F-actin associates with and regulates the formation of PVs. (A) F-actin is recruited to the PV. HeLa cells infected for 16 h with C. burnetii were incubated for 32 h with 0.05% DMSO (control) (a to d) or 10 μM latrunculin B (LatB) (e to h). After latrunculin incubation, cells were washed and incubated for 24 h with fresh medium without latrunculin (LatB + wo) (i to l). After cell fixation, F-actin and C. burnetii were stained with rhodamine-phalloidin (green) (b, f, and j) and a specific anti-C. burnetii antibody (red) (c, g, and k), respectively. Cells were analyzed by phase-contrast (PC) (a, e, and i) and fluorescence (b, c, f, g, j, and k) microscopy. Micrographs of representative cells are shown. PVs are indicated by arrows. The colocalization between PVs and F-actin is shown in the merge column (d, h, and l). Either small F-actin patches or F-actin rings surrounding PVs are indicated by arrowheads. Bars, 15 μm. (B) F-actin disassembly reduces PV size. Cells were treated and processed as described for panel A. The sizes of the vacuoles were determined by morphometric analysis using the ImageJ program. Large vacuoles correspond to the PV population of ≥22 μm². Results are expressed as means ± standard errors (SE) for at least three independent experiments. ***, P < 0.001. (C) F-actin disassembly increases the number of PVs. Cells were treated and processed as described for panel A, and the number of vacuoles per cell was determined by morphometric analysis with ImageJ software (see Materials and Methods). Results are expressed as means ± SE for at least three independent experiments. ***, P < 0.001. (D) Analysis of infected cells by transmission electron microscopy. HeLa cells infected for 16 h with C. burnetii were incubated for 32 h with 0.05% DMSO (control) (a and b) or 10 μM latrunculin B (LatB) (c and d) and then processed for transmission electron microscopy using conventional techniques. Electron micrographs of representative cells are shown. PVs are indicated by arrows. Bars, 1 μm.

F-actin regulates the formation of the PV without affecting its lysosomal characteristics. HeLa cells were infected for 1 h with C. burnetii and then incubated for 47 h with 0.05% DMSO (A [a to d] and B [i to l]) or 10 μM latrunculin B (LatB) (A [e to h] and B [m to p]). The cells were either incubated with Lysotracker (red) (b and f), a marker of acidic compartments, before fixation (A) or fixed to analyze the presence of the lysosomal enzyme cathepsin D (B). After fixation, cells were labeled with an anti-cathepsin D antibody (red) (B [j and n]), anti-C. burnetii (green) (A [c and g]), or Hoechst dye (blue) (B [k and o]). Cells were analyzed by phase-contrast (PC) (a, e, i, and m) and fluorescence (b, c, f, g, j, k, n, and o) microscopy. Micrographs of representative cells are shown. PVs are indicated by arrows. The colocalization between PVs and Lysotracker or cathepsin D is shown in the merge column (d, h, l, and p). Bars, 5 μm.

EGFP-Cdc42 V12, EGFP-RhoA V14, and F-actin are recruited to the PV. (A) EGFP-Cdc42 V12 and -RhoA V14, but not EGFP-Rac1 V12, associated with PVs. HeLa cells were infected with C. burnetii and then transfected with pEGFP (a to c), pEGFP-Cdc42 V12 (d to f), or pEGFP-RhoA V14 (g to i). After 24 h, the cells were fixed and processed for immunofluorescence, using a specific anti-C. burnetii antibody (red) (b, e, and h). Cells were analyzed by confocal microscopy. Micrographs of representative cells are depicted. PVs are indicated by arrows. The colocalization is shown in the merge column (c, f, and i). (B) Quantification of colocalization. Results are expressed as means ± SE for at least three independent experiments. **, P < 0.01; ***, P < 0.001. (C) EGFP-Cdc42 V12, EGFP-RhoA V14, and F-actin colocalize with PVs. Infected HeLa cells were transfected with pEGFP-Cdc42 V12 (a to d) or pEGFP-RhoA V14 (i to l). After 24 h, cells were fixed and processed for immunofluorescence, using phalloidin-rhodamine to label actin (red) (b and j) and a specific anti-C. burnetii antibody (blue) (c and k). Panels e to h and m to p represent a magnification of the insets. Cells were analyzed by confocal microscopy. Micrographs of representative cells are depicted. PVs are indicated by arrows. The colocalization is shown in the merge column (d, h, l, and p). Bars, 5 μm.

Overexpression of the dominant-negative mutant of RhoA does not modify the cargo and lysosomal characteristics of PVs. Infected HeLa cells were transfected with pEGFP-RhoA N19 (a, e, and i). After 24 h, the cells were incubated (see Materials and Methods) with either DQ-BSA (A, panel b) (red), Lysotracker (B, panel f) (red), or transferrin (C, panel j) (blue). Cells were fixed and processed for immunofluorescence, using a specific anti-C. burnetii antibody (blue [c and g] or red [k]). Cells were analyzed by confocal microscopy. Micrographs of representative cells are shown. PVs are indicated by arrows. The colocalization between PV, expressed proteins, and markers is shown in the merge column (d, h, and l). The inset in panel l shows the localization of transferrin (blue) in the delineated vacuoles harboring the bacteria (red). Bars, 5 μm.

F-actin is required for fusion between PVs and bead-containing phagosomes. Infected cells were incubated with either transferrin (A) (blue; b and f) or latex beads (B) (red; j and n) (see Materials and Methods) and treated as indicated in the legend to Fig. 1. C. burnetii cells were stained with a specific antibody (red [c and g] or green [k and o]). Cells were analyzed by phase-contrast (PC) (a, e, i, and m) and fluorescence (b, c, f, g, j, k, n, and o) microscopy. Colocalization of the different probes with the PVs is shown in the merge column (d, h, l, and p) and quantified in panel C. Micrographs of representative cells are shown. PVs are indicated by arrows. Insets in panels d and h show the localization of transferrin (blue) in the delineated vacuoles harboring the bacteria (red). Results are expressed as means ± SE for at least three independent experiments. **, P < 0.01. Bars, 5 μm.

PVs are decorated with the WT forms of EGFP-Cdc42 and -RhoA but not with EGFP-Rac1. (A) HeLa cells were infected as indicated in the legend to Fig. 1 and then transfected with pEGFP alone (a to c), pEGFP-Cdc42 WT (d to f), pEGFP-RhoA WT (g to i), or pEGFP-Rac1 WT (j to l) as described in Materials and Methods. After 24 h, cells were fixed and processed for immunofluorescence, using a specific anti-C. burnetii antibody (red) (b, e, h, and k). Cells were analyzed by confocal microscopy. Micrographs of representative cells are shown. PVs are indicated by arrows. Filipodia (d) and lamellipodia (g) are indicated by arrowheads. The colocalization between PVs and overexpressed proteins is shown in the merge column (c, f, i, and l). Bars, 5 μm. (B) Quantification of colocalization between PVs and EGFP fusion proteins. Results are expressed as means ± SE for at least three independent experiments. ***, P < 0.001. (C and D) Chloramphenicol treatment reduces the colocalization of PVs with EGFP-Cdc42 and EGFP-RhoA. Infected cells were transfected with pEGFP-Cdc42 WT (C) or pEGFP-RhoA WT (D) and then treated with chloramphenicol (Chl) as indicated in Materials and Methods. Colocalization between PVs and EGFP fusion proteins was quantified. Results are expressed as means ± SE for at least three independent experiments. *, P < 0.05; ***, P < 0.001.

The dominant-negative mutants of Cdc42, RhoA, and Rac1 are not recruited to the PV. (A) Infected HeLa cells were transfected with pEGFP (a to c), pEGFP-Cdc42 N17 (d to f), or pEGFP-RhoA N19 (g to i). After 24 h, the cells were fixed and processed for immunofluorescence, using a specific anti-C. burnetii antibody (red) (b, e, and h). Cells were analyzed by confocal microscopy. Micrographs of representative cells are shown. PVs are indicated by arrows. The colocalization is shown in the merge column (c, f, and i). Bars, 5 μm. (B) Quantification of colocalization. Results are means ± SE for at least three independent experiments. (C) Overexpression of the dominant-negative mutant EFGP-RhoA N19 reduces PV size. Cells were treated and processed as described for panel A. The sizes of the vacuoles were determined by morphometric analysis (see Materials and Methods). Results are expressed as means ± SE for at least three independent experiments. ***, P < 0.001.