(A) Transmission electron micrograph demonstrating layer structure of fungal cell wall (courtesy of C. Rondeau). The plasma membrane is tightly connected to a thick layer of β-glucan network. Mannoproteins are linked to β-glucan and protrude outside of this layer to make up a dense coat. Schematic adapted from [5].

(B–D) There is little β-glucan on live C. albicans or S. cerevisiae that is exposed and accessible to the anti-β-glucan antibody (B), the Dectin-CRD (C), or the Dectin-CRD-anti-Myc probe (D). The staining with anti-β-glucan and Dectin-CRD-anti-Myc is nearly indistinguishable, and is more specific than that with the directly labeled Dectin-CRD. The Dectin-CRD-anti-Myc has the same size as an antibody and the same specificity as Dectin-1. The difference in staining between the β-glucan-binding reagents (B and D versus C) is likely due to the size of the reagents (IgG has dimensions of approximately 95 Å × 171 Å [33], while the CRD of CD69, which is similar to the Dectin-1 CRD, has dimensions of 44 Å × 32 Å × 30 Å [34]) relative to the estimated pore size of the S. cerevisiae cell wall (58 Å [23]), and thus the monomeric Dectin-CRD likely has more access to smaller areas of exposed β-glucan.