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. 2018 Mar 19;7:e34798. doi: 10.7554/eLife.34798

Figure 1. Partial phagocytosis of C. albicans hyphae is associated with formation of an actin cuff.

Phagocytosis of C. albicans yeast (A) or hypha (B) by RAW-Dectin1 cells. After incubation with Candida-BFP, RAW-Dectin1 cells were fixed and extracellular C. albicans stained using Alexa594-conjugated concanavalin A (red). The fluorescence of the BFP is shown in white here and elsewhere to reveal the location of the Candida-BFP. Inset in (B): overexposure of the concanavalin A signal to show less intense, staining of the macrophage membrane (as in A). Scale bars: 5 μm and 10 μm, respectively. (C) Transmission electron micrograph of a RAW-Dectin1 cell with a partially internalized C. albicans hypha. Area of organelle clearance corresponding to the cuff structure is indicated in inset by arrows. Scale bar: 5 μm. (D) F-actin enrichment at the neck of partial phagosome. RAW-Dectin1 cells were allowed to internalize C. albicans hyphae, fixed and stained with fluorescent phalloidin (green). Actin cuff indicated with a bracket. Inset: overexposure to show the less intense cellular actin. Scale bar: 10 μm. (E–H) 3D rendering of a C. albicans hypha partially internalized by a RAW-Dectin1 cell. After incubation with Candida-BFP (white), RAW-Dectin1 cells were fixed and extracellular portions of the hyphae stained using Alexa647-conjugated concanavalin A (blue). Actin was stained with fluorescent phalloidin (red). Scale bar: 5 μm. (F) 3D rendering sliced near the middle of the tubular phagosome, (G) same as E showing only the hypha (white) and actin (red), and (H) same as E showing only the hypha (white) and concanavalin A (blue). (I) Stability of the actin cuff assessed by live cell imaging. RAW-Dectin1 cells expressing LifeAct-GFP were allowed to internalize C. albicans hyphae and imaged at defined intervals. Where indicated (105 min) 1 µM latrunculin A was added and recording continued. Actin cuff location indicated by bracket. Scale bar: 10 μm. Images are representative of ≥30 fields from ≥3 separate experiments of each type. In this and subsequent figures the outline of the phagocyte (when not readily apparent) is indicated by a dotted grey line.

Figure 1.

Figure 1—figure supplement 1. Actin cuffs are observed in both murine and human primary macrophages infected with C. albicans hyphae.

Figure 1—figure supplement 1.

Phagocytosis of Candida-BFP hyphae by murine BMDM (A) or human monocyte-derived macrophages (B). Following phagocytosis, cells were fixed and extracellular C. albicans stained using Alexa594 conjugated concanavalin A (red). F-actin was stained using fluorescent phalloidin (green). Scale bars: 5 μm.