Figure 3.

K8 and K18 assist actin depolymerization during later stages of internalization. (A,B) Kinetic analysis of actin, K8 and K18 recruitments during internalization of InlB-coated latex beads. (A) Stack projections of widefield microscopy images of HeLa cells incubated with InlB-coated latex beads for different periods of time, fixed, immunostained for K8 or K18 (green) and labeled for F-actin with TRITC-phalloidin (red). Scale bar, 3 μm. Insets show high-magnification images. Scale bar, 1 μm. (B) Quantification of beads positive for K8, K18, or actin recruitment. Results are expressed as the percentage of particles associated with either protein in relation to the total number of particles associated to cells. The total number of beads was determined in brightfield. Values are the mean ± S.E. of at least three independent experiments. For determination of beads internalization, extracellular beads were stained with anti-InlB before cell permeabilization and total beads number quantified in brightfield. Values are shown in percentage and are representative of two independent experiments. (C) Quantification of InlB-coated latex beads associated to polymerized actin in HeLa cells transfected with control (Ctr) or specific siRNA targeting K8 (K8-si) or K18 (K18-si). Cells were incubated with InlB-coated latex beads for 15, 30, and 60 min, fixed and stained for F-actin. Beads displaying actin recruitment were considered recruitment-positive. The total number of beads associated to cells was determined in brightfield. Values represent the mean ± S.E. of at least three independent experiments. Statistically significant differences are indicated: ^***p < 0.001.