Fig. 5. Vitronectin-mediated pneumococcal invasion requires the dynamics of the actin cytoskeleton. (A) Vitronectin-mediated infection of Detroit 562 cells (4 hours) with pneumococci was followed in the absence (none) or presence of inhibitors of the actin cytoskeleton, including cytochalasin D (Cyto D, 10 µM), latrunculin B (Lat B, 1 µM) and jasplakinolide (Jspk, 1 µM), by antibiotic protection assays. Results are presented as the mean ± s.d. of at least three independent experiments. ^*P<0.05. (B) Immunofluorescence microscopy of host-cell-attached and intracellular pneumococci. Host cells were pre-treated with vitronectin and infected with pneumococci. (Ba) Intracellular pneumococci were stained with Alexa Fluor 568 (red) and are indicated by arrows, whereas adherent bacteria appear yellow (green/red stain). Illustration of microspike-like structures (Bb,Bc) was achieved by phalloidin staining of the actin cytoskeleton (green). (Bd) Magnification of Bc, illustrating pneumococci attached to microspike-like structures on the cell surface. (C) Expression of the microspike-associated proteins frabin (upper row) and profilin (lower row) is induced upon vitronectin-mediated invasion of pneumococci. F-actin is stained with phalloidin (green), whereas profilin and frabin are stained with protein-specific antibodies and Alexa Fluor 568 (red). Boxed areas indicate frabin in microspikes; arrows indicate the association of profilin with microspike structures. (D) Scanning electron microscopy of Detroit 562 cells that were pre-treated with vitronectin (3.0 µg) and subsequently infected with pneumococci for 4 hours (a,c,e) or 6 hours (b,d,f). The inset in panel De shows pneumococci invading the host cell (arrow). (E) Scanning electron microscopy (top) and immunofluorescence microscopy (bottom) after staining the actin cytoskeleton of untreated Detroit 562 cells (a,d), Detroit 562 cells incubated with multimeric vitronectin (b,e) or host cells infected with pneumococci in the absence of vitronectin (c,f).