Fig. 8: Light-dependent translocation of heterologous cargo into eukaryotic host cells. | Nature Communications

Fig. 8: Light-dependent translocation of heterologous cargo into eukaryotic host cells.

From: LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

Fig. 8

a Fluorescence micrographs depicting cultured HEp-2 cells that were incubated with the indicated strains expressing either a heterologous T3SS substrate, YopE1-53-β-lactamase, or β-lactamase without a secretion signal as a negative control, for 60 min. Translocation of β-lactamase is detected by cleavage of the intracellular β-lactamase substrate CCF2 (leading to loss of FRET, and a transition from green to blue fluorescence emission). Scale bar, 50 μm. b Fraction of β-lactamase-positive HEp-2 cells in (a) (blue fluorescence). 2343/2423/2226/2694 cells from 26/28/25/27 fields of view from three independent experiments were analyzed for the LITESEC strains under the given conditions from left to right (809/671/995/823 cells from 8/8/10/9 fields of view from three independent experiments for the controls). Single data points (percentage of positive cells per field of view) indicated by circles; error bars display the standard error of the mean. ***p < 0.001 in a two-tailed homoscedastic t test; n.s., difference not statistically significant (exact values from left to right, 6 × 10−6/2 × 10−8/0.80/0.65). c Micrographs depicting cultured HEp-2 cells that have been incubated with the indicated strains expressing a heterologous T3SS substrate, YopE1-138-tBID15 for 60 min. Translocation of tBID induces apoptosis, which leads to a condensed star-shaped host cell morphology. Scale bar, 50 μm. d Visual classification of HEp-2 cells used in (c) after infection. 1522/1914/1510/1600/2299/1218/1468/1194 cells from 17/18/17/19/14/13/14/12 fields of view from five independent experiments were analyzed per strain and condition (from left to right). Single data points (percentage of apoptotic cells per field of view) indicated by circles; error bars display the standard error of the mean among fields of view. */***p < 0.05/0.001 in a two-tailed homoscedastic t test; n.s., difference not statistically significant (exact values from left to right, 2 × 10−25/1 × 10−15/0.40/0.038). e Translocation of tBID induces cleavage of poly (ADP-ribose) polymerase (PARP), which was monitored by Western blot analysis of HEp-2 cells used in (c). β-actin was used as a loading control. Left, molecular weigth in kDa. Source data for panels (b, d, e) are provided as a Source Data File.

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