Accepted Article

Cellular Microbiology

Organisation and function of an actin cytoskeleton in Plasmodium falciparum gametocytes1

Marion Hliscs 1,2,3, Coralie Millet 1,2, Matthew W. Dixon1,2, Inga Siden-Kiamos4, Paul

McMillan1,5, and Leann Tilley1,2* 1

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology

Institute, 2Australian Research Council Centre of Excellence for Coherent X-ray Science, and 3

School of Botany, The University of Melbourne, Melbourne, VIC 3010, Australia; 4Institute of

Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, 700 13 Heraklion, Crete, Greece; 5The Biological Optical Microscopy Platform, The University of Melbourne, Melbourne, VIC, 3010, Australia.

*To whom correspondence should be addressed

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cmi.12359

1 This article is protected by copyright. All rights reserved.

Accepted Article Abstract

In preparation for transmission to its mosquito vector, Plasmodium falciparum, the most virulent of the human malaria parasites, adopts an unusual elongated shape. Here we describe a previously unrecognised actin-based cytoskeleton that is assembled in maturing P. falciparum gametocytes.

Differential extraction reveals the presence of a highly stabilised population of F-actin at all stages of development. Super-resolution microscopy reveals an F-actin cytoskeleton that is concentrated at the ends of the elongating gametocyte but extends inward along the microtubule cytoskeleton. Formin-1 is also concentrated at the gametocyte ends suggesting a role in actin stabilisation. Immunoelectron microscopy confirms that the actin cytoskeleton is located under the inner membrane complex rather than in the sub-alveolar space. In stage V gametocytes the actin and microtubule cytoskeletons are reorganised in a co-ordinated fashion. The actin depolymerizing agent, cytochalasin D, depletes actin from the end of the gametocytes, while the actin-stabilizing compound, jasplakinolide, induces formation of large bundles and prevents late stage disassembly of the actin cytoskeleton. Long-term treatment with these compounds is associated with disruption of the normal mitochondrial organisation and decreased gametocyte viability.

Keywords: Plasmodium falciparum, Apicomplexa, gametocyte, actin, tubulin, formin-1,

cytoskeleton.

Abbreviations: Filamentous actin, F-actin; Actin-related protein, Arp; 3D-SIM, 3D-Structured Illumination Microscopy; EM, Electron Microscopy; Glideosome-Associated Protein, GAP; IMC, Inner Membrane Complex; RBC, red blood cell

2 This article is protected by copyright. All rights reserved.

Accepted Article Introduction

Actin is an important cytoskeletal element that contributes to a broad variety of biological processes in eukaryotic cells such as cell motility, cell division, vesicle trafficking, mitochondrial morphology, establishment of cell polarity and gene regulation (reviewed in (Pollard et al., 2009)). While most eukaryotic actins are highly conserved, the malaria-causing apicomplexan Plasmodium species express two actin isoforms that are quite divergent from conventional (mammalian and yeast) actins ( 25) and an emPAI score of 21.2, was the most abundant actin isoform in the detergent-resistant cytoskeleton fraction. Actin-II (PF3D7_1412500) was also identified but with a 15-fold lower emPAI score (1.4) and lower sequence coverage (38%). The actin-related protein-1 (Arp1), a component of the dynactin complex, was found with 18% sequence coverage and a 62-fold lower emPAI score (0.34). The actin cytoskeleton is located underneath the Inner Membrane Complex The P. falciparum gametocyte possesses a cisternal membrane compartment, the Inner Membrane Complex (IMC) that lies beneath the plasma membrane and interacts closely with structural microtubules (Dearnley et al., 2012, Sinden, 1982, Kudryashev et al., 2010). In Plasmodium zoites, actin is postulated to be located in the sub-alveolar space between the IMC and the plasma membrane (Baum et al., 2006, Schatten et al., 2003, Angrisano et al., 2012b). To study the

subcellular position of the F-actin structure relative to the IMC and the microtubule cytoskeleton we used parasites expressing a GFP chimera of IMC component, Glideosome-Associated Protein-50 (GAP50; (Dearnley et al., 2012, Yeoman et al., 2011)). As anticipated, tubulin is laid down at the cytoplasmic side of the IMC in elongating stage IV gametocytes (Figure 4A). We also detected well-defined actin labelling at the cytoplasmic side of the gametocyte IMC, most prominent at the parasite poles, but also evident along the gametocyte sides (Figure 4B, arrows). To verify the subcellular position of actin and to overcome the spatial resolution limitations of light microscopy we prepared samples of stage III/IV gametocytes for analysis by electron microscopy (EM). High pressure freezing and freeze-substitution enables good preservation of the cellular 11 This article is protected by copyright. All rights reserved.

Accepted Article

structure revealing the organisation of the microtubules and the IMC (Figure 4C). The nascent IMC is observed as flattened cisterna underneath the parasite plasma membrane. The microtubules are evident as ~25 nm hollow rods, organised underneath the IMC in groups of 5-10, separated by regions with fewer tubules (Figure 4C, arrowheads). Actin filaments are not readily visualised in these preparations, even in samples stabilised with jasplakinolide (data not shown). This suggests that P. falciparum F-actin structures do not survive the fixation methods employed.

We instead sought to use immunoEM to determine the location of the F-actin and employed the Tokuyasu method, which permits optimum epitope preservation, but less optimal preservation of structure. Samples were labelled with anti-ACT16-30 antibodies. Actin labelling is evident in a region underneath the IMC (Figure 4D-F; see larger field of view for 4D in Suppl Fig 4A). The organisation is best appreciated in sections of samples where the host RBC has separated from the gametocytes (Figure 4E). The actin labelling appears to be located on the cytoplasmic side of the microtubule layer (Figure 4E-F). No labelling was observed in samples prepared without the primary antibody (Suppl Figure S4B). Effect of cytoskeleton modulating agents on gametocyte morphology and viability Cytochalasin D is a cell-permeable mycotoxin that binds to the slow-growing end of actin filaments and inhibits both polymerisation and depolymerisation (Cooper, 1987). Treatment of schizontinfected RBCs with 1 µM cytochalasin D completely ablates merozoite invasion but does not affect maturation of ring stage parasites (McMillan et al., 2013, Srinivasan et al., 2011). By contrast

treatment of ring stage parasites with a higher concentration of cytochalasin D (10 M for 8 h) is associated with a substantial loss of parasite viability (McMillan et al., 2013). In this work we found that short-term (2 h, 37oC) treatment with cytochalasin D resulted in an apparent decrease of tip-associated actin labelling (Suppl Figure S5). In control samples 77% of stage III/IV gametocytes showed the typical bipolar tip-associated F-actin labelling. This was reduced to 4% and 0% in parasites treated with 1 µM and 10 µM cytochalasin D, respectively. By contrast 3D-SIM revealed that short-term cytochalasin D treatment was not sufficient to disrupt the F-actin population 12 This article is protected by copyright. All rights reserved.

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associated with microtubules or the organisation of the tubulin cytoskeleton in general (Suppl Figure S5C).

To examine the effect of long-term treatment we added 3 µM cytochalasin D (or an equivalent volume of carrier solvent, DMSO) to a P. falciparum gametocyte culture (at stage I). Drug pressure was maintained for 12 days with daily exchange of drug and media (Figure 5). Parasitemia and gametocyte development were monitored by Giemsa-stained smears, as previously described (Dearnley et al., 2012).

Normal gametocyte development was observed in control cultures with steadily increasing gametocyte numbers, reaching a maximum of ~1.8% on day 4, with ~1% still present on day 12 (Figure 5A). Cytochalasin D treatment (3 µM) led to a reduction in gametocyte numbers from ~1.2% on day 2 to 0.4% on day 12. Despite declining numbers, 3 µM cytochalasin D did not prevent surviving gametocytes from progressing through the developmental stages (Figure 5B, middle panel). Long-term treatment with a higher concentration of cytochalasin D (10 µM) was associated with a further drop in gametocyte numbers and more marked cellular deformations (data not shown). On day 6 of the culture, the cytoskeletal organisation was analysed by immunofluorescence microscopy. 3D-SIM showed that, as for the short-term treatment, the prominent tip-associated F-actin labelling was depleted (Figure 5C, middle panel, yellow arrowheads), but F-actin labelling remained associated with the body of the gametocyte (Figure 5C, middle panel, white arrowheads). This suggests that the microtubule-associated actin structures are very highly stabilised. We also examined the effect of the actin-stabilizing compound, jasplakinolide (Bubb et al., 1994). Jasplakinolide (0.3 µM) was previously shown to inhibit re-invasion but not maturation from the ring to trophozoite stage (Mizuno et al., 2002). We found that short-term (30 min) treatment of stage III/IV gametocytes with 1 M jasplakinolide was associated with the formation of very thick actin bundles spanning lengthwise through the parasite body (Suppl Figure S6). Treatment of stage V gametocytes resulted in sequestering of the F-actin into two to three thick actin shafts in the 13 This article is protected by copyright. All rights reserved.

Accepted Article

parasite cytoplasm. We found that long-term actin stabilisation by jasplakinolide is lethal for gametocytes. A gradual loss of gametocyte numbers was observed (Figure 5A), with complete loss of parasites by days 10 and 12, for 0.3 µM and 0.1 µM jasplakinolide, respectively (Figure 5A,B, and data not shown). Jasplakinolide treatment was also associated with an apparent increase in the rate of maturation of the parasites. For example, by day 6 of culture almost all parasites exhibited either stage IV or a deformed morphology. No gametocytes displaying stage V morphology were observed when cultures were treated with 0.3 µM jasplakinolide. This suggests failure of development at the point where the actin and tubulin cytoskeletons are normally disassembled. Immunofluorescence at day 8 revealed similar morphological effects to those observed during short-term drug treatment and confirmed the presence of thick actin bundles in the parasite cytoplasm (Figure 5C, bottom panel). Modified actin organisation is associated with mitochondrial disruption but does not alter apicoplast organisation Chemical or genetic perturbation of actin dynamics can induce defects in positioning of cellular organelles in P. falciparum and T. gondii as well as in higher organisms (Andenmatten et al., 2013,

Jacot et al., 2013, Mueller et al., 2013, Korobova et al., 2013). We examined the effect of long-term (8 days) exposure to jasplakinolide and cytochalasin D on the organisation of the mitochondrion and apicoplast. The apicoplast was visualised using an antibody against acyl carrier protein (Tonkin et al., 2004, Okamoto et al., 2009), and is evident as a focused slightly elongated organelle, located near the nucleus (Figure 6A, green). No obvious change in apicoplast morphology was observed upon treatment with the actin-modulating agents. Gametocyte mitochondria were visualised using the cell permeant MitoTracker® RedCMXRos probe. This dye accumulates in mitochondria with intact membrane potential, allowing the visualisation and classification of mitochondrial phenotypes by fluorescence microscopy. In control samples, the gametocyte mitochondrion shows a characteristic reticular structure (Figure 6A, red). To assess alterations to the morphology associated with drug treatment, we categorised the 14 This article is protected by copyright. All rights reserved.

Accepted Article

mitochondrial profiles as compact (extending 6 m) or fragmented, and examined the effects of the different treatments by live cell microscopy. Long-term treatment with cytochalasin D was associated with mitochondrial elongation in 46% of cells compared with 16% for controls. By contrast, long-term jasplakinolide exposure led to a substantial increase in the number of gametocytes with fragmented mitochondria, with complete loss of mitochondrial labelling also often observed (Figure 6). While secondary effects of parasite killing need to be considered, these data indicate that disruption of the actin cytoskeleton has a substantive effect on the organisation of mitochondria. Discussion

Early ultrastructural analyses revealed that the gross morphological restructuring of the gametocyte is coincident with the appearance of a tri-laminar membrane structure subtended by a layer of structural microtubules (Sinden, 1982, Sinden et al., 1978, Aikawa et al., 1969). More recent

studies have revealed the relationship between the sub-pellicular membrane complex of

gametocytes and the IMC of motile zoites (Dearnley et al., 2012, Gould et al., 2008). Until now, no role for actin in gametocyte architecture has been described. By contrast, actin polymerisation is assumed to be necessary for the motility of all apicomplexan parasites, with filamentous actin proposed to provide a rigid track along which the myosin heads walk to generate the power stroke. It is also needed for other roles in endocytosis, organelle positioning and gene regulation. Paradoxically, it has not been possible to visualise long actin filaments in Plasmodium zoites under physiological conditions (Wetzel et al., 2003, Skillman et al., 2011).

Recently two antisera have been developed, recognising different regions of actin, which preferentially bind to the filamentous form of P. falciparum actin (Siden-Kiamos et al., 2012, Angrisano et al., 2012b). Anti-ACT239–253 was previously shown to recognise structures associated with the apical end of zoites and the merozoite's tight junction. Anti-ACT16–30 recognised actin in short rods in ookinetes of P. berghei. Using these antibodies we made the surprising finding that prominent F-actin-containing structures are present in P. falciparum gametocytes. Super-resolution 15 This article is protected by copyright. All rights reserved.

Accepted Article

imaging reveals that the F-actin is organised into a cytoskeletal network that is concentrated at the ends of developing gametocytes but also radiates in striations along the body of the gametocyte. The F-actin structures appear to be assembled very early in gametocyte development (stage IIa), and F-actin puncta are first observed at the apex of a curved band of microtubules. F-actin structures appear to define both longitudinal poles of the elongating parasite but are also present in regions at the gametocyte periphery, where the IMC is established but microtubules are not yet present. Once the microtubules are organised into a sheath enclosing much of the parasite body (stage III/IV), F-actin becomes even more concentrated at the ends. Given that the microtubules do not extend to the very tips of the crescent-shaped stage IV gametocyte, the actin cytoskeleton may stabilise the IMC and facilitate bending into a shape that can be accommodated by the host RBC. Cross-talk between actin and microtubule cytoskeletons has been described in other eukaryote cells (reviewed by (Goode et al., 2000)). Microtubules often bridge long distances inside the cell, while actin is located closer to the cell periphery, with actin cables capturing, anchoring and guiding microtubules to the cell periphery. It has been shown that filamentous actin can associate lengthwise with microtubules and exert force by promoting microtubule bending and nicking in vivo and in vitro (Kaverina et al., 1998, Sider et al., 1999). We propose that cytoskeletal cross-talk

drives co-ordinated assembly and dismantling of microtubules and actin structures at different stages of gametocyte development. We found that lysis of stage III/IV gametocytes released a pool of actin that likely includes G-actin. Low speed centrifugation was sufficient to pellet a substantial proportion of the actin population, suggesting it is stabilised by incorporation into an actin cytoskeleton. The presence of a stabilised actin cytoskeleton is also indicated by the solubility profile of the pellet material. Surprisingly, the relative amounts of cytoplasmic and cytoskeletal actin are similar in stage III/IV and stage V gametocytes. This suggests that F-actin filaments are present as stabilised oligomers or short filaments in stage V gametocytes and not disassembled into their monomeric forms. These remnant

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structures may help provide the cellular deformability properties that are needed for survival in the host circulation (Dixon et al., 2012).

While asexual P. falciparum expresses only actin-I, gametocytes also express actin-II (Wesseling et al., 1989, Skillman et al., 2011), and this isoform plays a critical role in male gametogenesis in P. berghei (Deligianni et al., 2011). A previous study reported that anti-ACTII has a diffuse (cytoplasmic) labelling pattern in gametocytes (Rupp et al., 2011). The nature of its role is not clear

but it cannot be complemented by actin- I (Vahakoski et al., 2014). A very recent study showed that both actin-I and actin-II have unusual properties, hydrolysing ATP very efficiently, and rapidly forming short oligomers in the presence of ADP (Vahakoski et al., 2014). Crystal structures of the isoforms identify structural features that are responsible for the unusual polymerisation properties. Of the two isoforms actin-II was found to form filaments with a length and structure more similar to canonical actins, although both isoforms can form filaments if stabilised by actin-binding proteins (Vahakoski et al., 2014). We were interested to determine which actin isoform is responsible for the F-actin cytoskeleton. Using mass spectrometry we confirmed that actin-I is by far the most abundant isoform in detergent-solubilised gametocytes, with actin-II present at much lower levels. Similarly the mass spectrometric analysis revealed the presence of low levels of Arp1, a protein that shares 73% sequence similarity with actin-I. Arp1 is part of the dynactin complex, known to be involved in vesicular transport and control of gene expression; it appears to be an essential protein (SidenKiamos et al., 2010, Gordon et al., 2005). Given the much lower levels of actin-II and Arp1 and the

fact that both the anti-ACT16–30 and the anti-actin(Dicty) antibodies recognise recombinant actin-II

and Arp1 much less strongly or not at all by Western analysis, we conclude actin-I is that the major component of the F-actin cytoskeleton. Given that gametocytes have no documented motility, even upon activation, we conclude the Factin is likely to be part of a structural cytoskeleton, and would need to be stable rather than

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Accepted Article

dynamic. To overcome the inherent instability of plasmodial actin, the filaments would need to be stabilised and organised into a higher order structure.

The Plasmodium genome encodes only a very few homologues of actin-binding proteins that promote filament nucleation, stabilisation and bundling. Thus far, the only identified proteins with nucleation activity in apicomplexan parasites are the formin-like proteins – with Plasmodium

encoding two formins and T. gondii, three. In higher eukaryotes, formins act by recruiting profilin-

bound G-actin to the barbed end of F-actin via a proline-rich FH-1 domain (Kovar et al., 2006,

Skillman et al., 2012). T. gondii formins have been shown to dramatically enhance polymerisation

of T. gondii actin-I and to induce bundling of actin filaments in a process that does not rely on profilin (Skillman et al., 2012). In asexual blood stages, formin-1 is concentrated at the tips of P. falciparum merozoites and appears to track with the tight junction during invasion. Attempts to delete the gene were not successful (Baum et al., 2008). We found that formin-1 is concentrated at the tips of gametocytes, but also shows some labelling along the length of the parasite. While further work on the dynamics of the interaction is required, this finding is consistent with formin-1 playing a role in F-actin nucleation, stabilisation or bundling. Previous studies have suggested that actin might be located in the sub-alveolar space in zoites, i.e. between the IMC and the plasma membrane. Preparation of samples for EM by high pressure freezing enabled good preservation of membranes and microtubules and showed that microtubules are initially laid down in groups of 5 – 10 rods, which likely gives rise to the striated appearance observed by immunofluorescence, when stage IV gametocytes are labelled with anti-tubulin. It was not possible to visualise actin filaments in these EM preparations, even when the filaments were stabilised with jasplakinolide. This suggests that the F-actin filaments do not survive the harsh fixation methods. In a further effort to determine the location of the F-actin structures and their organisation relative to the microtubule cytoskeleton, we employed the Tokuyasu method, which is designed to preserve antigenicity. Using the F-actin specific anti-ACT16-30 antibody, we showed that

F-actin is present underneath the microtubule layer. Similarly immunofluorescence microscopy 18 This article is protected by copyright. All rights reserved.

Accepted Article

showed that F-actin is located on the cytoplasmic face of the IMC, as marked by a GAP50-GFP chimera.

We attempted to disrupt the actin cytoskeleton using cytochalasin D. Cytochalasins block actin turnover but do not cause disassembly of stabilised actin filaments (Lin et al., 1980, Cooper, 1987). We found that both short- and long-term treatment with cytochalasin D depleted F-actin from the ends of the gametocytes but did not appear to disassemble the microtubule-associated F-actin in the body of gametocytes. This provides further support for the suggestion that these structures are highly stabilised, perhaps by capping at both ends or by lengthwise association with stabilising proteins. Consistent with this, short-term treatment with cytochalasin D at concentrations up to 3 M did not result in dramatic changes in morphology. Nonetheless long-term treatment with cytochalasin D did result in some deformation of shape and loss of viability. Jasplakinolide is a potent inducer of actin polymerisation and filament bundling (Bubb et al., 1994). Treatment with jasplakinolide led to the formation of thick F-actin structures at all stages of development. Long-term jasplakinolide treatment led to accelerated production of gametocytes with stage IV morphology, followed by loss of gametocytes from the culture. This suggests that actinstabilised parasites are unable to transition to stage V of development. Chemical or genetic perturbation of actin dynamics in T. gondii causes defects in positioning of the apicoplast and secretory organelles (Mueller et al., 2013, Shaw et al., 2000, Jacot et al., 2013). Gametocytes have no obvious anterior-posterior polarity and the nucleus, apicoplast and mitochondrion are roughly centrally placed. Untreated gametocytes show a small slightly elongated apicoplast, as previously reported (Okamoto et al., 2009), with no obvious morphological change upon treatment with actin-modulating reagents. By contrast we found that long-term cytochalasin D treatment was associated with an extension of the mitochondrial network, while treatment with jasplakinolide was associated with fragmentation of the mitochondrion and, in some cells, loss of mitochondrial labelling. In mammalian systems, F-actin has been shown to be involved in the recruitment of dynamin-related protein-1 to the mitochondrion, which in turn controls 19 This article is protected by copyright. All rights reserved.

Accepted Article

mitochondrial fission (De Vos et al., 2005, Korobova et al., 2013). Disruption of mitochondrial morphology and normal function may be responsible for the loss of gametocyte viability observed during long-term treatment with cytochalasin D or jasplakinolide. Taken together our data provide strong evidence for an actin cytoskeleton in P. falciparum gametocytes. In this non-motile stage we propose that F-actin plays an important structural role, providing a template for the positioning of microtubules that helps both to initiate and stabilise the tubulin cytoskeleton. The concentration of the actin at the tips of the gametocyte is interesting. Factin has been shown to accumulate at the apical end of zoite stages in Plasmodium and T. gondii

upon treatment with jasplakinolide (Wetzel et al., 2003, Angrisano et al., 2012b, Siden-Kiamos et al., 2012), indicating the presence of pre-existing short actin filaments at this location. These Factin structures may have structural functions during invasion. Gametocytes can persist in symptomless carriers for many weeks and are responsible for transmission and endemicity of disease. Unfortunately gametocytes are refractory to treatment with current antimalarials, which can accelerate the spread of drug-resistant parasites (Ecker et al., 2011). Thus new strategies to prevent the spread of malaria by targeting this stage are badly needed. This work identifies a novel feature of gametocyte cell biology that could potentially be targeted with new antimalarial strategies.

Materials and Methods Recombinant Proteins The complete ORFs of P. falciparum ACTI, ACTII and Arp1 were cloned in plasmid pET23d and

expressed in E. coli BL21(DE3)pLysS containing the plasmid pMICO as described previously (Andreadaki et al., 2014). Whole cell extracts were used to characterise the anti-ACTI16-30 and antiDictyostelium actin antibodies. Parasite culture and gametocyte enrichment We used a high gametocyte producing P. falciparum 3D7 isolate, which was recovered from a trial

volunteer (Lawrence et al., 2000) and has been cultured for only a limited time. Parasite-infected 20 This article is protected by copyright. All rights reserved.

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RBCs were cultured in RPMI-HEPES supplemented with 5% human serum and 0.25% AlbuMAX II as previously described (Foley et al., 1994). Briefly a culture of mainly ring stage parasites (6-8%

parasitemia, 5% haematocrit) was treated with 5% sorbitol (Lambros et al., 1979) and subsequently

enriched for ring stages by Percoll density gradient centrifugation (Knight et al., 1982). Parasites were cultured until they reached 8-10% trophozoite stage and divided to 2% parasitemia, keeping the haematocrit at 5%. The culture was maintained for 12 days in the presence of 62.5 mM N-acetyl glucosamine to inhibit merozoite invasion and thus asexual replication (Hadley et al., 1986). The

medium was changed daily without substitution of RBCs. Parasite growth was monitored by Giemsa-stained blood smears. At the desired stages of development gametocytes were purified by magnetic separation (Fivelman et al., 2007). Gametocyte drug assays For short-term drug treatment gametocyte stages III/IV were magnet-purified and incubated in media containing 1 – 10 µM cytochalasin D (Sigma-Aldrich), or 1 µM jasplakinolide (Santa-Cruz)

from 30 min to 2 h, at 37oC. All drugs were dissolved in DMSO and control samples contained an

equivalent volume of DMSO or media only. The samples were washed, fixed and processed for immunofluorescence. Long-term drug treatment was initiated at stage I of gametocyte development, three days after sorbitol-Percoll synchronisation. Parasites were cultured in a volume of 2 ml in 12 well culture dishes under constant drug pressure for 12 days. Cultures were supplemented with fresh drugs and media daily. Gametocyte development was assessed by microscopy on Giemsastained thin blood smears. Parasitemia was determined by assessing at least 30-50 random fields of view (3000-8000 uninfected RBCs). Gametocyte morphology was classified as previously described (Dearnley et al., 2012). In total seven independent long-term drug treatment experiments were performed. Microscopy and image analysis Parasite-infected RBCs were washed in 37°C warm RPMI media containing glucose and gentamicin (wash media) and settled on Concanavalin A-coated cover slips for 10 min at 37°C. 21 This article is protected by copyright. All rights reserved.

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Samples were fixed for 20 min at room temperature (RT) with 2% PFA in 1 x microtubule stabilisation buffer (MTSB, 10 mM MES, 150 mM NaCl2, 5 mM EGTA, 5 mM glucose, 5 mM

MgCl2) (Deligianni et al., 2011) containing 0.0025% EM-grade glutaraldehyde. Cells were washed, permeabilised with 0.2% Triton-X100 for 20 min at RT and blocked for 1 h with 3% BSA/PBS.

Sample were incubated for 1 h in 3% BSA/PBS with the following primary antibodies: rabbit antiACT239-253 (1:300, (Angrisano et al., 2012b)), rabbit anti-ACT16–30 (1:300, (Siden-Kiamos et al.,

2012)), mouse anti-Dictyostelium actin (1:300, (Westphal et al., 1997), mouse anti-β-tubulin (1:300, Sigma Aldrich), mouse anti-GFP (1:300, Roche), rabbit anti-formin-1 (1: 300, (Baum et al., 2008)).

We used less stringent permeabilisation (0.1% Triton X-100 for 10 min) for the following antibodies: mouse anti-β-actin (1:300, Sigma Aldrich) and anti-acyl carrier protein (ACP, 1: 1000, (Tonkin et al., 2004)). Secondary anti-mouse and anti-rabbit IgG conjugated Alexa Fluor 488 and 568 (Life Technologies) were incubated for 1 h at room temperature in PBS. Parasite DNA was labelled with 1 µg/ml DAPI before mounting in 90% glycerol containing anti-fading agent pphenylene-diamine (PPD) in PBS, pH 8.6. Microscopy was performed using a DeltaVision Elite deconvolution system (Applied Precision). Zstacks (0.15-0.2 µm steps) were deconvolved using the default settings in the SoftWoRx 5.0 acquisition software. Images were further processed using NIH ImageJ version 1.47c (rsbweb.nih.gov/ij). 3D-Structured Illumination Microscopy (3D-SIM) (Schermelleh et al., 2008)

was implemented on a DeltaVision OMX V4 Blaze™ (Applied Precision). Samples were excited using 488 and 568 lasers and imaged using 528/48 nm, 608/37 nm and with a 60x oil immersion lens (1.42 NA) as previously described (McMillan et al., 2013). The diameters of F-actin structures were analysed by measuring the intensity profiles of ten filaments using FIJI (NIH). The resulting curves were averaged and background corrected and the FWHM was measured using Excel. Tubulin filaments were traced using a segmented line and linearised using the selection/linearise tool in Image J, then intensity profiles for the tubulin and actin labelling were assessed using ImageJ. 22 This article is protected by copyright. All rights reserved.

Accepted Article

Actin pelleting and solubility studies Standard gametocyte cultures were sorbitol-treated at day three after induction to eliminate asexual stages. Gametocytes were harvested on day 4 or 5 of culture (mainly stages III and IV) and day 10 (mainly stage V). Magnet-purified gametocytes (10 x 106 per sample) were treated with 0.15%

saponin/PBS on ice for 10 min to release host cell contents. Samples were washed and diluted in 50 µl Tris acetate buffer (TA) modified from (Hu et al., 2006) (10 mM Tris acetate, pH 7.4, 150 mM NaCl2, 10 mM EGTA, 2.5 mM MgCl2) supplemented with 1x EDTA free protease inhibitor complete and 2 µg/ml RNaseA and 10 µg/ml DNAseI (Roche) and sonicated (25s, 3x at 4 oC). Sonicated parasites where subjected to low speed centrifugation (15,000g, 10 min, 4oC). The

resulting supernatant representing the cytosolic fraction was collected. The pellet was washed three times in TA buffer and either dissolved in an equivalent volume of 1% SDS loading dye for SDSPAGE or further used in solubility studies. For solubility assays pellets were treated for 1 h at 4°C in 50 µl TA buffer containing 1-2% Triton X-100, 6 M urea or 100 mM Na2CO3, pH 11.5. Soluble and insoluble fractions were separated by centrifugation (15,000g, 10 min at 4°C). The pellet was washed in TA buffer and boiled in 1xSDS loading dye (5 min at 95°C). Samples were subjected to Western blot analysis. Blots were blocked overnight in 5% milk TBS (0.25% Tween20) and probed with primary antibodies in 1% milk in TBS-T: mouse anti-Dictyostelium actin (1:1000, (Westphal

et al., 1997)), mouse anti-β-tubulin (1:1000, Sigma Aldrich). Proteins were detected by secondary anti-mouse and anti-rabbit horseradish peroxidase conjugate (HRP) antibodies (1:10,000, Millipore). Chemiluminescence was visualised via Clarity Western-ECL reagent (BioRad) and detected using a luminescence image analyser, LAS3000 (Fuji Film). Total parasite lysate, HFF cell lysate and ghosts of uninfected RBCs were dissolved in RIPA buffer as described previously (Yeoman et al., 2011).

Mass spectrometry The Triton X-100-insoluble pellet was subjected to 4-12% SDS-PAGE (Life Technologies) and stained with Coomassie G-250 (SimplyBlue SafeStain, Invitrogen). A gel band at the size of actin 23 This article is protected by copyright. All rights reserved.

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(42 kDa) was excised and further processed by tryptic in-gel digestion. In brief, the gel plug was distained (50 mM TEAB, 50% acetonitrile 1:1, 3 h), dehydrated (100% acetonitrile, 30 min), reduced (10 mM TCEP/ 50 mM TEAB, 45 min at 55°C) and alkylated (55 mM iodoacetamide/ 50 mM TEAB) at room temperature for 30 min. The sample was washed, rehydrated and digested overnight with trypsin (Sigma). The tryptic digest was analysed by LC-MS/MS using a LTQ Orbitrap Elite (Thermo Scientific) with a nanoelectrospray interface coupled to an Ultimate 3000 RSLC nanosystem (Dionex). Data were analysed using Proteome Discoverer (Thermo Scientific version 1.4) with Mascot (Matrix Science version 2.4) against the Uniprot database and searched against the P. falciparum proteome. High pressure freezing (HPF) – freeze substitution

Infected RBCs were high-pressure frozen in 20% BSA using EM PACT2 (Leica Microsystems) at 2000 bar. The vitreous samples were perfused, with slow exchange of bound water, using anhydrous acetone containing 0.2% uranyl acetate. Freeze substitution was initiated at -90°C and the temperature increased to -50°C over 6 h. Exchange with Lowicryl resin was performed at -50°C over 97.5 h and temperature was then raised to +20°C over 12 h. Sections of 70 nm were cut at room temperature and examined on a FEI Tecnai F30 at an accelerating voltage of 300 kV. ImmunoEM

For Tokuyasu sections, the cells were fixed in 2% paraformaldehyde in 1x MTSB buffer and 0.008% glutaraldehyde for 2 h at room temperature, embedded in gelatine, perfused in 2.3 M sucrose overnight, and subsequently frozen in liquid nitrogen. Frozen blocks were sectioned (70 nm or 100 nm) in a cryo-microtome (Cryo Ultra Microtome, Leica UC7/FC7) at –110°C, thawed on

sucrose and labelled with rabbit anti-ACT239–253 (1:20, 1:50 or 1:65 for 1 h at room temperature) followed by either 6 nm protein A. Samples were examined on a FEI Tecnai F30 at an accelerating voltage of 300 kV. Mitochondria and apicoplast analysis

24 This article is protected by copyright. All rights reserved.

The mitochondria and apicoplast morphology was examined at day 8 of gametocyte drug cultures.

Accepted Article

Cells were washed and stained with MitoTracker®Red CMXRos (Molecular Probes) (20 nM, 5 min,

37°C). Mitochondria were either processed for immunofluorescence and apicoplast detection as described above or directly examined by live cell imaging. Cells were washed after incubation with MitoTracker®Red CMXRos in wash media, diluted to 1% haematocrit and settled on Concanavalin A-coated coverslips. Samples were sealed with Vaseline and imaged for 10 min. Sample preparation for each condition was performed just before imaging to prevent cellular stress. A DeltaVision Elite deconvolution microscope was used for imaging. Quantification of mitochondria length was performed on deconvolved z-stack projections using NIH ImageJ version 1.47c (rsbweb.nih.gov/ij). For data processing and presentation Microsoft Excel was used.

Acknowledgements We thank Ching-Seng Ang from the Mass Spectrometry and Proteomic Facility Bio21 Institute, Shannon Kenny, Bio21 Institute, and Lefteris Spanos, Foundation for Research and Technology, Hellas, for technical support. We thank Jake Baum from Imperial College, London, for generously providing the anti-ACT239-253 and anti-formin-1 antibodies and for helpful comments on the

manuscript. We thank Geoff McFadden from the School of Botany University of Melbourne for providing the ACP antibody, Martin Blume and Malcom McConville from Bio21 Institute for sharing equipment, and David Sibley for useful discussions. Microscopy was performed at the Melbourne Advanced Microscopy Facility and the Biological Optical Microscopy Platform, University of Melbourne (www.microscopy.unimelb.edu.au).We thank Dr Eric Hanssen for imaging contributions. This work is supported by the Deutsche Forschungsgemeinschaft (HL65/11) (to MH) and by grants from the Australian Research Council and the Australian National Health & Medical Research Council. LT is an ARC Australian Professorial Fellow.

25 This article is protected by copyright. All rights reserved.

Accepted Article Figure legends

Figure 1. Unusual F-actin localisation in P. falciparum gametocytes. Immunofluorescence deconvolution microscopy images of different P. falciparum gametocyte stages (II – V) labelled

with anti-F-actin (anti-ACT239–253 or anti-ACT16–30; red), anti--tubulin (green) and DNA stain (DAPI, blue). Brightfield images are shown in the third column. F-actin accumulates at the gametocyte tips and at the parasite periphery (arrows). Scale bars: 5 m. Higher magnification images highlighting accumulation of actin at the ends of gametocytes are shown at the right. Figure 2. Close association of the F-actin and tubulin cytoskeletons and co-labelling with formin-1. (A,B). 3D-Structured Illumination Microscopy (3D-SIM) of actin in P. falciparum

gametocytes (stages II to IV) immunolabelled with anti-ACT239–253 and anti-ACT16-30 (red). (B) Tipassociated actin bundles extend into the body of the gametocyte, juxtaposed to microtubules (anti-tubulin; green, arrows). Higher magnification images show tip-associated actin (yellow arrows) and actin-microtubule structures (blue arrows). (C) Deconvolution images of stage IV gametocytes labelled with anti-actin(Dicty) (red) and anti-formin-I (green). Higher magnification images highlighting accumulation of formin-I at the ends of gametocytes (arrows) are shown at the right. Scale bars: 5 m. Figure 3. Parasite actin and tubulin are present in soluble and insoluble fractions in stage IV and V gametocytes. Parasites were saponin-treated, sonicated, and centrifuged at low speed (15,000g, 10 min, 4°C). The cytoplasmic fraction (supernatant) and the pellet were collected and analysed by Western blot (left panels). The pellet was further incubated (1 h, 4°C) under different conditions (2% Triton X-100, 6 M urea, 100 mM Na2CO3, pH 11.5) and again subjected to

centrifugation (15,000g) (right panels). The supernatant (soluble) and pellet (insoluble) material were prepared for Western blotting and probed using anti-actin(Dicty) (A) and anti--tubulin antibodies (B).

Figure 4. The actin cytoskeleton is located on the cytoplasmic side of the IMC. (A,B) 26 This article is protected by copyright. All rights reserved.

Accepted Article

Immunofluorescence microscopy was performed on gametocytes expressing GAP50-GFP as an IMC marker. Samples were labelled with anti-GFP (green) and anti--tubulin (A, stage IV, purple)

or anti-ACT16-30 (B, stage IV, red). Higher magnification images of regions marked 1-3 are shown at

right. (C) A thin section (70 nm) of a high pressure frozen freeze-substituted sample prepared for EM showing the host RBC, the parasite plasma membrane (PPM), the parasitophorous vacuole membrane (PVM), the IMC and microtubules (MT; arrowheads) in a stage IV gametocyte. (D-F) Samples of stage III/IV gametocytes were plunge frozen, and cryosections (80 nm) were prepared for immunolabelling using the Tokuyasu method. The thawed sections were labelled with antiACT16-30, probed with 6 nm gold-protein-A and prepared for EM. The RBC, PPM, IMC and MT are indicated.

Figure 5. Modulation of the actin cytoskeleton compromises gametocyte development. P. falciparum gametocyte were cultured in the presence of 3 µM cytochalasin D (CytoD), 0.3 µM jasplakinolide (Jas) or an equivalent level of the carrier solvent (0.3% DMSO) continuously for 12 days. Parasitemia and the gametocyte stages were determined every second day by Giemsa-stained blood smears. (A) Sexual stage parasitemia is shown for controls (grey), 3 µM cytochalasin D (blue) and 0.3 µM jasplakinolide (red). (B) Different gametocyte stages are presented as a percentage of the total number of infected RBCs at the indicated day of culture. Colour coding as shown in the top right panel is used to distinguish asexual trophozoite stages (grey) and gametocyte stages: II, blue; III, green; IV, yellow; V, red; rounded, purple; deformed, white. The absence of parasites at days 10 - 12 of jasplakinolide treatment is indicated with a cross. Shown is one representative experiment (n = 7). (C) Immunofluorescence at day 6 of continuous drug exposure showing the labelling of the actin (red) and tubulin (green) cytoskeletons. Controls and cytochalasin D treated samples were imaged by 3D-SIM and jasplakinolide-treated samples by widefield deconvolution microscopy. Tip-associated (yellow arrows) and parasite body-associated (white arrows) F-actin labelling is indicated. Scale bars: 5 µm Figure 6. Modulation of the actin cytoskeleton compromises mitochondrial organisation. 27 This article is protected by copyright. All rights reserved.

Accepted Article

Gametocyte cultures maintained in the presence 3 µM cytochalasin D, 0.3 µM jasplakinolide, or 0.3% DMSO (controls) were assessed at day 8 of culture. (A) Immunofluorescence images showing apicoplast labelling (acyl carrier protein, green), mitochondria labelling (MitoTracker® RedCMXRos; red), DNA staining (DAPI; blue) and brightfield images. Scale bars: 5 µm. (B) Mitochondrial length was determined and categorised into compact (extending 6 m, blue), or fragmented (grey) morphology. Mitochondrial phenotypes are presented as a percentage of the total number of gametocytes examined for controls (n = 55), 3 µM cytochalasin D (n = 48) and 0.3 µM jasplakinolide (n = 20) from two independent experiments, one performed in duplicate.

Supplementary Figures Figure S1. Actin profiles recognised by anti-P. falciparum actin antibodies. (A) Recombinant P.

falciparum actin-I (ACTI), actin-II (ACTII) and Arp1 were expressed in E. coli and crude extracts were subjected to SDS-PAGE. Blots were probed with anti-ACT16–30 (top) or anti-actin(Dicty) (middle) antiserum or stained with Ponceau (bottom). (B) Aliquots of RBCs infected with P. falciparum mixed asexual stage parasites (Asex) or stage IV gametocytes (Gam IV) were subjected to SDS-PAGE. Blots were probed with anti-ACT16–30 or anti-ACT239–253. (C) Immunofluorescence microscopy of a schizont-infected RBC labelled with anti-ACT16–30 and anti--tubulin, with nuclear co-labelling (DAPI). Labelling of punctate F-actin structures at the periphery of the nascent daughter merozoites is evident. Scale bar: 3 µm. Figure S2. Actin profiles recognised by anti-Dictyostelium and anti--actin antibodies. (A) Immunofluorescence microscopy of stage III-IV gametocytes labelled with anti-actin(Dicty) showing labelling of gametocyte tips and cytoplasm. (B) Immunofluorescence microscopy of stage III-V gametocytes (left) and uninfected RBCs (right) labelled with anti--actin. Labelling of the gametocyte cytoplasm is evident, as well as the RBC membrane (arrow). In uninfected RBCs the RBC membrane is labelled. Brightfield images at right. (C,D) Aliquots of a mammalian cell line 28 This article is protected by copyright. All rights reserved.

Accepted Article

(human foreskin fibroblast, HFF), uninfected RBCs (URBCs) and RBCs infected with P. falciparum mature asexual stage parasites (asex) or stage III/IV gametocytes (sex) were subjected to SDS-PAGE. Blots were probed with anti-actin(Dicty) (C) or anti--actin (D). The actin band is

indicated with an arrow. The higher bands in (D) may represent actin oligomers that are not fully dissociated during the sample preparation. Figure S3. Analysis of overlapping F-actin and tubulin cytoskeletal structures. (A)

Fluorescence 3D-SIM micrograph showing labelling of F-actin (red) and tubulin (green) in a stage IV gametocyte. Scale bar: 5 µm. The intensity profile was analysed along the region indicated with the line (right hand side). (B) The intensity profiles were linearised and the red, green and overlayed signals are presented. (C) Graphical depiction of the intensity profiles. Figure S4. F-actin detection by immuno-electron microscopy in stage III/IV gametocytes. Samples of stage III/IV gametocytes were plunge frozen, and cryosections (80 nm) were prepared for immunolabelling using the Tokuyasu method. The thawed sections were labelled with antiACT16-30 (A) or without (B) and probed with 6 nm gold-protein-A and prepared for EM. The RBC, PPM and IMC are indicated.

Figure S5. Effect of short-term cytochalasin D treatment on F-actin organisation in gametocytes. Stage IV gametocytes were exposed for 2 h (37oC) to cytochalasin D at 1 or 10 M or

an equivalent volume of solvent (final concentration < 1% DMSO) and assessed by immunofluorescence

microscopy

using

anti-ACT16-30

antibody.

(A)

Representative

immunofluorescence images show controls with actin association at two tips (upper panel, arrows) and treated samples (1 µM cytochalasin D) with F-actin accumulation at one or neither tip (lower panels, arrow). (B) Quantification of tip-associated actin labelling as a percentage of analysed gametocytes (controls, n = 180; 1 µM cytochalasin D, n = 190; 10 µM cytochalasin D, n = 192). Actin association with two tips is shown in dark grey, one and zero tip association in light grey and white, respectively. (C) 3D-SIM imaging confirms tip- (yellow arrows) and body-associated actin (red) labelling (white arrows), which aligns lengthwise with microtubules (green) in controls (upper 29 This article is protected by copyright. All rights reserved.

Accepted Article

panel). Cytochalasin D treatment (1 µM) treatment is associated with the loss of F-actin tip association but not the F-actin population associated with the parasite body (lower panel, white arrows). The treatment had no gross effect on β-tubulin organisation. Scale bars: 5 µm. Figure S6. Effect of short-term jasplakinolide treatment on F-actin organisation in gametocytes. Stage III-V gametocytes were exposed for 30 min (37oC) to jasplakinolide at 1 M and assessed by fluorescence microscopy using anti-ACT239–253 (red) and anti--tubulin (green)

antibodies. Large shafts of F-actin were observed in the parasite cytoplasm. Scale bars: 5 µm.

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CMI_12359_f6

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