Fig. 2.

Biogenesis, structure and topology of ejectosomes. Paraformaldehyde-fixed cells infected with GFP-expressing M. marinum (green), stained for F-actin (red). (A) A phagocytic cup lined with F-actin (arrows). A single (B) or multiple bacteria (C, D) spanning the host cell plasma membrane (asterisks) through ejectosomes (white arrowheads) with the intracellular part of the bacterium devoid of actin-labeling (black arrowhead). (E) Cell-to-cell transmission of bacteria from a donor (do) to an acceptor (ac) cell through an ejectosome into a phagocytic cup (arrows). (F) Movement of cytosolic bacteria (blue) through ejectosomes (F-actin, green) induces a plasma membrane bulge (small arrows) that ruptures at the tip (F, asterisk). The intracellular part of the ejecting bacterium is devoid of p80, a plasma membrane marker (F), and vacuolin, a niche marker (asterisk, G). Some extracellular bacteria were positive for p80 (small arrows, H). (I–K) Live infected cells (M. marinum, blue, actin, green) were incubated in the presence of an anti-M. marinum serum. The extracellular parts of ejecting (small arrows, I, K) or outside (small arrow, J) bacteria were accessible to the antibody and labeled red, in contrast to intracellular bacteria (black arrowhead, J), confirming partial loss of plasma membrane integrity. Scale bars 1 µm.