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. 2012;7(4):e35663.
doi: 10.1371/journal.pone.0035663. Epub 2012 Apr 24.

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis

Youhei Egami  1 , Nobukazu Araki
Affiliations

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis

Youhei Egami et al. PLoS One. 2012.

Abstract

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Live-cell imaging of GFP-Rab20 in RAW264 macrophages during phagocytosis of IgG-Es.
Live RAW264 cells expressing GFP-Rab20 were put into contact with IgG-Es and observed by phase-contrast and fluorescence microscopy. Time-lapse images were acquired using the MetaMorph imaging system. Phase-contrast images are shown (upper panels). The elapsed time is indicated at the top. The binding of IgG-Es to the cell surface was set as time 0. It is noteworthy that Rab20 was associated with newly formed phagosomes (arrows). At least four cells were examined in four independent experiments. Similar results were obtained from four independent experiments. Scale bars: 5 µm. The corresponding video is Movie S1.
Figure 2
Figure 2. The association of Rab20 with phagosomal membranes persists following the loss of Rab5.
Live RAW264 cells co-expressing CFP-Rab20 (red) and GFP- Rab5 (green) were incubated with IgG-Es and monitored by phase-contrast and fluorescence microscopy. The elapsed time is indicated at the top. The binding of IgG-Es to the cell surface was set as time 0. It was found that Rab20 and Rab5 were transiently colocalized on formed phagosomes and that Rab20 remained associated with phagosomal membrane from which Rab5 was dissociated (arrows). Representative images from three independent experiments are shown. Scale bar: 5 µm. The corresponding video is Movie S2.
Figure 3
Figure 3. Live-cell imaging of Rab20 and Rab7 in RAW264 macrophages during phagocytosis of IgG-Es.
RAW264 macrophages co-expressing CFP-Rab20 (red) and YFP-Rab7 (green) were allowed to interact with IgG-Es and observed by phase-contrast and fluorescence microscopy. Although Rab20 was colocalized with Rab7 on phagosomes, the recruitment of Rab20 to phagosomes occurred earlier than that of Rab7 (arrows). Similar results were obtained from four independent experiments. Scale bar: 5 µm. The corresponding video is Movie S3.
Figure 4
Figure 4. Time-lapse imaging showing the spatiotemporal relationship between Rab20 and Lamp1 during phagocytosis.
RAW264 cells were co-transfected with GFP-Rab20 (green) and CFP-Lamp1 (red). Time-lapse images of phase-contrast, GFP-Rab20 and CFP-Lamp1 were taken by phase-contrast and fluorescence microscopy. The association of Rab20 with phagosomes was followed by Lamp1 accumulation (arrows). Similar results were obtained from fourindependent experiments. Scale bar: 5 µm. The corresponding video is Movie S4.
Figure 5
Figure 5. Time-lapse images showing different dynamics of Lamp1 between a Rab20-T19N-expressing cell and a non-expressing cell during ingestion of IgG-Es.
RAW264 macrophages co-expressing GFP-Rab20-T19N and CFP-Lamp1 were exposed to IgG-Es and observed by phase-contrast and fluorescence microscopy. The delivery of Lamp1 to formed phagosomes was inhibited in cells expressing Rab20-T19N (arrows) as compared to non-expressing controls (arrowheads). Similar results were obtained from four independent experiments. Scale bar: 5 µm. The corresponding video is Movie S5.
Figure 6
Figure 6. Expression of GDP-locked mutant Rab20-T19N abrogates phagosomal acidification.
Quantification of LysoTracker colocalization with internalized phagosomes in cells expressing Rab20 wt or Rab20-T19N was conducted. The number of formed phagosomes and LysoTracker-positive phagosomes was counted under a microscope. The results are expressed as a percentage of LysoTracker-positive phagosomes. The data are means ± SEM of four independent experiments. Student's t-test was used for statistical analysis. *P<0.05.
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