Dynamic Changes in the Spatiotemporal Localization of Rab21 in Live RAW264 Cells during Macropinocytosis

Rab21, a member of the Rab GTPase family, is known to be involved in membrane trafficking, but its implication in macropinocytosis is unclear. We analyzed the spatiotemporal localization of Rab21 in M-CSF-stimulated RAW264 macrophages by the live-cell imaging of fluorescent protein-fused Rab21. It was demonstrated that wild-type Rab21 was transiently associated with macropinosomes. Rab21 was recruited to the macropinosomes after a decrease in PI(4,5)P2 and PI(3,4,5)P3 levels. Although Rab21 was largely colocalized with Rab5, the recruitment of Rab21 to the macropinosomes lagged a minute behind that of Rab5, and preceded that of Rab7. Then, Rab21 was dissociated from the macropinosomes prior to the accumulation of Lamp1, a late endosomal/lysosomal marker. Our analysis of Rab21 mutants revealed that the GTP-bound mutant, Rab21-Q78L, was recruited to the macropinosomes, similarly to wild-type Rab21. However, the GDP-bound mutant, Rab21-T33N, did not localize on the formed macropinosomes, suggesting that the binding of GTP to Rab21 is required for the proper recruitment of Rab21 onto the macropinosomes. However, neither mutation of Rab21 significantly affected the rate of macropinosome formation. These data indicate that Rab21 is a transient component of early and intermediate stages of macropinocytosis, and probably functions in macropinosome maturation before fusing with lysosomal compartments

Rab20 Regulates Phagosome Maturation in RAW264 Macrophages during Fc Gamma Receptor-Mediated Phagocytosis

<div><p>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.</p> </div

Time-lapse imaging showing the spatiotemporal relationship between Rab20 and Lamp1 during phagocytosis.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035663#pone.0035663.s004" target="_blank">Movie S4</a>.</p

Expression of GDP-locked mutant Rab20-T19N abrogates phagosomal acidification.

<p>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.</p

Live-cell imaging of Rab20 and Rab7 in RAW264 macrophages during phagocytosis of IgG-Es.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035663#pone.0035663.s003" target="_blank">Movie S3</a>.</p

Time-lapse images showing different dynamics of Lamp1 between a Rab20-T19N-expressing cell and a non-expressing cell during ingestion of IgG-Es.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035663#pone.0035663.s005" target="_blank">Movie S5</a>.</p

Live-cell imaging of GFP-Rab20 in RAW264 macrophages during phagocytosis of IgG-Es.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035663#pone.0035663.s001" target="_blank">Movie S1</a>.</p