Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Abstract

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.

Gliding motility powers invasion and egress in Apicomplexa

“…Although erythrocyte actin is more dynamic than previously assumed 118 , merozoite entry does not involve host actin reorganization at the moving junction 119 . During invasion, the concentration of parasite actin has been visualized as a ring posterior to the moving junction, which supports a role for gliding during invasion 29,38,39 . Invasion crucially depends on the discharge of rhoptries 120 , and concordantly, Plasmodium spp.…”

“…Recently, the use of chromo bodies has enabled the visualization of F-actin in T. gondii 28 for the first time, whereas F-actin in invading merozoites was described in an earlier study 29 .…”

“…In invading merozoites, P. falciparum FRM1 (PfFRM1) localizes to the apical pole and follows the moving junction 36 . In addition, F-actin is highly concentrated posterior to the moving junction, where the parasite is attached to the host cell surface receptors to sustain its progression into the cell, which is powered by the myosin motor 29,38,39 . TgFRM1 was also recently shown to be located exclusively at the tip of the tachyzoites, but it does not follow the moving junction in invading parasites 40 .…”

Anopheles Midgut FREP1 Mediates Plasmodium Invasion

“…The absence of the detectable fluorescence signal further supports that FREP1 binds to the sexual stage of P. falciparum parasites or mosquito invasion stage ookinetes. Finally, to confirm that our non-permeabilized fixation approaches maintain cell membrane integrity, we stained parasite preparations with antibodies against Plasmodium actin (31). Cells containing gametocytes were fixed separately with either 4% paraformaldehyde or 100% methanol on dry ice for 5 min.…”

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

“…Furthermore, parasites become readily motile upon induced egress and stop moving equally fast immediately after sealing the parasitophorous vacuole. A rather unique feature of apicomplexan actin is that it is largely maintained in an un-polymerized state (Angrisano et al, 2012;Dobrowolski et al, 1997). Filamentous actin is hence predictably highly dynamic, short, and the rate of polymerization and depolymerization must be tightly controlled.…”