sDectin binds bud scars and birth scars. (A) sDectin binding to S. cerevisiae depends on proper bud separation. Wild-type (top) or endochitinase (cts1)-deficient (middle and lower panels) S. cerevisiae yeast were stained with sDectin and imaged by confocal microscopy. (B) Dectin-1 binds S. cerevisiae and C. albicans equivalently. C. albicans and S. cerevisiae yeast stained with sDectin (solid lines) and analyzed by flow cytometry demonstrate equivalent laminarin-sensitive (dashed line) binding compared to controls (filled). (C) S. cerevisiae bud scars and birth scars were stained with calcofluor white, and staining colocalized with sDectin as assessed by fluorescence microscopy.

Dectin-1 mediates phagocytosis of C. albicans yeast. (A) Yeast trigger phagocytosis through Dectin-1. HEK293 cells stably expressing Dectin-1 were fed FUN-1-labeled Candida yeast. Internalization of live yeast was detected by flow cytometry. Internalization was completely inhibited by soluble β-glucan (laminarin), as indicated. (B) Dectin-1 is recruited to yeast phagosomes. Bone marrow-derived macrophages were stained with anti-Dectin-1 antibody, showing plasma membrane staining (top), which was blocked by recombinant Dectin-1 (middle). Dectin-1 was enriched on early phagosomes in macrophages fed Candida yeast (bottom, arrow), and staining was diminished on later phagosomes (arrowhead). (C) C. albicans yeast were preincubated with FITC-labeled wheat-germ agglutinin (WGA-FITC) to identify bud and birth scars, and fed to bone marrow macrophages for 10 min. Dectin-1 recruitment to phagosomes was assessed by immunofluorescence microscopy with anti-Dectin-1 antibody. (D) Bone marrow macrophages were pretreated as indicated, and fed Candida yeast for 30 min. Internalization was quantified by microscopy and is expressed as phagocytic index (particles internalized per 100 macrophages).

sDectin stains C. albicans yeast. (A) Zymosan particles were labeled with sDectin, and binding was assessed by flow cytometry. sDectin strongly stained zymosan particles (unfilled line and inset confocal image) compared to control-stained particles (filled). (B) Binding of sDectin to zymosan is β-glucan dependant. sDectin binding in the presence of the indicated doses of yeast cell wall components was assessed by flow cytometry and expressed as % mean fluorescence of maximal binding. (C) Binding of sDectin to C. albicans was assessed by confocal microscopy. C. albicans yeast was stained with sDectin, either in the presence (bottom) or absence (top) of laminarin (soluble β-glucan). (D) Binding of sDectin to C. albicans yeast was assessed by flow cytometry. As indicated, yeast were pretreated with proteinase K, or β(1,3)-glucanase. (E) Live (left panel) or heat-killed (right panel) C. albicans yeast were stained with sDectin and imaged by confocal microscopy.

Dectin-1 recognizes C. albicans yeast, but does not bind filaments. (A) Candida yeast (top) or filaments (bottom) stained with sDectin were imaged by confocal microscopy. (B) Binding of sDectin to yeast and filaments was quantified using an enzyme-linked assay. Strong laminarin (β-glucan)-sensitive binding to yeast was detected, but binding to filaments could not be detected over the background (control). (C) Yeast, but not filaments, activate Dectin-1. RAW264.7 cells stably expressing epitope-tagged Dectin-1 were incubated with the indicated stimuli for 15 min. Lysates were prepared, and Dectin-1 expression was confirmed by immunoblotting for the tag. Tyrosine-phosphorylated protein was immunoprecipitated with antiphosphotyrosine antibodies and Dectin-1 was detected by immunoblotting. (D) Candida filaments do not block Dectin-1 activation by zymosan. Cells were stimulated with zymosan in the presence or absence of filaments, as indicated, and Dectin-1 activation was assessed as above. (E) C. albicans filaments formed in vivo do not bind sDectin. C. albicans yeast (left panels) were injected intraperitoneally and filaments were recovered 24 h later (right panels), and stained with calcofluor white (top panels) and sDectin (bottom panels).

Candida yeast activate production of ROS through Dectin-1, but filaments do not. (A–F) Production of ROS by bone marrow-derived macrophages was measured by luminol-based chemiluminescence. (A) Candida yeast (50 μg/ml) activate production of ROS, while Candida filaments (200 μg/ml) fail to activate. (B) Filaments do not actively inhibit ROS production, since zymosan (30 μg/ml)-induced ROS production was unaffected by coincubation with filaments (200 μg/ml). (C) β-Glucan recognition is critical for ROS production induced by Candida yeast (100 μg/ml) since laminarin (1 mg/ml) blocked the response. (D) Stimulation of ROS production by sDectin-coated yeast (100 μg/ml) was blocked compared to uncoated yeast (100 μg/ml). (E) Preincubation of macrophages with anti-Dectin-1 antibody blocked ROS production induced by yeast (100 μg/ml). Control cells were treated with preimmune rabbit serum. (F) Pretreatment with anti-Dectin-1 serum had no effect on the induction of ROS by S. aureus (250 μg/ml), a stimulus that does not contain β-glucan.