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

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

An apicomplexan parasite-specific anti-actin antibody.

<p><b>A</b>) Sequence comparison between human non-muscle actin amino acids 237–251 (the basis of anti-Gly₂₄₅<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188-Varma1" target="_blank">[39]</a>) and apicomplexan actin I orthologues over the amino acids 239–253 (the basis for anti-Act_239–253). <b>B</b>) Surface representation of the structures of rabbit G-actin (PDB:1J6Z; A) and a protomer in rabbit F-actin (PDB:3G37; B) showing anti-Gly₂₄₅ epitope. Residues in yellow indicate polymorphisms between mammalian and <i>P. falciparum</i> actin. <b>C</b>) Representative immunoblot showing reactivity of rabbit® anti-Act_239–253 serum with human erythrocytes (hRBC), asexual <i>P. falciparum</i> (3D7), mouse erythrocytes (mRBC), asexual <i>P. berghei</i> (ANKA), human foreskin fibroblasts (HFF) and <i>T. gondii</i> tachyzoites (RH). Lower panel shows same hRBC and 3D7 sample probed with mouse (m) anti-Act_239–253 serum. <b>D</b>) As C but using generic anti-actin monoclonal C4.</p

Concentration of actin labelling in the nucleus and around the nuclear periphery.

<p>Widefield IFA of representative <i>P. berghei </i><b>A</b>) ookinetes and <b>B</b>) sporozoites that show pronounced nuclear labelling using rabbit anti-Act_239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s009" target="_blank">Movie S5</a>. <b>C</b>) Widefield IFA of <i>P. falciparum</i> rings labelled with rabbit anti-Act_239–253 (Red) and DAPI (Blue). <b>D</b>) As <b>C</b> but following 6 hour JAS treatment. <b>E</b>) Two colour widefield IFA using rabbit anti-Act_239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 µM JAS. All scale bars = 5 µm.</p

The tight junction is composed of dynamic actin filaments that localise posterior to the junction during invasion.

<p><b>A</b>) Widefield IFA with deconvolution and <b>B</b>) 3D reconstruction of <i>P. berghei</i> merozoites incubated with and without 1 µM JAS and labelled with anti-Act_239–253 (Green) and DAPI (Blue). Scale bar = 2 µm. Arrows show direction of invasion. <b>C</b>) Graphic representation of actin labelling in <i>P. berghei</i> merozoites with and without the addition of JAS. <i>n</i> = 124 merozoites for each of three replicates, mean is shown. <b>D</b>) 3D structured illumination microscopy (3D SIM) of three separate invading <i>P. falciparum</i> merozoites labelled with rabbit (upper row) and mouse (lower row) anti-Act_239–253. Labelling shows actin (Red), RON4 (Green) and DAPI (Blue). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s010" target="_blank">Movie S6</a>. Gamma settings were altered in 3D reconstructions.</p

The spatial distribution of actin in invading merozoites and sporozoites.

<p><b>A</b>) Transmission electron micrograph with anti-Act_239–253 (rabbit) immunogold labelling (arrowheads) of invading <i>P. falciparum</i> merozoite. Arrows show direction of invasion. <b>B</b>) Widefield IFA with deconvolution of invading <i>P. falciparum</i> merozoites labelled with mouse anti-Act_239–253 (Red) or rabbit PfRON4 (Green) and DAPI (Blue). Scale bar = 2 µm. <b>C</b>) Widefield IFA with deconvolution of invading <i>P. berghei</i> merozoites labelled with rabbit anti-Act _239–253 (Green) and DAPI (Blue). Scale bar = 2 µm. Gamma settings were altered in 3D reconstruction. <b>D</b>) Widefield IFA with deconvolution of invading <i>P. berghei</i> sporozoites labelled with rabbit anti-Act_239–253 (Green), anti-PbCSP (Red, exterior only) and DAPI (Blue). Scale bar = 5 µm, arrowhead shows presumed site of tight junction.</p