Simultaneous Nanoscale Imaging of Chemical and Architectural Heterogeneity on Yeast Cell Wall Particles

Abstract

Particles extracted from yeast cell walls are naturally occurring immunomodulators with significant therapeutic applications. Their biological function has been thought to be a consequence of the overall chemical composition. In contrast, here we achieve direct nanoscale visualization of the compositional and structural heterogeneity of yeast cell wall particles and demonstrate that such nanoscale heterogeneity directly influences the receptor function of immune cells. By combining peak force infrared (PFIR) microscopy with super-resolution fluorescence microscopy, we achieve simultaneous chemical, topographical, and mechanical mapping of cell wall particles extracted from the yeast Saccharomyces cerevisiae with ≈6 nm resolution. We show that polysaccharides (β-glucan and chitin) and proteins are organized in specific nonuniform structures, and their heterogeneous spatial organization leads to heterogeneous recruitment of receptors on immune cell membranes. Our findings indicate that the biological function of yeast cell wall particles depends on not only their overall composition but also the nanoscale distribution of the different cell wall components.

Figure 1.

Identification of chemical heterogeneity on the surface of zymosan particles. (A) Schematic illustration showing the structure features (cell wall, bud scar, and nucleus core) of a zymosan particle. (B) SEM image of a zymosan particle. (C) Schematic illustration of the signal generation of PFIR microscopy. An AFM is operated in the peak force tapping mode, synchronized external laser pulses are focused to the tip and the sample region, and the deflection of the cantilever (force curve) is monitored in real time through a built-in position-sensitive detector (PSD). Mechanical information and IR absorption signal are obtained from the regular force curve (blue) and the force curve with a laser pulse (red). (D) Topography image of a zymosan particle. Dots marked as P1–P4 indicate the four spots where IR signals were measured. (E) PFIR spectral scans at the four different positions marked as P1–P4 in (D). (F) Bright-field and fluorescence images of a zymosan particle labeled with Alexa Fluor 568-tagged wheat germ agglutinin (WGA). (G) Maximum projection of three-dimensional (3-D) SIM images of bud scars on WGA-labeled zymosan particles. (H) Bright-field and fluorescence images of an Alexa Fluor 568-labeled zymosan particle. Scale bars in all images: 2 μm.

Figure 2.

Simultaneous mapping of structural, mechanical, and chemical images of zymosan particles. (A) SEM image of a zymosan particle after air drying. (B–F) Corresponding topography (B), PFIR signal at 1030 cm⁻¹ (C) and 1630 cm⁻¹ (D), Young’s modulus (E), and adhesion (F) images of the same zymosan particle. (G, H) The line scan profiles of height, PFIR intensities at 1030 and 1630 cm⁻¹, Young’s modulus, and adhesion along the dash line indicated in (B). (I) Schematic illustration showing the component distribution on zymosan particles. Scale bars in all images: 2 μm.

Figure 3.

Nanoscale mechanical and chemical mapping of zymosan bud scars. (A) Topography image of a zymosan bud scar and PFIR spectral scans at seven different positions marked as P1–P7. Scale bar: 500 nm. (B) Topography, PFIR signal at 1030 and 1630 cm⁻¹, Young’s modulus, and adhesion images of a bud scar. Scale bars: 500 nm. (C, D) Zoom-in images of merged PFIR signals (red for 1030 cm⁻¹ and green for 1630 cm⁻¹), tracked mesh skeletal structure, and line scan profiles of PFIR intensities inside the bud scar (C) and outside the bud scar (D), areas outlined in the white boxes in (B). Scale bars: 100 nm.

Figure 4.

Heterogeneous recruitment of Dectin-1 receptors on phagosomes containing zymosan particles. Fluorescently labeled zymosan particles (red) were internalized by RAW264.7 macrophages expressing GFP-Dectin-1 (green). (A) Schematic illustration of phagosomes encapsulating zymosan particles inside a macrophage cell. Three-dimensional (3-D) SIM images (B) and constructed 3-D surface models (C) show the distribution of GFP-Dectin-1 on the membrane of phagosomes encapsulating zymosan particles. Scale bars in all images: 1 μm.