Fig. 2. Depletion of ROP2 produces aborted parasite vacuoles and alters rhoptry shape. (A-G) Transmission electron microscopy of ROP2AS-7 and ROP2AS-20 revealed that a small proportion of ROP2-deficient parasites were able to invade Vero cells and establish a vacuole with a delimiting membrane. The majority of the vacuoles contained large multinucleated (N) parasites (A) and the network membrane (NM) was significantly increased (A,B). Altered parasite morphology was because of an arrest in the formation of the cytokinetic furrow during cell division (endodyogeny) (arrows in A,B). Although dense granules (DG) and micronemes (M) appeared to be unaffected, the formation of mature rhoptries (R) was blocked in the majority of parasites (C-F). Defective rhoptries were most often no longer polarized to the apical half of the cell (A) and their categorical flask shape was neither formed nor maintained (C-F). Whereas the formation of the honeycombed basal portion containing packaged lumenal membranes was disrupted (arrowhead in F) in some rhoptries, the condensation of lumenal contents to form the electron-dense rhoptry distal tip (arrowheads in D,E) was severely disrupted in the majority of aberrant organelles. Bars: 1.0 µM (A-B,G), 0.2 µM (C-F,H-J). (H-J) Ultrathin cryoimmuno electron microscopy of ROP2AS-1. Serial cryosections of an unsegregated rhoptry cluster in ROP2AS-1 contains ROP2/3/4 (arrows) as immunolabelled with T34A7 monoclonal antibody. (K-R) Immunofluorescent microscopy of extracellular ROP2AS-7 immunostained with an antiserum specific to ROP2, as reported previously (Sadak et al., 1988). In up to one-third of parasites, rhoptries appeared normal, even when rhoptries in adjacent parasites in the same vacuole were distorted. This was evident both at the electron microscopic level (G, normal rhoptries, arrows; abnormal rhoptries, arrowheads) and by immunofluorescence (K-R, see text for description).

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