Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics.DOI: http://dx.doi.org/10.7554/eLife.24119.001

The reaction of o-ethynylphenols 3 with a wide variety of unsaturated halides or triflates 6 in the presence of Pd(OAc)2(PPh3)2, CuI, and Et3N (procedure A) gives 2-vinyl- and 2-arylbenzo[b]furans 7, in good to high yield, through a palladium-catalyzed coupling followed by an in situ cyclization step. Small amounts of 2,3-disubstituted-benzo[b]furans 8 are usually isolated as side products. In some cases, however, compounds 8 are generated in significant yield or even as the main products. The formation of 8 can be prevented by employing alternative procedures (B and C) that use o-((trimethylsilyl)ethynyl)phenyl acetates 5 as starting building blocks. Procedure B is based on the palladium-catalyzed reaction of 5 with 6 in the presence of Pd(PPh3)4, Et3N, and n-Bu4NF, followed by the hydrolysis of the resultant coupling derivative 12 under basic conditions. Procedure C affords 7 through an in situ coupling/cyclization of 5 with 6 in the presence of Pd(PPh3)4 and KOBut. The utilization of o-alkynylphenols 9 as the starting alkynes in the palladium-catalyzed reaction with 6 leads to the formation of 2,3-disubstituted-benzo[b]furans 13 through an annulation process promoted by σ-vinyl- and σ-arylpalladium complexes generated in situ. The best results in this case are obtained by using KOAc and Pd(PPh3)4. In the presence of KOAc and Pd(PPh3)4, and under an atmosphere of carbon monoxide, the reaction of o-alkynylphenols with 6 provides 2-vinyl- and 2-aryl-3-acylbenzo[b]furans 14.

In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions.