Figure 1.
To search for binding partners of SEPT9, we performed immunoprecipitation experiments using antibodies for SEPT9 and lysates from untreated HeLa cells (−InlB), or HeLa cells stimulated for 2 minutes with 5 nM (total) of purified InlB (+InlB). Mass spectrometry was subsequently performed on samples analyzed by SDS-PAGE, where in both cases SEPT2, SEPT7, and SEPT11 were identified to coimmunoprecipitate with SEPT9.
Figure 2.
Septin expression, localization, and recruitment at the bacterial entry site in human non-phagocytic cells.
(A) Western blots (WB) of septins in non-phagocytic cell lines. HeLa and JEG-3 cells were harvested, and cell lysates were separated by 10% SDS-PAGE before immunoblotting. Blots were probed with antibodies specific to GAPDH, SEPT2, SEPT9, SEPT11, and actin. Blots for GAPDH are shown as a loading control. (B) Septin filaments partially colocalize with actin filaments in HeLa cells. Endogenous α-tubulin, F-actin, and SEPT2, SEPT9, or SEPT11 were visualized by immunostaining with anti-α-tubulin (grey and in Merge blue), anti-F-actin (green), and anti-SEPT2, anti-SEPT9, or anti-SEPT11 antibodies (red). Representative confocal images for α-tubulin, F-actin, and SEPT11 distribution in HeLa cells are here displayed, and similar images were obtained labeling for SEPT2 or SEPT9. Scale bars indicate 10 µm. (C) Septin filaments partially colocalize with microtubules in JEG-3 cells. Endogenous α–tubulin, F-actin, and SEPT2, SEPT9, or SEPT11 were visualized for immunostaining with anti-α-tubulin (grey and in Merge blue), anti-F-actin (green), and anti-SEPT2, anti-SEPT9, or anti-SEPT11 antibodies (red). Representative confocal images for α-tubulin, F-actin, and SEPT11 distribution in JEG-3 cells are here displayed, and similar images were obtained labeling for SEPT2 or SEPT9. Scale bars indicate 10 µm. (D) Septin recruitment at the site of Listeria entry in JEG-3 cells. Cells were infected with L. monocytogenes BUG 1641 for 5, 10, or 15 minutes and then fixed for microscopy. Endogenous septin was stained with anti-SEPT2, anti-SEPT9, or anti-SEPT11 antibodies (red). Actin was stained with anti-F-actin (green), and Listeria was marked using DAPI (blue). Representative photos are here displayed, where inset images highlight the septin collar-like recruitment around Listeria to which the white arrows are pointing. Scale bars indicate 1 µm. (E, F) Septin recruitment to the site of Shigella entry in (E) JEG-3 cells or (F) HeLa cells. Cells were infected with S. flexneri M90T for 15 minutes and then fixed for microscopy. Endogenous septin was stained with anti-SEPT2, anti-SEPT9, or anti-SEPT11 antibodies (red). Actin was stained with anti-F-actin (green), and Shigella was marked using DAPI (blue). Representative photos showing SEPT11 (JEG-3 cells) and SEPT2 (HeLa cells) are here displayed, where inset images highlight the septin collar-like recruitment around Shigella to which the white arrows are pointing. In JEG-3 cell and HeLa cells, similar recruitment was obtained when labeling for SEPT2, SEPT9, or SEPT11. Scale bars indicate 1 µm.
Figure 3.
The impact of SEPT2-depletion in HeLa and JEG-3 cells on bacterial entry.
(A, B) Western blot (WB) of (A) HeLa cells or (B) JEG-3 cells transfected with siRNA against control (CTRL) or SEPT2. Cell lysates were separated by 10% SDS-PAGE before immunoblotting. The blots were probed with antibodies specific to GAPDH, SEPT2, and actin. GAPDH is shown as a loading control. The red box outlines depleted protein levels for SEPT2. (C–F) SEPT2 regulates the invasion of L. monocytogenes and S. flexneri. Gentamicin survival assays for (C and E) L. monocytogenes EGD or (D and F) S. flexneri M90T were performed in (C and D) HeLa cells or (E and F) JEG-3 cells treated with control (CTRL) siRNA, or siRNA targeted against SEPT2. Graphs represent the relative number of intracellular bacteria found inside siRNA-treated cells after the survival assay, where CFU counts obtained from septin-depleted cells were normalized to CTRL siRNA-treated cells. On the graph CTRL siRNA is figuratively presented as 1, and data represents the mean from n ≥9 experiments. Results were analyzed for statistical significance using the Student's t-test.
Figure 4.
SEPT2 inactivation impairs the entry of InlB-coated beads.
(A) Quantification of InlB-induced phagocytosis in control (CTRL) and SEPT2-depleted HeLa cells. For internalization assays, beads were analyzed by immunofluorescence (see Materials and Methods) for being extracellular or intracellular in at least 50 host cells counted for each of n≥2 separate experiments per siRNA treatment. The left graph depicts the total % of internalized 1 µm InlB-coated beads at 5 minutes post-incubation of siRNA-treated cells, calculated from the (total number of internalized beads) / (total number of cell-associated beads) ×100. Results were analyzed for statistical significance using the z-test for percentages. The right graph depicts the average number of internalized 1 µm InlB-coated beads per cell at 5 minutes post-incubation of siRNA-treated cells. Graphed data represent this average value ±SEM, where results were analyzed for statistical significance using the two sample z-test. (B) Representative scanning electron microscopy images of siRNA-treated HeLa cells to depict the membrane surface of control (CTRL) and SEPT2-depleted cells. Magnification = 1500×, where scale bars indicate 10 µm. (C) Uptake of InlB-beads by siRNA-treated cells. Control (CTRL) and SEPT2-depleted HeLa cells were incubated with 1 µm InlB-beads for 5 minutes, and cells were processed for scanning electron microscopy. Depicted here are representative images of InlB-beads internalized (i.e. CTRL cells), or only adherent (i.e. SEPT2-depleted cells), for siRNA treatments. Hollow arrows indicate internalized beads, solid white arrows indicate extracellular adherent beads. Magnification = 15000×, where scale bars indicate 1 µm.
Figure 5.
SEPT2-depleted cells respond less to InlB stimulation.
(A) Representative phase and FRET efficiency images of control (CTRL) and SEPT2-depleted HeLa cells expressing YFP-AktPH and CFP-AktPH stimulated with InlB. FRET efficiency at 2 time points are presented for each siRNA-treatment: T0 (frame 16) and Tmax (frame corresponding to the maximum induction of FRET efficiency) after stimulation. Pseudocolor scale represents the range of FRET efficiency values from original signal ranging from blue (low) to red (high). Scale bars indicate 10 µm. Movies for the entire timecourse of InlB stimulation for these cells can be observed as Movies S2 and S3. (B) Quantification of FRET response in siRNA-treated cells. Processed FRET response was plotted against time (i.e. frame per 15 seconds), for the representative siRNA-treated cells depicted in Figure 5A. Values for the control (CTRL) cell are plotted in black, and the SEPT2-depleted cell in red. (C) Percentage of cells that respond to InlB treatment. Processed FRET response was determined for 18 or more experiments for each siRNA treatment. Cells were classified as not responding to InlB stimulation if the slope of processed FRET response could not be distinguished above baseline values (i.e. the SEPT2-depleted cell in Figure 5B). The percentage of SEPT2-depleted cells classified as not responding to InlB stimulation was statistically compared to expectations as derived from control cells by Chi-Square test.