Electron Microscopic Visualization of Receptor-mediated Endocytosis of DiI-labeled Lipoproteins by Diaminobenzidine Photoconversion

Materials and Methods

Results

We attempted to visualize DiI-labeled HDLp in fat body tissue of young adult locusts 4 days after imaginal ecdysis, a developmental stage for which we have previously shown that the fat body cells internalize HDLp (Dantuma et al. 1997). In an initial experiment, fat body tissue that had been incubated with 0.3 mg/ ml DiI-labeled HDLp for 90 min was used for DAB photoconversion. As a control, fat body tissue was incubated with DiI-labeled HDLp in the presence of an excess of unlabeled HDLp. During the 1–hr photoconversion of these samples, strong bleaching of the fluorescent signal was established, which coincided with the appearance of dark-brown punctate staining located in the cytoplasm of the fat body cells. To our surprise, we observed similar punctate staining in the control sample, in which an excess of unlabeled HDLp was included (Figure 1A). We have previously demonstrated, using fluorescence microscopy, that under this condition internalization of the DiI-labeled HDLp was almost completely abrogated by the competition between labeled and unlabeled HDLp (Dantuma et al. 1997). The punctate staining in the control was not caused by a higher sensitivity of the photoconversion technique, because we found in a subsequent control experiment that fat body tissue that had not been incubated with DiI-labeled HDLp also showed punctate staining on DAB photoconversion (not shown). On electron microscopic examination of the stained spots in the negative control, we observed dark-stained smooth vesicles with diameters ranging from 200 to 400 nm (Figure 2A). Illumination of fat body tissue appeared to stimulate this apparent background staining, because it was almost (but not completely) absent in fat body tissue that had been treated identically except for the illumination (not shown).

OPEN IN VIEWER

Figure 1

Light micrographs of semithin sections of photoconverted fat body stained with methylene blue. Fat body tissue was incubated with 0.3 mg/ml DiI-labeled HDLp in the presence of a 50-fold excess of unlabeled HDLp and was photoconverted in the absence of ATA (A) or in the presence of ATA (B). Fat body was incubated with 0.3 mg/ml DiI-labeled HDLp and photoconverted in the presence of ATA (C). LD, lipid droplet; N, nucleus. Bars = 10 μm.

OPEN IN VIEWER

Figure 2

Electron micrographs of control fat body tissue that had not been incubated with DiI-labeled HDLp after photoconversion with DAB in the absence (A) or presence (B) of ATA and H₂O₂. Dark staining of putative peroxisomes was observed when ATA and H₂O₂ were omitted (arrows in A), which was completely inhibited by ATA and H₂O₂ (arrow in B indicates a structure that is morphologically similar to the stained structures in A). LD, lipid droplet. Bars = 300 nm.

In the samples that had been incubated with DiIlabeled HDLp, in addition to the background-stained vesicles typical endosomal structures were found to contain DAB reaction product, as discussed below. Because the background-stained vesicles could not be distinguished from putative late endosomal or lysosomal structures containing DiI-labeled HDLp, it was a prerequisite to reduce the background staining.

No punctate autofluorescence was observed in nonphotoconverted control fat body samples that had not been incubated with DiI-labeled HDLp or had been incubated with DiI-labeled HDLp in the presence of an excess of unlabeled HDLp when examined with fluorescence microscopy (Dantuma et al. 1997). This implies that the aforementioned background staining in photoconverted samples cannot be due to the conversion of DAB by an endogenous fluorescent substance. We considered the possibility that the background staining was caused by a DAB reaction product generated by endogenous catalase activity. Therefore, we suspected that the stained vesicles in the negative control were peroxisomes because these organelles contain catalase, an enzyme that converts DAB in the presence of H₂O₂ in a light- and electron-dense product (Herzog and Fahimi 1974). The tissue was fixed at initiation of the incubation in the DAB-containing buffer, which favors our hypothesis because fixation activates the potency of catalase to convert DAB (Fahimi 1968). However, H₂O₂, which has been reported to be essential for DAB staining of peroxisomes (Fahimi 1968), was omitted in our initial experiments. We repeated the DAB photoconversion of fat body samples but now the tissue was preincubated with ATA, which inactivates the peroxisomal catalase (Margoliash and Novogrodsky 1958). In this experiment, H₂O₂ was included because this is also required to establish inactivation of catalase by ATA. In addition, ATA was supplemented during the preincubation with DAB and illumination of the tissue. In the presence of ATA, no staining was observed on photoconversion of fat body tissue incubated with DiI-labeled HDLp in the presence of an excess of unlabeled HDLp (Figure 1B). At the electron microscopic level, no DAB reaction product was observed in control fat body tissue (Figure 2B).

In contrast to the complete abrogation of the punctate background staining by ATA, this catalase inhibitor did not affect the actual DAB photoconversion induced by the DiI-labeled HDLp. Fat body tissue that had been incubated with DiI-labeled HDLp still revealed an apparent punctate staining in the presence of ATA (Figure 1C).

Transmission electron microscopy of photoconverted fat body samples incubated with DiI-labeled HDLp in the presence of ATA revealed that the punctate staining represents labeling of intracellular vesicles, which is in good agreement with receptor-mediated uptake of HDLp (Figure 3A). Occasionally, in some of these vesicles spherical structures with dimensions identical to those of lipoprotein particles could be distinguished. These putative lipoproteins were visible either as dark spherical particles (Figure 3B) or as typically negative-stained particles (Figure 3C), similar to those observed after negative staining of HDLp (Van Antwerpen et al. 1988). Putative lipoproteins were observed at the cell surface and in structures resembling early endosomes (not shown). In a distinct type of vesicle that resembled late endosomes, a more homogeneous luminal staining was representative.

Apparently, the emitted light of DiI converted DAB only in a small area surrounding the fluorescent dye. Moreover, this area did not exceed the lumen of the labeled vesicles. Even when vesicles were heavily stained, no labeling was observed outside the lumen.

Discussion

Visualization of a fluorescent signal by photoconversion of DAB into an electron-dense product by the emitted light of fluorescent dyes has been used mainly in neuroanatomical studies, in which different dyes, such as Lucifer Yellow (Maranto 1982), DiI (Von Bartheld et al. 1990; Lübke 1993), FITC, and TRITC (Sandell and Masland 1988) were all shown to be capable of producing a dark electron-dense staining on bleaching of these dyes by illumination in the presence of DAB.

OPEN IN VIEWER

Figure 3

Electron micrographs of photoconverted fat body tissue that had been incubated with 0.3 mg/ml DiI-labeled HDLp. (A) Overview of labeled structures (arrows) in fat body cells. The intercellular space (arrowheads) and the superficial cell surface (asterisk) are indicated. (B) The spherical shapes of putative lipoprotein particles (arrow) can be observed in invaginations of the plasma membrane that borders the intercellular space. (C) Negative staining of putative lipoprotein particles in an endocytic vesicle (arrow) located just beneath the plasma membrane (arrowheads). LD, lipid droplets. Bars: A = 500 nm; B,C = 100 nm.

To our knowledge, only a single study has been published (Tabas et al. 1990) in which the usefulness of the photoconversion technique for detailed intracellular localization of a fluorescently labeled ligand is attempted. Tabas and co-workers used the classical DAB photoconversion method to stain DiI-labeled, lipoprotein- containing endosomes in macrophages. In addition to the expected staining of endosomes, they observed stained vesicles that were present even in cells that had not been incubated with DiI-labeled lipoproteins. They mention that because of the presence of these stained vesicles, which resembled multivesicular bodies or lysosomes, only the putative early endosomes, with characteristic electron-lucent centers, could be definitely identified as endosomal structures containing DiI-labeled lipoproteins. We also observed DAB staining in smooth vesicles present in photoconverted fat body samples that had not been incubated with DiI-labeled HDLp. Because this staining appeared to be completely inhibited by photoconversion of DAB in the presence of ATA, which inhibits peroxisomal catalase (Margoliash and Novogrodsky 1958), the background-stained structures are most likely peroxisomes. In retrospect, this is not very surprising if we consider the reported activation of peroxisomal catalase by fixation (Herzog and Fahimi 1974) and the fact that staining with DAB is routinely used to identify peroxisomes (Fahimi 1968). Apparently, the illumination of the tissue during the photoconversion compensated sufficiently for the absence of H₂O₂, which is known to be a prerequisite for peroxisomal staining (Fahimi 1968). Indeed, we found only very faint DAB staining in the supposed peroxisomes in tissue that had not been photoconverted and from which H₂O₂ had been omitted.

The background staining of peroxisomes on DAB photoconversion appears not to be as general as might be expected, because in a previous study (Pagano et al. 1989), in which DAB photoconversion was used to investigate the cellular distribution of a fluorescent ceramide, no punctate staining was observed in photoconverted control cells that had not been incubated with the fluorescent probe. It is unlikely that this is due to the conditions of the illumination, because we observed weak but distinct peroxisomal staining after the DAB preincubation. We consider it most probable that the staining intensity reflects variations in the amount of endogenous catalase. Therefore, inhibition of catalase by ATA may also be a prerequisite for study of other intracellular fluorescent dyes with DAB photoconversion in cells that contain substantial amounts of this enzyme. As a consequence of the similar enzymatic activities of catalase and peroxidase, we expect that substantial amounts of the latter enzyme might also cause background staining during DAB photoconversion. It can be anticipated, on the basis of the present study, that inclusion of a peroxidase inihibitor during the photoconversion may reduce such background staining.

ATA did not affect the photoconversion of DAB because the distribution of the electron-dense DAB product, present on photoconversion by internalized DiI-labeled HDLp, corresponded with the previously reported staining pattern observed with fluorescence microscopy (Dantuma et al. 1997). In the presence of an excess of unlabeled lipoproteins and ATA, no DAB staining was observed, as is expected for saturable processes such as receptor-mediated endocytosis.

Photoconversion was established only in close proximity of the labeled lipoproteins according to the occasionally observed individual staining of lipoproteins and the fact that staining of vesicles was always restricted to the lumen and never diffused into the cytosol. This demonstrated that the DAB reaction product marked the precise location of the internalized fluorescently labeled lipoproteins.

In conclusion, we have demonstrated that DAB photoconversion can be used to localize a fluorescent ligand in the endosomal compartment at the electron microscopic level. Addition of ATA during DAB photoconversion is required for appropriate identification of labeled organelles in tissues that express high levels of catalase. Because ATA did not affect the true DAB photoconversion, it may be advisable to include this agent routinely when visualization of an intracellular dye is pursued.