Unraveling the Elusive Rhoptry Exocytic Mechanism of Apicomplexa Parasitology

Apicomplexan parasites are unicellular eukaryotes that invade the cells in which they proliferate. The development of genetic tools in Toxoplasma , and then in Plasmodium , in the 1990s allowed the ﬁ rst description of the molecular machinery used for motility and invasion, revealing a crucial role for two different secretory organelles, micronemes and rhoptries. Rhoptry proteins are injected directly into the host cytoplasm not only to promote invasion but also to manipulate host functions. Nonetheless, the injection machinery has remained mysterious, a major conundrum in the ﬁ eld. Here we review recent progress in uncovering structural components and proteins implicated in rhoptry exocytosis and explain how revisiting early ﬁ ndings and considering the evolutionary origins of Apicomplexa contributed to some of these discoveries. sortilin micronemebiogenesis. trichocyst biogenesis of mucocysts/trichocysts, common with of micronemes and rhoptries. rhoptries, at the and on homologous exocytic machinery, in similar mechanisms are used to specialized secretory organelles in Ciliata and Apicomplexa. Such secretory compartmentshavedifferentiatedduringevolutiontoful ﬁ lltheverydifferentrequirementsforparasiticvsfree-livingprotists but maintain clear molecular traces of their common origin. PtNd9 interacts with both the PM and tricho- cyst membranes to assemble the rosette and connectingmaterial[108]. PtNd9 has two domains. The N-terminal domain is predicted to be important for direct or indirect membrane binding, while the armadillo repeat-containing C-terminal do- main may moderate protein – protein interactions [62]. The analysis of dominant negative forms of PtNd9 suggested that PtNd9 might act as a homopolymeric bridge between the trichocyst and PM. Studies on double nd mutants revealed in- teractions between PtNd9 with two other cytosolic factors, PtNd16 and PtNd18. These have not been characterized but are both implicated in rosette assembly [59,62]. These proteins may form a multimeric complex that contributes to the connecting material beneath the plasma membrane. Another gene involved in Paramecium exocytosis is calmodulin ( cam ), the most ubiquitous Ca 2+ mediator in eukaryotic cells. A screen of mutants affected in swimming behavior identi ﬁ ed several mutations in the calmodulin gene (named cam mutants) [110]. One of them, cam1 , was shown to be also defective in rosette assembly and formation of underlying connecting material, and thus in regulated exocytosis [111]. These observations argue for a role of calmodulin in the exocytic step, which may account in part for the well established role of Ca 2+ in trichocyst exocytosis [53]. (RLD) of Nd6 might act as guanine exchange factor towards small GTPases, as previously seen for proteins involved in vesicular membrane fusion [109]. Via its armadillo-like repeats, the Nd9 protein might facilitate protein – protein interactions with other Nd members at the exocytic site in order to assemble the rhoptry secretion apparatus and to stabilize the intramembranous particles (IMPs) of the rosette. The C2 domain-containing proteins NdP2 and Fer2 might contribute to the Ca 2+ -dependent fusion of the AV at the parasite's membrane. Upon fusion and rhoptry discharge, a new exocytosis machinery might be rebuilt at the parasite apex, by recruiting to the PPM a new apical vesicle from the reservoir of vesicles (V) inside the conoid. This event would stimulate the formation of another rosette and anchoring of a new rhoptry selected from the pool of undocked organelles present at the apex of the parasite. Rearming the secretion apparatus would ensure successive secretion of rhoptries, and thus successive injections of rhoptry effectors into the host, in order to hijack cells that the parasite does not invade.