erythrocytes (as seen in free merozoites in Fig. 2A) was found to be similar for both treatments, no actin ring labelling was seen for any merozoite following JAS treatment (Fig. 7C). These data demonstrate that the ring like formation of actin at the tight junction, and its maintenance through invasion [41], is not a static structure but instead is dependent on filament turnover. To resolve the architecture of the actin ring at the junction further, P. falciparum merozoites labelled with both rabbit (r) and mouse (m) anti-Act239–253 serum were imaged using three- dimensional 3D-SIM, an approach that we have recently used to provide insight into the structures formed during P. falciparum erythrocyte infection [41]. In all instances Anti-Act239–253 labelling was concentrated in a ring lying posterior to the tight junction during merozoite invasion, defined as the edge of the junction towards the posterior of the parasite (Fig. 7D, Movie S6). However, labelling was rarely seen overlapping with the junction plane. Furthermore, on occasion the distribution of fluorescent signal clearly showed short filament-like structures around the circumfer- ence of the tight junction, with each fibre running approximately parallel to the plane of merozoite invasion. Although accurate sizing of these filaments is beyond the resolution limits of 3D-SIM, their size (less than 500 nm, Fig. 7D, Movie S6) is entirely consistent with the in vitro determined length of actin filaments from apicomplexan parasites [31,32,33]. These images may represent the first time that actin filaments (or bundles thereof) have been seen under conventional and drug free imaging conditions in motile apicom- plexan parasites. Combined with the results following JAS treatment, these data suggest dynamic actin filaments are a critical component of the tight junction. However, contrary to expectations, filaments reside behind and not directly in the plane of the junction, in contrast with labelling of apical membrane antigen (AMA) 1 [41]. This suggests that the driving force for motility (via actin-myosin) and the architecture of the tight junction (via AMA1-RON complex interaction) are discrete entities during invasion. Discussion Understanding of the role of actin in malaria parasite development has focussed in the most part on its core function during motility, though other auxiliary roles in development are being increasingly explored [33,34,47,48]. The current accepted model for apicomplexan motility and the role actin plays in cell movement [8] draws much of its support from immunoprecipi- tation of core components of the gliding motor and associated proteins [8,9,11,13,51,52] with some topological support from microscopic studies [8,9,11,13,18,28,53]. Although actin filaments form an essential dynamic component of the active parasite motor [5,15,22,23,24,26,28,54,55,56], much of the evidence so far has been based on experiments that lack the ability to dissect, on a fine scale, precisely where the microfilaments localise in the cell. This is also true for studies exploring the role of actin in hemoglobin uptake [33] and vesicle trafficking [34]. Until now, the definitive localisation of filaments under native imaging conditions has not been possible [19,20,28]. Here, utilising a parasite specific actin antibody that demonstrates preferential labelling of actin fila- ments, we show that F-actin is directly associated with several key compartments of the parasite indicative of separate functions. These include the nuclear periphery and F-actin association with gene regulation, pellicular membranes of zoite forms and cell motility, and the host-parasite tight junction and the essential role played by actin in merozoite invasion. Nuclear localisation of dynamic actin is consistent with recent studies that have demonstrated a role for actin and myosin in the movement of chromosome ends to the nuclear periphery in Actin Filaments in Plasmodium Development human cells [45,46]. Movement of active genes to sub-compart- ments within the nucleus has been shown to function in regulating antigenic variation in blood stage malaria parasites [57,58]. Indeed, two recent studies have demonstrated a key role of actin in both the spatial repositioning of genes in the nucleus and in binding to a regulatory nuclear histone methyltransferase [47,48]. As such, dynamic actin is clearly a key factor involved in mediating parasite antigenic variation and gene activation. Further investi- gation of the parasite nucleus in high definition and the effects of actin inhibitors on gene regulation will likely prove significant areas of interest for probing this possible function. Association of actin filaments with the supra-alveolar space and its association with pellicular membranes is clearly in line with the current model for gliding motility. At present, however, it is not clear which, if either, pellicular membrane provides the native binding surface. A compelling model would be for filaments of actin to directly associate with the plasma membrane. In other systems membrane bound glycosyl-phosphatidylinositol anchored proteins play a key role in linking F-actin directly to cortical membranes [59]. If validated in an apicomplexan cell, such a scenario would negate the need for a linear relationship linking actin filaments to the surface via tetrameric aldolase and the cytoplasmic tails of secreted thrombospondin related anonymous protein (TRAP)-family adhesins [11]. Of note, two-dimensional rafts of actin filaments associated with positively charged lipid layers form an ordered array when mixed with aldolase in vitro [60,61]. Indeed, were such an organisation to exist underlying the plasma membrane (as might be suggested by EM data [20,28]), this would directly provide the key rigid element upon which myosin bears its traction, transferring the entire plasma membrane raft rewards. External adhesins/invasins that cluster to this raft, perhaps anchored through interactions with aldolase [11,13], would then also be drawn rearwards. This might also imply that no singular adhesin/invasin-host receptor interaction necessarily mediates motility but instead it is the movement of the entire cluster of proteins through the membrane, and its interaction with the external milieu that moves an apicomplexan cell. The behaviour of sporozoite adhesion foci during motility would certainly favour the existence of raft- or patch-like traction [15]. Such a raft-like model linking the IMC bound motor with external adhesins is consistent with current data, and would only challenge the terminal link between actin and the extra-cellular milieu [11]. An important refinement to our current model of gliding mechanics during invasion may be required in light of the placement of actin filaments behind the tight junction and not directly beneath it. Recent evidence for the in vitro interaction of AMA1 with aldolase has been used to argue that AMA1 may interact directly with the gliding motor, leading to the notion that force generation and attachment are one contiguous structure [62,63]. Our imaging evidence supports an alternative model where force from the actomyosin motor is transmitted indirectly to the tight junction [64] (Fig. 8). If verified, such a model would raise several key questions. First, how is actin polymerisation spatially restricted to such a localised site? Since apicomplexan parasites possess a markedly reduced repertoire of identifiable actin regulators [12] this dramatically shortens the list of potential actin-binding proteins that might govern filament spatiotemporal localisation. A likely factor may be Formin1, which in malaria parasites has been tentatively localised to the tight junction [38]. However, this does not entirely agree with recent work in T. gondii, which demonstrates that of the two conserved formins (Formin1 and 2) both localise to the pellicle of the motile or invasive zoite but not specifically to the junction during invasion [65]. Discrepancies between the focussed ring of actin seen during PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32188