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. 2009;1(3):244-53.
doi: 10.1159/000173694. Epub 2008 Nov 12.

Actin and phosphoinositide recruitment to fully formed Candida albicans phagosomes in mouse macrophages

Sigrid E M Heinsbroek  1 , Lynn A Kamen, Philip R Taylor, Gordon D Brown, Joel Swanson, Siamon Gordon
Affiliations

Actin and phosphoinositide recruitment to fully formed Candida albicans phagosomes in mouse macrophages

Sigrid E M Heinsbroek et al. J Innate Immun. 2009.

Abstract

Candida albicans is a dimorphic yeast that enters macrophages (Mphi) via the beta-glucan receptor dectin-1. Phagocytosis of C. albicans is characterized by actin polymerization, Syk kinase activation and rapid acquisition of phagolysosomal markers. In mice, C. albicans are able to resist the harsh environment of the phagosome and form pseudohyphae inside the phagolysosomal compartment, eventually extending from the Mphi. In this study, we investigated these unique C. albicans phagosomes and found that actin localized dynamically around the phagosomes, before disintegrating. Membrane phosphoinositides, PI(4,5)P(2), PI(3,4,5)P(3), PI(3,4)P(2), and PI(3)P also localized to the phagosomes. Localization was not related to actin polymerization, and inhibitor studies showed that polymerization of actin on the C. albicans phagosome was independent of PI3K. The ability of mature C. albicans phagosomes to stimulate actin polymerization could facilitate the escape of the growing yeast from the Mphi.

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Figures

Fig. 1
Fig. 1
Actin polymerization on C. albicans (pseudo)hyphae growing inside phagosomes. a Images of a RAW264.7 Μϕ 2 h after yeast phagocytosis stained with FITC-phalloidin. The growing (pseudo)hypha was marked with increased actin. b FITC-phalloidin staining of primary bone marrow-derived Μϕ 2 h after C. albicans yeast phagocytosis. Actin polymerization can be observed around the (pseudo)hypha. c RAW264.7 Μϕ stained for LAMP-1 (green) and actin (red) 2 h after phagocytosis of C. albicans yeast. The two (pseudo)hyphae growing inside the Μϕ are marked with LAMP-1, but only 1 phagosome exhibited actin polymerization. d Three-dimensional reconstruction of actin polymerization around a C. albicans phagosome. RAW264.7 Μϕ 2 h after phagocytosis of C. albicans yeast. The first panel is a transmission image of a single C. albicans (pseudo)hypha growing from one Μϕ into the next (arrow). The second panel is the equivalent fluorescent image showing actin in red and the lysosomes in green. The last two panels show three-dimensional reconstructions of a Z-series taken through the cells. Scale bar corresponds to 5 μm.
Fig. 2
Fig. 2
Live confocal microscopy with GFP-actin-transfected RAW264.7 Μϕ. Transmission image (a) and fluorescent images (b) of C. albicans (pseudo)hyphae growing inside a Μϕ (arrow). Fluorescent images were taken at different time points and show actin movement around the (pseudo)hyphae. See also movie 1. Scale bar corresponds to 5 μm.
Fig. 3
Fig. 3
Recruitment of PI(4,5)P2 during C. albicans growth inside phagosomes. Scale bar corresponds to phase contrast, YFP and CFP images showing RAW Μϕ infected with C. albicans. After a 2-hour incubation, Plδ1PH-YFP increased on the C. albicans phagosomes, along with actin-CFP. The three rows illustrate three different cells from different experiments. Arrows indicate the localization of the fluorescent chimera to the phagosome. Plcδ1PH is a marker for PI(4,5)P2. Scale bar corresponds to 5 μm.
Fig. 4
Fig. 4
Recruitment of actin and phosphoinositides at the C. albicans phagosomes. Average of maximum IP/IC for actin, Plcδ1PH, AktPH and FYVE-YFP. Plot showing the increased localization of either actin-CFP or the PH domain-YFP fusion on C. albicans phagosomes. For each PH domain proxy, the maximum level of localization (IP/IC) on the phagosome was averaged for each movie. This value was then plotted. Error bars reflect standard error of the mean.
Fig. 5
Fig. 5
Recruitment of PI(3,4,5)P3 and PI(3,4)P2 to growing C. albicans phagosomes. Phase contrast, YFP and CFP images showing RAW Μϕ transfected to express AktPH-YFP and actin-CFP, and infected with C. albicans. After a 2-hour incubation, AktPH-YFP increased on the C. albicans phagosomes, along with actin-CFP. The 3 rows illustrate 3 different cells from different experiments. Arrows indicate the localization of the fluorescent chimera to the phagosome. AktPH is a marker for PI(3,4,5)P3 and PI(3,4)P2. Scale bar corresponds to 5 μm.
Fig. 6
Fig. 6
Recruitment of PI(3)P to growing C. albicans phagosomes. Phase contrast, YFP and CFP images showing RAW Μϕ expressing 2xFYVE-YFP, a marker for PI(3)P, and actin-CFP, and infected with C. albicans. After a 2-hour incubation, FYVE-YFP increased on the C. albicans phagosomes, along with actin-CFP. The 3 rows illustrate 3 different cells from different experiments. Arrows indicate the localization of the fluorescent chimera to the phagosome. Scale bar corresponds to 5 μm.
Fig. 7
Fig. 7
Recruitment of actin to fully formed C. albicans phagosomes during PI3K inhibition. Phase contrast and fluorescence images showing actin-mCherry recruited to C. albicans phagosomes after treatment with LY294002. Scale bar corresponds to 5 μm.
Fig. 8
Fig. 8
Proposed model for events on growing C. albicans phagosomes. During phagosome formation, PI(3,4,5)P3, PI(3,4)P2 and PI(4,5)P2 form at the site of phagocytosis. These PIs are converted during maturation; at later stages PI(3)P is predominantly found at the phagosome. C. albicans growth inside the phagosome is accompanied by large amounts of dynamic actin polymerization and association of the four PIPs.
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