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. 2021 Jun 1;10:e67789. doi: 10.7554/eLife.67789

Figure 4. Super-resolution imaging of GCE-labeled SifA.

(Ai) SifA colocalizes with SseJ. Spinning-disk SIM image of an infected HeLa cell, 16 hr after invasion, fixed with PFA and stained for SifA (green) and SseJ-HA (red). (Aii) Magnified view of SifA and SseJ in the boxed region at the far left (arrows indicate colocalization). (iii) Colocalization was analyzed by calculating Mander’s M1 coefficient (SifA colocalizing with SseJ) for the original dual-color image (x = y = 0) and for control images generated by translating the spectral channels with respect to each other in x and y directions. This analysis generated a colocalization map, with a peak intensity at x = y = 0, indicating true colocalization (n = 3 cells). (iv) Colocalization analysis of mock-infected cells (n = 3 cells) stained for SifA-E52AzF and SseJ-HA showed no peak intensity at x = y = 0, indicating no colocalization of anti-HA antibodies and SifA. Bars, 10 µm (i), 5 µm (ii). (B) SifA colocalizes with kinesin. (i) Spinning-disk SIM image of an infected HeLa cell fixed with PFA at 16 hr post-infection and stained for kinesin (red) and SifA (green). (ii) Magnified view of SifA and kinesin in the boxed region (arrows indicate colocalization). (iii and iv) Colocalization analysis was performed as described in (A). The same color map scaling was used for comparison. px, pixels. See Figure 4—figure supplement 1 for statistical analysis. Scale bar, 5 µm (i), 2 µm (ii). (C) Bioorthogonal fluorescence imaging of SseJ-Y10TCO-HA with tetrazine fluorophores versus immunofluorescence staining. HeLa cells expressing SseJ-Y10TCO-HA in the presence of TCO*Lys were labeled with BDP-Tz under physiological conditions, briefly washed, and subjected to anti-HA immunofluorescence staining. Images were acquired by confocal microscopy. The data are representative of at least three independent experiments. Scale bar, 10 µm.

Figure 4—source data 1. Source data for Figure 4(iii-iv)..
Figure 4—source data 2. Source data for Figure 4(iii-iv).

Figure 4.

Figure 4—figure supplement 1. The proportion of tubules with labeled SifA and its colocalization with SseJ (n = 33) and kinesin (n = 35); n is the total number of cells analyzed.

Figure 4—figure supplement 1.

Error bars represent the standard deviation across three data sets pooled from separate infections.
Figure 4—figure supplement 1—source data 1. Source data for Figure 4—figure supplement 1.
Figure 4—figure supplement 2. GA fixation preserves microtubules.

Figure 4—figure supplement 2.

(A) HeLa cells were infected with sifA mutants harboring pSifA52TAG and pEVOL-AzF. At 10 hr post infection, excess AzF was washed out and replaced with fresh growth media without AzF for 2 hr. At 14 hr post infection, HeLa cells were incubated with 2.5 μM BDP-DBCO in DMEM with 10% FBS growth media for another 2 hr, followed by extenstive removal of excess dye with fresh growth media as described in Materials and methods. At 16 hr post-infection, cells were extracted for 1 min using a microtubule-stabilizing buffer (MTSB). The buffer was exchanged with MTSB containing 0.2% GA, and cells were fixed for 10 min at room temperature. Excess GA was quenched for 7 min using 0.2% sodium borohydride in PBS. After several washes with PBS, cells were immunostained for microtubules (green). Scale bar, 10 µm. (B) Prior to immunostaining, GA-fixed HeLa cells were imaged for SifA labeled with BDP-DBCO using Spinning-disk SIM. Note the punctate labeling of SifA, indicating that while SseJ forms continuous filaments, SifA does not. The results presented in each panel were obtained in at least three independent experiments. Scale bar, 5 µm.
Figure 4—figure supplement 3. Functional complementation of SIFs by expression of fluorescent SifA-E52Hco.

Figure 4—figure supplement 3.

(A) HeLa cells were infected with the sifA null strain of Salmonella expressing SifA-E52Hco and SseJ-HA for 16 hr, fixed and immuno-stained for LAMP1 (green), SseJ (red). The SifA-E52Hco strain was capable of forming SIFs and colocalized with SseJ. Images were acquired on a spinning-disk SIM as described in Materials and methods. (B) Direct visualization of secreted SifA. HeLa cells were infected with a sifA null strain of Salmonella expressing SifA-E52Hco in the presence of 1 mM Hco and fixed 16 hr post infection with 4% PFA. Hco-tagged secreted SifA-E52Hco was directly imaged by two photon microscopy. Immuno-stained SseJ-HA was also imaged in the same cell. The data are representative of at least three independent experiments. Scale bar, 10 µm (A), 20 µm (B).