Compared with nonimmune controls, anti-Gpnmb antibodies specifically identified Gpnmb on the cell surface (Fig. 4B). Figure 4. Gpnmb localizes to autophagosomes in toward epithelial cells and is expressed at low levels at the plasma membrane. A) Gpnmb-GFP is localized to membranes of a network of intracellular vesicles when expressed in kidney epithelial cells in vitro. B) Cell surface … To identify the intracellular Gpnmb-containing compartment in resting cells, we stained Gpnmb-GFP-expressing LCC-PK1 epithelial cells with the lysosomal markers LAMP-1 and LysoTracker Red. However, we failed to detect overlap between the extensive Gpnmb staining and either of these lysosomal markers (Fig. 4C, D). Similarly, antibodies to the early endosomal marker EEA1 (Fig.

4E) and the peroxisomal marker catalase (not shown) also failed to colocalize with the tagged Gpnmb. Thus, Gpnmb did not localize to lysosomal, early-endosomal, and/or peroxisomal compartments. However, Gpnmb colocalized with both filipin (Fig. 4F) and oil red O (not shown), demonstrating that Gpnmb localized to cholesterol-rich vesicular membranes. Since the identity of Gpnmb+ vesicles was not apparent and since autophagosomes can form during cellular stress in the kidney epithelium (Supplemental Fig. S2A), we explored whether Gpnmb was detected in autophagosomes. A GFP-tagged form of the autophagy protein Atg8 (LC3) was stably expressed in epithelial cells. In cells not expressing recombinant Gpnmb, GFP-LC3 was localized diffusely in the cytoplasm and nucleus (Fig.

4G), but in Gpnmb+ cells or Gpnmb-RFP+ cells, GFP-LC3 was relocalized to intracellular compartments (Fig. 4G) and colocalized with Gpnmb-RFP. This pattern of GFP-LC3 was similar to its reorganization induced by amino acid starvation (not shown). Gpnmb+ epithelial cells were quantified for autophagy by scoring the presence of LC3+ vesicles (Fig. 4I), revealing a clear difference compared with the control cells. Thus, Gpnmb colocalized with LC3 to a vesicular compartment, suggesting that it may localize to autophagosomes. Gpnmb-expressing cells but not control cells exhibited many double membrane vesicles by electron microscopy (EM) consistent with autophagosomes (Fig. 4H). Analysis of random EM fields (��20,000) Carfilzomib of Gpnmb+ LLCPK1 cells revealed 1.0 �� 0.3 autophagosomes/field, whereas no autophagosomse were found in control cells. In rapamycin-treated cells, we observed 0.5 �� 0.2 autophagosomes/field. When Gpnmb+ cells were incubated with chloroquine to inhibit autophagsome degradation, Gpnmb+ vesicles rapidly expanded in size (Supplemental Fig. S2C). Moreover, the Gpnmb-RFP fusion protein accumulated rapidly in response to chloroquine treatment (Supplemental Fig. S2D).