Figure 1.
Actin and microtubules inhibitors decrease C. neoformans internalization by macrophages.
Quantification of the attachment to macrophages and the internalization of C. neoformans yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the presence of the actin polymerization inhibitors cytochalasin D or latrunculin B (A and B) or the microtubule stabilizers nocodazole or paclitaxel (C and D). Graphs show normalized mean values and standard deviation from three experiments. *p<0.05; **p<0.01; ***p<0.001.
Figure 2.
Treatment with both cytochalasin D and nocodazole did not increase the inhibitory effect.
Quantification of the internalization (A) and the attachment (B) to macrophages of C. neoformans yeast cells from capsular (H99 and B3501) and acapsular (CAP67 and CAP 59) strains, in the absence of cytoskeletal inhibitors or in the presence of cytochalasin D and nocodazole. The metabolic viability of C. neoformans strains H99 and CAP59 was measured using the FUN®-1 dye (C) and the metabolic viability of macrophages was measured by MTS/PMS (D) after incubation with cytoskeletal inhibitors for 2 h. Yeast cells fixed with 70% ethanol, and macrophages with 4% formaldehyde, were used as a positive control for the loss of cell viability in each method. Graphs show normalized mean values and standard deviation from three experiments (A–B) and mean and standard deviation from absolute values of fluorescence intensity (C) and absorbance (D).*p<0.05; **p<0.01; ***p<0.001.
Figure 3.
Involvement of the cytoskeleton in the yeast-macrophage interaction.
Scanning electron microscopy of membrane extracted macrophages interacting with C. neoformans strains H99 (A) and CAP59 (B and C), showing cytoskeletal filaments associated with yeasts in untreated samples (A–B). After 5 µm nocodazole treatment (C) the area surrounding yeast cells appeared mostly devoid of cytoskeletal components but association with yeast still occurred (inset in C). Scale bars, 2 µm.
Figure 4.
Actin is recruited to the phagosome area during C. neoformans internalization.
Confocal laser scanning microscopy (z-stack series of confocal planes) of interacting macrophages and C. neoformans yeast cells from strains H99 (A and B) and CAP59 (C and D). Internalized yeasts identified by DIC (arrows in A and C) can be visualized in the context of host cell actin (red) and microtubule (green) cytoskeletons (B and D). Host cell DNA is labeled with DAPI (blue, indicated by the letter ‘N’) and yeast is labeled with calcofluor (blue, indicated by arrows). Actin, but not tubulin, is recruited to sites of yeast internalization. Scale bars, 5 µm.
Figure 5.
Actin recruitment is inhibited by cytochalasin D.
Confocal laser scanning microscopy of C. neoformans capsular strain H99 interacting with macrophages (single confocal plane). DIC showing internalized yeasts (arrows); and confocal images showing actin filaments (red), microtubules (green), yeast (blue) and host DNA (blue, indicated by ‘n’). Actin is recruited to the site of phagocytosis in untreated cells (A), and actin recruitment was inhibited by 0.5 µM cytochalasin D (B). In contrast, treatment with 5 µM nocodazole (C) or with a combination of nocodazole and cytochalasin D (D) did not inhibit actin recruitment to the phagosome area. Scale bars, 5 µm.
Figure 6.
Uptake of Cryptococcus strains by trigger-like and zipper-like structures.
Scanning electron microscopy of C. neoformans capsular strain H99 (A–E) and acapsular strain CAP59 (F–G) interacting with peritoneal macrophages. Improved preservation of macrophage membranes was obtained with routine SEM fixation (A–B; F–G), although post-fixation in the presence of sucrose provided better capsule preservation and allowed visualization of direct interactions between the capsule and host cell membranes, prior to internalization (C–E). Both trigger-like (arrow in A and F) and zipper-like (arrow-head in B and G) uptake structures were observed. Scale bars, 1 µm (A–C; F–G) and 0.5 µm (D–E).
Figure 7.
Transmission electron microscopy of C. neoformans strain H99 interacting with peritoneal macrophages.
Phagosomes of different sizes containing yeast cells were observed in the cytoplasm of macrophages, including ‘loose’ vacuoles typical of ‘triggered’ phagocytosis (asterisk in A), as well as ‘tight’ vacuoles typical of receptor-mediated phagocytosis (arrow in B). We observed direct interactions between the capsule of C. neoformans and the macrophage cell membrane (C, and higher magnification in D and E). Scale bars, 1 µm (A–C) and 0.5 µm (D and E).
Table 1.
Quantification of the uptake mechanisms during C. neoformans-macrophage interaction.