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. 2017 Mar 10;8:14729. doi: 10.1038/ncomms14729

Figure 1. Laser-mediated inclusion rupture triggers chlamydial egress.

Figure 1

(a) Time-lapse videomicroscopy of GFP-expressing mCherry-Rab25 and CFP-H2B stable HeLa cells imaged from 36 h p.i. with CTL2 (MOI∼0.5). Presented is a single egress event captured from 49 h p.i. Arrows highlight the nucleus and asterisks the inclusion. Interval of capture was 5 min. (b) Diagram of inclusion ablation system and time-lapse videomicroscopy of the system applied on GFP-expressing and mCherry-Rab25 stable HeLa cells 48 h p.i. with CTL2 (MOI∼0.5). Arrows highlight the nuclei, asterisks the inclusion and arrow heads blebbing of the plasma membrane. Interval of capture is as indicated. (c) Time-lapse videomicroscopy of mCherry-Rab25 stable HeLa cells ablated 24 h p.i. with GFP-CTL2 (MOI∼0.5). Arrows highlight the nuclei and arrow heads blebbing of the plasma membrane. Interval of capture is as indicated. (d) Quantification of cell survival of ablated and unablated cells as described in the methods under the conditions as indicated. Ten independent movies with at least one ablated cell each for each condition were examined. (e) Time-lapse videomicroscopy of mCherry-Rab25 stable HeLa cells ablated either 24 h p.i. with GFP-CTL2 (MOI∼0.5) in calcium-free Ringer's solution and 50 μM BAPTA-AM. Interval of capture is as indicated. Movie is representative of at least 10 independent movies, and endosomes of dimensions similar to ablated inclusions were targeted for comparison. (f) Time-lapse videomicroscopy of two mCherry-Rab25 stable HeLa cells ablated 24 h p.i. with GFP-CTL2 in the presence of 800 nM YM201636 to swell endosomal compartments. Numbers highlight nuclei of individual cells. Insets highlight a ruptured Rab25-positive chlamydial inclusion denoted by the prominent GFP-expressing bacterial fluorescence within (cell 2) and a ruptured Rab25-positive endosome (cell 1). Asterisks indicate the inclusion and arrow heads highlight blebbing of the plasma membrane. Movie is representative of at least 10 independent movies, and endosomes of dimensions similar to ablated inclusions were targeted for comparison.