nanomolar concentrations. Furthermore, the LysoTracker probes are available in several fluorescent colors (Table 12.7, Figure 12.34), making them especially suitable for multicolor applications.

The LysoTracker probes, which comprise a fluorophore linked to a weak base that is only partially protonated at neutral pH, are freely permeant to cell membranes and typically concentrate in spherical organelles (, , , ). We have found that the fluorescent LysoTracker probes must be used at very low concentrations — usually about 50 nM — to achieve optimal selectivity. Their mechanism of retention has not been firmly established but is likely to involve protonation and retention in the organelles' membranes, although staining is generally not reversed by subsequent treatment of the cells with weakly basic cell-permeant compounds. Furthermore, the larger acidic compartments of cells stained with LysoTracker Red DND-99 (L7528; , ) usually retain their staining pattern following fixation with aldehydes. Simultaneous staining of lysosomes by two LysoTracker dyes — LysoTracker Yellow HCK-123 (L12491) and LysoTracker Red DND-99 (L7528) — yields identical staining patterns when viewed through either the bandpass filter set appropriate for fluorescein or a longpass filter set appropriate for rhodamine ().

LysoTracker Green DND-26 (L7526) was used to identify acidic compartments in a study of a membrane protein that facilitates vesicular sequestration of zinc, to visualize acidic organelles labeled with rhodamine B in denervated skeletal muscle and to assess acrosomal integrity in cryopreserved bovine spermatozoa. This LysoTracker probe also proved useful in a continuous assay for the secretion of pulmonary surfactant by exocytosis of lamellar bodies. LysoTracker Red DND-99 provided researchers with a probe for examining lysosome damage in Trypanosoma brucei after specific uptake of cytokine tumor necrosis factor-α, for studying apoptosis in organogenesis-stage mouse embryos and for determining the subcellular localization of receptor and channel proteins.

Our kinetic studies on the internalization of LysoTracker Green DND-26 (L7526) indicate that the dye is taken up by live cells within seconds. Unfortunately, these lysosomal probes can exhibit an alkalinizing effect on the lysosomes, such that longer incubation with these probes can induce an increase in lysosomal pH. Therefore, we recommend incubating cells with these probes for only one to five minutes before imaging.

The LysoTracker probes were principally developed for fluorescence microscopy applications. The lysosomal fluorescence in LysoTracker dye–stained cells may constitute only a portion of total cellular fluorescence due to cellular autofluorescence or nonspecific staining. Consequently, successful application of these probes for quantitating the number of lysosomes by flow cytometry or fluorometry will likely depend on the particular cell lines and staining protocols used.

Image-iT LIVE Lysosomal and Nuclear Labeling Kit

The Image-iT LIVE Lysosomal and Nuclear Labeling Kit (I34202) provides two stains — red-fluorescent LysoTracker Red DND- 99 dye (excitation/emission maxima ~577/590 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm) — for highly selective staining of lysosomes and the nucleus, respectively, in live, green-fluorescent protein (GFP)–transfected cells (). When used according to the sample protocol, cell-permeant LysoTracker Red DND-99 dye provides highly selective lysosomal staining with minimal background. A significant amount of specific staining is retained after formaldehyde fixation, although some cytoplasmic background staining may be seen. Hoechst 33342 dye, a cell-permeant nucleic acid stain that is selective for DNA and spectrally similar to DAPI, is UV excitable and emits blue fluorescence when bound to DNA. This dye should not interfere with GFP fluorescence and is retained after fixation and permeabilization. It is not recommended that the dyes be combined into one staining solution; they should instead be used in separate labeling steps, with Hoechst 33342 staining first. Each Image-iT LIVE Lysosomal and Nuclear Labeling Kit contains:

• LysoTracker Red DND-99 dye
• Hoechst 33342 dye
• Labeling protocols (Image-iT(R) LIVE Lysosomal and Nuclear Labeling Kit)

Each kit provides enough staining solution for 500 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.

LysoSensor Probes: Acidic Organelle–Selective pH Indicators

For researchers studying the dynamic aspects of lysosome biogenesis and function in live cells, we have developed the LysoSensor probes — fluorescent pH indicators that partition into acidic organelles. The LysoSensor dyes (LysoTracker(R) and LysoSensor Probes) are acidotropic probes that appear to accumulate in acidic organelles as the result of protonation. This protonation also relieves the fluorescence quenching of the dye by its weakly basic side chain, resulting in an increase in fluorescence intensity. Thus, the LysoSensor reagents exhibit a pH-dependent increase in fluorescence intensity upon acidification, in contrast to the LysoTracker probes, which exhibit fluorescence that is not substantially enhanced at acidic pH.

We offer five LysoSensor reagents that differ in color and pK_a (Table 12.7). Because these probes may localize in the membranes of organelles, it is probable that the pK_a values listed in Table 12.7 will not be equivalent to those measured in cellular environments and that only qualitative and semiquantitative comparisons of organelle pH will be possible. The blue- and green-fluorescent LysoSensor probes are available with optimal pH sensitivity in either the acidic or neutral range (pK_a ~5.2 or ~7.5 in aqueous buffers). With their low pK_a values, LysoSensor Blue DND-167 (L7533) and LysoSensor Green DND-189 (L7535) are almost nonfluorescent except when inside acidic compartments, whereas LysoSensor Green DND-153 (L7534) is brightly fluorescent at neutral pH. LysoSensor Yellow/Blue DND-160 (L7545, ) is unique in that it exhibits both dual-excitation and dual-emission spectral peaks that are pH dependent (Figure 12.40). LysoSensor Yellow/Blue DND-160 exhibits predominantly yellow fluorescence in acidic organelles, and in less acidic organelles it exhibits blue fluorescence. Dual-emission measurements facilitate ratio imaging of the pH in acidic organelles such as lysosomes, myeloid leukemic cells and acidic vacuoles of plant cells. LysoSensor Yellow/Blue DND-160 has been used in conjunction with fluo-3 AM (F1241, F1242, F23915; Section 19.3) for simultaneous fluorescence measurements of H⁺ and Ca²⁺ in rabbit gastric glands in order to determine the kinetics of acid secretion upon activation of the cholinergic and the cAMP-dependent pathways.

We have prepared a 10,000 MW dextran conjugate of the LysoSensor Yellow/Blue dye (L22460). As this labeled dextran is taken up by the cells and moves through the endocytic pathway, the fluorescence of the LysoSensor dye changes from blue fluorescent in the near-neutral endosomes to longer-wavelength yellow fluorescent in the acidic lysosomes. The greatest change in fluorescence emission occurs near the pK_a of the dye at pH ~3.9. Unlike the cell-permeant LysoSensor dyes, LysoSensor Yellow/Blue dextran allows measurement of pH in lysosomes using either fluorescence microscopy () or flow cytometry.

The cell-permeant probes in this section can be used singly (or potentially in combination) to investigate the acidification of lysosomes and alterations of lysosomal function or trafficking that occur in cells. For example, lysosomes in some tumor cells have a lower pH than normal lysosomes, whereas other tumor cells contain lysosomes with higher pH. In addition, cystic fibrosis and other diseases result in defects in the acidification of some intracellular organelles, and the LysoSensor probes may prove useful in studying these aberrations. LysoSensor Green DND-189 has been used to selectively label acidic compartments within granule cell neurites and, along with LysoSensor Green DND-153, to examine the acidification of endosomes and lysosomes in a mutant CHO cell line. LysoSensor Yellow/Blue DND-160 was employed in a study that demonstrated the involvement of lysosomes in the acquired drug-resistance phenotype of a doxorubicin-selected variant of human U-937 myeloid leukemia cells.

Kinetic studies on the internalization of LysoSensor Yellow/Blue DND-160 indicate that the probe is taken up by live cells within seconds. Unfortunately, this lysosomal probe can exhibit an alkalinizing effect on the lysosomes, such that longer incubation with this probe can induce an increase in lysosomal pH. Therefore, it is a useful pH indicator only when incubation times are kept short; we recommend incubating cells for only one to five minutes before imaging.

As with the LysoTracker probes, the cell-permeant LysoSensor probes were originally developed for fluorescence microscopy applications. The lysosomal fluorescence in LysoSensor dye–stained cells may constitute only a portion of total cellular fluorescence due to cellular autofluorescence or nonspecific staining. Therefore, the successful application of these probes for quantitating the number of lysosomes or their pH by flow cytometry or fluorometry will likely depend on the particular cell lines and staining protocols used.

DAMP and Other Lysosomotropic Probes

DAMP

The reagent DAMP (N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, dihydrochloride; D1552; ) is a weakly basic amine that is taken up in acidic organelles of live cells. This cell-permeant acidotropic reagent can be detected with anti-DNP antibodies (Section 7.4), including those labeled with fluorochromes, ferritin, colloidal gold (as well as our NANOGOLD reagents for light and electron microscopy) or enzymes, making DAMP broadly applicable for detecting acidic organelles by electron and light microscopy. For example, DAMP has been used to investigate:

• Acidification of yolk platelets in sea urchin embryos
• Defective acidification of intracellular organelles in cells from cystic fibrosis patients
• Dependence on pH of the conversion of proinsulin to insulin in beta cells
• Development of autophagic vacuoles
• Location of intracellular acidic compartments during viral infection

As alternatives to DAMP, our cell-permeant fluorescent LysoTracker and LysoSensor probes described above have significant potential in many of these applications. Because they can be visualized directly without any secondary detection reagents, the LysoTracker and LysoSensor reagents enable researchers to study acidic organelles and follow their dynamic processes in live cells.

RedoxSensor Red CC-1 Stain

The fluorescence localization of our RedoxSensor Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060) appears to be based on a cell's cytosolic redox potential. Once it passively enters live cells, the RedoxSensor Red CC-1 stain may be oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes where it is oxidized. The differential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol. In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal (). The best method we have found to quantitate the distribution of the oxidized product is to use the mitochondrion-selective MitoTracker Green FM stain (M7514) in conjunction with the RedoxSensor Red CC-1 stain.

Other Lysosomotropic Probes

BODIPY FL histamine (B22461) combines the pH-insensitive, bright green-fluorescent BODIPY FL dye with the weakly basic imidazole moiety of histamine. When used at low concentrations, this probe selectively stains lysosomes ().

As with the LysoTracker and LysoSensor probes, the weak basicity of the amine group in Dapoxyl (2-aminoethyl)sulfonamide (D10460) leads to its accumulation in acidic organelles. Dapoxyl (2-aminoethyl)sulfonamide () uptake by the acidic lumen of the intact acrosome of mouse sperm is accompanied by significant enhancement of this probe's fluorescence. The fluorescence of Dapoxyl (2-aminoethyl)sulfonamide is considerably reduced upon loss of the pH gradient at the onset of the acrosome reaction.

Our high-purity neutral red (N3246) is a common lysosomal probe that stains lysosomes a fluorescent red. It has also been used to determine the number of adherent and nonadherent cells in a microplate assay and to stain cells in brain tissue.

In addition, dansyl cadaverine (D113) and the DNA intercalator acridine orange (A1301, A3568) have been reported to be useful lysosomotropic reagents. Dansyl cadaverine has been shown to selectively label autophagic vacuoles, at least some of which had already fused with lysosomes; it did not, however, accumulate in early or late endosomes.

Ligands for Receptor-Mediated Endocytosis

In addition to these lysosomotropic probes, Molecular Probes prepares a wide variety of low-density lipoproteins (LDL) and fluorescent transferrin conjugates. Once internalized, LDL dissociates from its receptor and ultimately accumulates in lysosomes. The contrasting fluorescence of DiI LDL (L3482, Section 16.1) and fluorescein transferrin (T2871, Section 16.1) permits their simultaneous use to follow the lysosomally directed pathway and the recycling pathways, respectively. The pH sensitivity of fluorescein transferrin has been exploited to investigate events occurring during endosomal acidification. Likewise, dextrans labeled with fluorescent pH indicators (Section 14.5, Section 20.4) can be used to monitor the uptake and internal processing of exogenous materials in acidic organelles through endocytosis.

SelectFX Alexa Fluor 488 Peroxisome Labeling Kit

Peroxisomes, single membrane–bound vesicles found in most eukaryotic cells, function to enzymatically oxidize fatty acids and to subsequently catalyze the breakdown of H₂O₂, a by-product of fatty acid degradation. The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit (S34201) provides all the reagents required for labeling peroxisomes in fixed cells, including cell fixation and permeabilization reagents. To specifically detect peroxisomes, this kit uses an antibody directed against peroxisomal membrane protein 70 (PMP 70), which is a high-abundance integral membrane protein in peroxisomes, 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. PMP 70 is significantly induced by administration of hypolipidemic agents, in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid β-oxidation enzymes. Each kit contains:

• Rabbit IgG anti–peroxisomal membrane protein 70 (PMP 70) antibody
• Highly cross-adsorbed Alexa Fluor 488 goat anti–rabbit 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 Peroxisome Labeling Kit)

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

Cell-Permeant Probes for Yeast Vacuoles

Biogenesis of the yeast vacuole has been extensively studied as a model system for eukaryotic organelle assembly. Using a combination of genetic and biochemical approaches, researchers have isolated a large collection of yeast vacuolar protein sorting (vps) mutants and characterized the vacuolar H⁺-ATPase (V-ATPase) responsible for compartment acidification. To facilitate the investigation of yeast vacuole structure and function, Molecular Probes offers membrane-permeant reagents and a Yeast Vacuole Marker Sampler Kit (Y7531, ), as well as several monoclonal antibodies directed against vacuolar proteins, including V-ATPase.

FUN 1 and FUN 2 Cell Stains: Vital Stains for Yeast

The FUN 1 () and FUN 2 cell stains (F7030, F13150) exploit endogenous biochemical processing mechanisms that appear to be well conserved among different species of yeast and other fungi. When used at micromolar concentrations, the FUN 1 and FUN 2 stains (Probes for Yeast Viability) are freely taken up by several species of yeast and fungi and converted from a diffusely distributed pool of yellow-green–fluorescent intracellular stain into compact red-orange– or yellow-orange–fluorescent intravacuolar structures, respectively (). This conversion requires both plasma membrane integrity and metabolic capability. Only metabolically active cells are marked clearly with fluorescent intravacuolar structures, while dead cells exhibit extremely bright, diffuse, yellow-green fluorescence (Figure 15.39, ). FUN 1 staining has been utilized to detect antifungal activity against Candida species and to measure susceptibility of fungi to fungicides by flow cytometry. The FUN 1 cell stain is also available as a component in our LIVE/DEAD Yeast Viability Kit (L7009, Section 15.3).

FM 4-64 and FM 5-95

One of our FM dyes, FM 4-64 has been reported to selectively stain yeast vacuolar membranes with red fluorescence (excitation/emission maxima ~515/640 nm). This lipophilic styryl dye is proving to be an important tool for visualizing vacuolar organelle morphology and dynamics, for studying the endocytic pathway and for screening and characterizing yeast endocytosis mutants. We offer FM 4-64 in 1 mg vials (T3166) or specially packaged in 10 vials of 100 µg each (T13320). The increasing number of successful applications for our FM dyes has prompted us to synthesize FM 5-95 (T23360), a slightly less lipophilic analog of FM 4-64 with essentially identical spectroscopic properties.

Yeast Vacuole Marker Sampler Kit

The Yeast Vacuole Marker Sampler Kit (Y7531, Probes for Yeast Vacuoles) contains sample quantities of a series of both novel and well-established vacuole marker probes that show promise for the study of yeast cell biology:

• 5-(and 6-)Carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA)
• CellTracker Blue CMAC
• Aminopeptidase substrate Arg-CMAC ()
• Dipeptidyl peptidase substrate Ala-Pro-CMAC
• Yeast vacuole membrane marker MDY-64 (, )

Our experiments have demonstrated that several cell-permeant derivatives of 7-amino-4-chloromethylcoumarin (CMAC) are largely sequestered within yeast vacuoles. The corresponding 7-amino-4-methylcoumarin derivatives are known to be substrates for yeast vacuolar enzymes. This sampler kit's three coumarin-based vacuole markers selectively stain the lumen of the yeast vacuole. To complement the blue-fluorescent staining of the lumen, we provide a novel green-fluorescent membrane marker MDY-64 for staining the yeast vacuole membrane. Membrane staining can also be accomplished using the red-fluorescent probe FM 4-64, as described below. The commonly used vacuole marker 5-(and 6-)carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) is supplied for use as a standard. Three of the components in the Yeast Vacuole Marker Sampler Kit — CellTracker Blue CMAC (C2110, Section 14.2), the proprietary yeast vacuole membrane marker MDY-64 (Y7536) and carboxy-DCFDA (C369, Section 15.2) — are also available separately for those researchers who find that one of these dyes is well suited for their application.

Monoclonal Antibodies for Yeast Cell Biology

In addition to the anti–yeast COX (OxPhos Complex IV) monoclonal antibodies described in Section 12.2, Molecular Probes offers several other immunoreagents that have proven useful for studying many aspects of cell biology with the yeast Saccharomyces cerevisiae (Table 12.8). These products include antibodies directed against yeast vacuole, cytosolic and endosomal proteins (described below) and antibodies directed against yeast endoplasmic reticulum and Golgi proteins (Section 12.4). We have selected this set of monoclonal antibodies because they are compatible with both Western blotting of denatured proteins and protein immunolocalization in fixed yeast cells. Other potential uses of these antibodies include the development of ELISAs to determine either the level of enrichment of a particular yeast organelle or the level at which the organelle contaminates a subcellular fraction. Detailed information regarding the IgG isotype and recommended working concentrations is provided with each product (Monoclonal Antibodies for Yeast Cell Biology). For detection of these monoclonal antibodies, Molecular Probes offers anti–mouse IgG secondary antibodies labeled with biotin, enzymes, NANOGOLD and Alexa Fluor FluoroNanogold 1.4 nm gold clusters, Captivate ferrofluid and a wide range of fluorophores and enzymes (Section 7.2).

Monoclonal Antibodies Specific for Yeast Vacuolar Membranes

For the detection of yeast vacuolar membranes, we offer four monoclonal antibodies directed against integral or peripheral membrane proteins. Two of the antibodies are specific for integral membrane proteins of the yeast vacuoles: Monoclonal 1D3 (A6458) is specific for the 70,000-dalton vacuolar alkaline phosphatase (ALP), the product of the PHO8 gene; this antibody is ideal for indirect immunofluorescence detection of vacuolar membranes in fixed yeast cells, as well as for detection of ALP by Western blotting. Monoclonal 10D7 (A6426) recognizes the 100,000-dalton vacuolar H⁺-ATPase (V-ATPase) subunit (the product of the VPH1 gene ) and is especially useful for Western blotting.

The other two antibodies are directed against peripheral membrane proteins of the yeast vacuoles, specifically two subunits of the V-ATPase complex that are associated with the cytosolic face of the yeast vacuolar membrane: monoclonal 13D11 (A6427) recognizes the 60,000-dalton subunit (the B-subunit, the product of the VMA2 or VAT2 gene), whereas monoclonal 8B1 (A6422) binds the 69,000-dalton subunit (the A-subunit, the product of the VMA1 or TFP1 gene). Both of these antibodies are useful for indirect immunofluorescence analysis, as well as for Western blotting.

Monoclonal Antibody Specific for the Yeast Vacuolar Lumen

For the detection of yeast vacuolar lumen, we offer monoclonal 10A5 (A6428). This antibody is specific for carboxypeptidase Y (CPY), a 61,000-dalton soluble glycoprotein located in the vacuolar lumen. Monoclonal 10A5 is useful for indirect immunofluorescence, as well as for Western blotting.

Monoclonal Antibody Specific for the Yeast Endosomal Compartment

For the detection of the yeast endosomal compartment, we have available anti-Pep12p monoclonal 2C3 (A21273). Yeast Pep12p, also known as Vps6p, is a 37,000-dalton yeast endosomal protein of the t-SNARE or syntaxin family. Pep12 is localized via a C-terminal transmembrane domain, and the epitope recognized by monoclonal 2C3 is located in the N-terminal cytosolic domain.

Monoclonal Antibody Specific for the Yeast Cytosol

For the detection of yeast cytosol, we offer a monoclonal antibody directed against the 44,700-dalton yeast cytosolic protein phosphoglycerate kinase (PGK). Monoclonal 22C5 (A6457) can be used for the detection of yeast PGK by Western blotting (Section 9.4).