Requirement of the SPI-2 T3SS and effector SseG for SCV association with the MTOC

<p><b>Copyright information:</b></p><p>Taken from "The SPI-2 type III secretion system restricts motility of -containing vacuoles"</p><p></p><p>Cellular Microbiology 2007;9(10):2517-2529.</p><p>Published online 07 Jun 2007</p><p>PMCID:PMC2062534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A. Representative images of HeLa cells infected for 2 h and 8 h with mutant Typhimurium. Infected cells were fixed in ice-cold methanol and triple labelled for (green in merged images), giantin (blue in merged images) and γ-tubulin (red in merged images). Cells were analysed by confocal microscopy and images represent combined projections of multiple z-sections. Scale bars, 5 μm. B. Percentage of mutant, mutant and wt Typhimurium bacteria within 5 μm from the MTOC over an 8 h time-course. Error bars not apparent if less than 0.1; -values are indicated above bars, compared with corresponding wt values

Analysis of wt, and vacuole directionality and speed between 6 h and 8 h p

<p><b>Copyright information:</b></p><p>Taken from "The SPI-2 type III secretion system restricts motility of -containing vacuoles"</p><p></p><p>Cellular Microbiology 2007;9(10):2517-2529.</p><p>Published online 07 Jun 2007</p><p>PMCID:PMC2062534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p>i. SCVs were imaged every 5 s for 5 min. A. Trajectories of small and large displacing vacuoles containing wt, and mutant bacteria. Trajectories were reoriented to start at = 0 with the first displacement vector pointing in the same direction (+x, +y) in a standardized grid. The number of SCVs examined and the average cosine are indicated for each category. B. Mean speeds of small and large displacing vacuoles containing wt, and bacteria. C. Scatter plot in which the speeds of individual 5 s movements of vacuoles containing wt, and bacteria undergoing large displacements were plotted against the distance of the vacuole from the Golgi centroid at the end of its 5 s movement. Spearman correlation -value and -values are indicated

Time-lapse video microscopy analysis of the movement of SCVs towards the Golgi in HeLa cells

<p><b>Copyright information:</b></p><p>Taken from "The SPI-2 type III secretion system restricts motility of -containing vacuoles"</p><p></p><p>Cellular Microbiology 2007;9(10):2517-2529.</p><p>Published online 07 Jun 2007</p><p>PMCID:PMC2062534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> To allow visualization of the Golgi, HeLa cells were transiently transfected with a vector encoding MannII-EGFP. Transfected cells were infected with wt Typhimurium expressing the red fluorescent protein (DsRed). A. Graphical representation of the distance between red fluorescence centroid () and green fluorescence centroid (Golgi) in every frame of six time-lapse sequences; images were taken at 1 min intervals. Each colour represents a single SCV. B. In the upper left panel an SCV trajectory (corresponding to the blue curve in ), is superimposed on the fluorescent and DIC image of the infected HeLa cell. The boxed region is magnified in subsequent panels, which show merged fluorescent images at 30 min intervals (see ). Scale bar, 5 μm. C. Change of speed over time is shown for two distinct SCVs. Data are from time-lapse sequences where images were acquired at 5 s intervals. One SCV (blue line), had an average speed of 0.045 ± 0.004 μm; the other (red line) had an average speed of 0.017 ± 0.001 μm s

Effect of nocodazole on the redistribution of vacuoles containing mutant bacteria

<p><b>Copyright information:</b></p><p>Taken from "The SPI-2 type III secretion system restricts motility of -containing vacuoles"</p><p></p><p>Cellular Microbiology 2007;9(10):2517-2529.</p><p>Published online 07 Jun 2007</p><p>PMCID:PMC2062534.</p><p>© 2007 The Authors; Journal compilation © 2007 Blackwell Publishing Ltd</p> A. Representative images of HeLa cells infected with wt Typhimurium, either left untreated or exposed to nocodazole at 2 h p.i., and fixed 6 h later. Cells were labelled for (green in merged images), giantin (blue in merged images) and γ-tubulin (red in merged images) and analysed by confocal microscopy. Images represent combined projections of multiple z-slices; scale bars correspond to 5 μm. B. Percentage of mutant bacteria within 5 μm of the MTOC following addition of nocodazole; -value is indicated above bars