Legionella effector LpPIP recruits protein phosphatase 1 to the mitochondria to induce dephosphorylation of outer membrane proteins
Fig 4
Downstream consequences of LpPIP-PP1 dephosphorylation events.
(A) Time course trace of oxygen consumption rate (OCR) in wild-type Flp-In T-REx 293 cells (16 technical replicates) and stable cells expressing FLAGLpPIP (15 technical replicates) or FLAGRVxFdead (15 technical replicates), following injections of oligomycin, FCCP, rotenone, and antimycin A. Data represents mean ± SD. Corresponding raw data are available in the Supporting Information (S3 Data). (B) Time course trace of extracellular acidification rate (ECAR) for the cells described in Fig 4A, following incubation with oligomycin and 2-deoxy-d-glucose. Data represents mean ± SD. Corresponding raw data are available in the Supporting Information (S3 Data). (C) Wild-type Flp-In T-REx 293 cells and stable cells expressing FLAGLpPIP or FLAGRVxFdead were induced with 1 µg/mL tetracycline for 24 h, followed by treatment with 1 µM staurosporine (STS) for 24 h. Whole cell samples were processed for SDS-PAGE and immunoblots. Corresponding raw images are available in the Supporting Information (S1 Raw Images). (D) Quantification of mitochondrial mean aspect ratio in wild-type HeLa cells and those transfected with FLAGLpPIP or FLAGRVxFdead (n = 3 independent experiments, 10 cells per experiment). Data are presented as mean ± SEM of the three experiments. Ordinary one-way ANOVA was performed on the means, showing a significant difference of 0.2273 between wild-type and FLAGLpPIP-transfected cells, with a p-value of 0.0309. Corresponding raw data are available in the Supporting information (S1 Data). (E) Quantification of mitochondrial mean form factor in HeLa cells and those transfected with FLAGLpPIP or FLAGRVxFdead (n = 3 independent experiments, 10 cells in each experiment). Data are presented as mean ± SEM of the three experiments. Ordinary one-way ANOVA was performed on the means, showing no significant differences among the groups. Corresponding raw data are available in the Supporting information (S1 Data). (F) HeLa cells transiently transfected with FLAGLpPIP, or FLAGRVxFdead were stained with 100 nM MitoTracker Deep Red FM (mitochondria; MTDR) and immunostained with antibodies against FLAG (green) and Drp1 (magenta). Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. (G) Proportion of Drp1 localized to mitochondria in HeLa cells transiently transfected with FLAGLpPIP or FLAGRVxFdead, calculated using raw integrated density in Fiji. Representative images are shown in Fig 3F. Data are presented as mean ± SD from 13 cells in one experiment. Unpaired t test showed no significant difference between the groups. Corresponding raw data are available in the Supporting information (S1 Data). (H) [35S]-Tim23 was imported into mitochondria isolated from wild-type Flp-In T-REx 293 cells and stable cells expressing FLAGLpPIP or FLAGRVxFdead. Following import mitochondria were isolated and treated with PK. Isolated mitochondria were solubilized in 1% [w/v] digitonin and the separated by BN-PAGE and observed by phosphor image analysis. Densitometric quantification was calculated as the percentage of import at 60 min in mitochondria from wild-type Flp-In T-REx 293 cells, normalized to SDHA loading control. Data are presented as mean ± SD of three experiments. Ordinary one-way ANOVA showed no significant difference among the three cell lines. Corresponding raw data are available in the Supporting Information (S1 Raw Images and S4 Data). (I) [35S]-GC1 was imported into mitochondria isolated from wild-type Flp-In T-REx 293 cells and stable cells expressing FLAGLpPIP or FLAGRVxFdead as described in Fig 4H. Densitometric quantification and ordinary one-way ANOVA showed no significant difference among the three cell lines. Corresponding raw data are available in the Supporting information (S1 Raw Images and S4 Data). (J) Quantification of mitophagy from live-cell imaging of Flp-In T-REx 293 cell lines constitutively expressing mt-Keima. Experimental conditions include wild-type Flp-In T-REx 293 cells cultured in normal media or media adjusted to pH 4 (positive control), and stable cells induced to express either FLAGLpPIP or FLAGRVxFdead. The 561/488 mt-Keima signal (red-to-green fluorescence ratio) was quantified using the Ratio Plus plugin in Fiji and normalized to that of wild-type cells in normal media. Data represent mean ± SEM from two to three independent experiments (>30 images per experiment). Statistical analysis by ordinary one-way ANOVA showed no significant differences among the cell lines, except for the pH 4 control. Corresponding raw data are available in the Supporting information (S1 Data).