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

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

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

Rac1-Dependent Lamellipodial Motility in Prostate Cancer PC-3 Cells Revealed by Optogenetic Control of Rac1 Activity

<div><p>The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P₃ and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.</p></div

Effect of LY294002 on the extended lamellipodial motility in PC-3 cells expressing constitutively active Rac1Q61L.

<p>PC-3 cells were transfected with pmCitrine-Rac1Q61L. The time-lapse images of phase-contrast and mCitrine-Rac1Q61L fluorescence were captured before (left) and after (right) the addition of LY294002. Kymographs were created to show the changes in length of a lamellipodium at the position of the two-headed line. The mCitrine-Rac1Q61L-expressing cell had an extended lamellipodium around its entire circumference. After the addition of 50 µM LY294002, the extended lamellipodium had shrunk only slightly, but the peripheral ruffling was pronounced. The kymographs show dynamic changes in length due to enhanced ruffling after the addition of LY294002. Scale bars, 10 µm.</p

Local and reversible control of lamellipodial dynamics by photomanipulation of PA-Rac1 activity.

<p>Time-lapse images of a PC-3 cell expressing mCherry-PA-Rac1 were acquired during PA-Rac1 photo-manipulation by local laser irradiation of different areas. Selected phase-contrast and mCherry fluorescence images are shown. First, region 1 was irradiated for 10 min. The irradiation was then moved to region 2. At 25 min, the irradiation was turned off. Selected time-lapse images of phase-contrast and mCherry fluorescence are shown. The extending and retracting lamellipodia are outlined in red and yellow, respectively. Scale bar, 10 µm.</p

PI3K-dependent WAVE2 recruitments to the leading edge of the extending lamellipodium during PA-Rac1 activation.

<p>PC-3 cells were co-transfected with pTriEx/mCherry-PA-Rac1 and pEGFP-N1-WAVE2. Phase-contrast, mCherry-PA-Rac1 (red fluorescence), and EGFP-WAVE2 (green fluorescence) images were acquired before and after PA-Rac1 photoactivation. PA-Rac1 photoactivation was repeated in the same cell region in the absence (control) or presence of 50 µM LY294002. The yellow arrowheads indicate that WAVE2 was recruited to the leading edge of the extending lamellipodium. In the presence of LY294002, WAVE2 was not recruited to the periphery of the cells where PA-Rac1 was photoactivated. The blue-dotted rectangle indicates the photoactivation area. Scale bar, 10 µm.</p