Research Article 2053 Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes

Megan K. Dearnley1,2, Jeffrey A. Yeoman3, Eric Hanssen4, Shannon Kenny1,2, Lynne Turnbull5, Cynthia B. Whitchurch5, Leann Tilley1,2 and Matthew W. A. Dixon1,2,* 1Department of Biochemistry, Bio21 Institute, 30 Flemmington Road, Melbourne, Vic 3010, Australia 2ARC Centre of Excellence for Coherent X-ray Science, The University of Melbourne, Vic 3010, Australia 3La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Vic 3086, Australia 4Electron Microscopy Unit, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Vic 3010, Australia 5The ithree Institute, University of Technology Sydney, NSW 2007, Australia *Author for correspondence ([email protected])

Accepted 12 December 2011 Journal of Cell Science 125, 2053–2063 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.099002

Summary The most virulent of the human malaria parasites, Plasmodium falciparum, undergoes a remarkable morphological transformation as it prepares itself for sexual reproduction and transmission via mosquitoes. Indeed P. falciparum is named for the unique falciform or crescent shape of the mature sexual stages. Once the metamorphosis is completed, the mature gametocyte releases from sequestration sites and enters the circulation, thus making it accessible to feeding mosquitoes. Early ultrastructural studies showed that gametocyte elongation is driven by the assembly of a system of flattened cisternal membrane compartments underneath the parasite plasma membrane and a supporting network of microtubules. Here we describe the molecular composition and origin of the sub-pellicular membrane complex, and show that it is analogous to the inner membrane complex, an organelle with structural and motor functions that is well conserved across the apicomplexa. We identify novel crosslinking elements that might help stabilize the inner membrane complex during gametocyte development. We show that changes in gametocyte morphology are associated with an increase in cellular deformability and postulate that this enables the gametocytes to circulate in the bloodstream without being detected and removed by the mechanical filtering mechanisms in the spleen of the host.

Key words: Gametocyte, Inner membrane complex, Malaria, Plasmodium falciparum Journal of Cell Science

Introduction stage III of development, leading to the characteristic crescent Malaria affects more than 240 million people annually and causes shape in stage IV, with the gametocyte ends becoming more about 781,000 deaths (WHO World Malaria Report 2010; http:// rounded in stage V parasites (Dixon et al., 2008; Sinden, 1982; www.who.int/malaria/world_malaria_report_2010/world_malaria_ Sinden et al., 1978). report_2010.pdf). The apicomplexan parasite Plasmodium Like mature asexual stage parasites, stage I–IV gametocytes falciparum is responsible for the most deadly form of the disease sequester away from the peripheral circulation (Day et al., 1998; in humans. The parasite has a complex lifecycle involving asexual Rogers et al., 1996), thereby avoiding passage through and and sexual reproduction within a human host and an Anopheles potential clearance by the spleen. It has been suggested that stage mosquito vector. To prepare itself for life in these different I–II gametocytes adhere to CD36 before undergoing a switch in intracellular niches, the parasite undergoes a remarkable series of stage III–IV that permits sequestration in the bone marrow, morphological transformations, changing its own shape and whereas the mature stage V gametocytes lose the ability to remodelling its host cell environment. The link between asexual cytoadhere (Rogers et al., 2000). Indeed, stage V gametocytes multiplication in the red blood cells (RBCs) of the human host and reappear in the peripheral circulation and this is the only stage sexual reproduction in the midgut of the mosquito is the formation that can complete sexual development after ingestion by a of a specialized sexual blood stage parasite called the gametocyte. mosquito. Gametocyte maturation thus represents a ‘bottle-neck’ Late stage P. falciparum gametocytes are characterized by in parasite development; inhibition of this process ablates disease their unique crescent or falciform shape. Development of these transmission. Despite the importance of this stage in efforts to specialized cells takes at least 7 days and can be divided into five eradicate malaria, relatively little is known of the mechanisms distinct stages (Baton and Ranford-Cartwright, 2005; Carter controlling gametocyte shape, form and function. and Miller, 1979; Dixon et al., 2008; Hawking et al., 1971). Coincident with the adoption of the unique crescent shape of Early gametocytes (stage I and IIa) are morphologically the late stage gametocyte is the appearance of a tri-laminar indistinguishable from early asexual parasites. Changes in membrane structure known as the sub-pellicular membrane cellular architecture are first observed during late stage II to complex. Early electron microscopy studies on mammalian 2054 Journal of Cell Science 125 (8)

malarial parasites identified this structure as a double- membraned cisternal compartment underlying the parasite plasma membrane (Sinden and Smalley, 1979). In this work, we describe some of the molecular components of the sub- pellicular complex and show that the complex is closely related to the inner membrane complex (IMC) of the invasive stages of the parasite. In the motile (zoite) stages of Plasmodium and other apicomplexa, the IMC is connected to an actin–myosin motor that drives cellular invasion through a process called ‘gliding motility’. The glideosome proteins include myosin-A (MyoA), myosin-A tail domain interacting protein (MTIP) (Bergman et al., 2003; Herm-Go¨tz et al., 2002) and the glideosome-associated proteins 45 and 50 (GAP45 and GAP50) (Gaskins et al., 2004). P. falciparum GAP50 is an integral membrane protein that anchors pre-complexed GAP45–MTIP–MyoA (Johnson et al., 2007). Host cell invasion is thought to require binding of the actin– myosin complex to adhesive proteins such as the merozoite thrombospondin-related adhesive protein (mTRAP). mTRAP is Fig. 1. Trilaminar complex of the gametocyte is closely related to the embedded in the parasite plasma membrane and binds to inner membrane complex of invasive stages of P. falciparum. (A) Electron receptors on the host cell, thereby providing the force that tomogram showing the membrane and microtubule organization in a stage IV drives invasion (Baum et al., 2006a). Underlying the IMC is a gametocyte. (Ai) Selected virtual tomogram section (22 nm). (Aii) Rendered microtubule scaffold that forms a major structural element of the model generated from four serial sections through the region shown in Ai. invasive stages of apicomplexan parasites (Kudryashev et al., The rendered model shows the RBC membrane (red), PVM (blue), PPM 2010; Sinden, 1982). (yellow), IMC (green) and microtubules (gray). Osmophillic bodies are First, we show that the sub-pellicular membranes of represented in gold. (B) Stained thin section through a dividing schizont showing a nascent merozoite (Bi) and a selected virtual section (7 nm) from a gametocytes have the same core components as in the invasive tomogram of an individual merozoite (Bii). Rhoptry (R), micronemes (m), stages and that these components are in complex. Second, we apicoplast (a), nucleus (N), microtubule (MT). Scale bars: 100 nm. provide evidence that a coordinated recruitment of the IMC to the parasite periphery and assembly of a network of microtubules Although previous studies have described the sub-pellicular drives elongation of the P. falciparum gametocyte, confirming at membrane complex of the P. falciparum gametocyte at the a molecular level suggestions from early electron microscopy ultrastructural level, there are few reports on the composition or studies (Sinden, 1982). This shape change is associated with an origin of this organelle. In an effort to determine whether the increase in cellular deformability and might explain how late gametocyte membrane structure is indeed related to the IMC, we Journal of Cell Science stage gametocytes can survive circulation through the blood performed fixed cell immunofluorescence microscopy using stream of the host. antibodies recognizing known components of the merozoite Results IMC. Fig. 2A–D shows the presence of GAP50, GAP45, MTIP and MyoA at the periphery of stage IV gametocytes, consistent IMC proteins are present in complex at the periphery of with a location in the sub-pellicular membrane complex. At this mature P. falciparum gametocytes The late stage P. falciparum gametocyte is characterized by its level of resolution, the profile was similar to that of the P. unique crescent shape. Coincident with the adoption of this shape falciparum PVM protein, Pfs16, which has been shown to be is the appearance of a tri-laminar structure known as the gametocyte-specific (Baker et al., 1995). Protein solubility and sub-pellicular membrane complex, consisting of a cisternal expression profiling by western blotting of gametocytes and compartment underlying the parasite plasma membrane (Sinden schizont stage parasites confirmed the presence of the IMC and Smalley, 1979). We performed high-resolution serial section components (supplementary material Fig. S1, and below). electron tomography of this region in a mature stage gametocyte. We have previously generated transfectants expressing a GFP Fig. 1A shows a virtual section from the tomogram and a chimera of P. falciparum GAP50 as a tool to assess IMC genesis rendered model of the membrane complex. The RBC membrane and reorganization during the development of merozoites (red), parasitophorous vacuole membrane (PVM, blue), parasite (Yeoman et al., 2011). We transfected the P. falciparum plasma membrane (PPM, yellow), the sub-pellicular membranes GAP50–GFP gene construct into a high gametocyte producing (green) and a layer of sub-pellicular microtubules (MT, gray) are clone of 3D7 and found that the GAP50–GFP chimera is evident. correctly expressed and present at the periphery of mature stage We were struck by the ultrastructural similarities between this gametocytes, co-locating with endogenous GAP45 (Fig. 3A). To sub-pellicular complex (Fig. 1A) and the IMC of merozoites determine whether components of the IMC are in complex, (Fig. 1B). In the merozoite, the IMC is a cisternal compartment we performed immunoprecipitation experiments. Stage III–IV that is flattened against the parasite plasma membrane (Fig. 1Bi). gametocytes expressing the GAP50–GFP chimera were It is supported by two or three longitudinally running magnet-enriched and then solubilized using RIPA detergent. microtubules that extend along one side of the merozoite The cleared lysate was incubated with rabbit antibodies against (Fig. 1Bii, arrows), as reported previously (Bannister et al., P. falciparum GAP45, and immunoprecipitated using Protein A/ 2000). G Sepharose. Precipitated samples were separated by SDS-PAGE The inner membrane complex of gametocytes 2055

gametocyte IMC we examined the location of GAP50–GFP in live gametocytes that were co-labelled with the membrane probe, BODIPY-TR-ceramide. During the initial stages of elongation (stage III), when the gametocyte first becomes morphologically distinguishable, GAP50 was associated with a reticular structure in the parasite as well as along one side of the elongating parasite (Fig. 4Ai). Fixed cell immunofluorescence microscopy confirmed that GAP50–GFP labels internal structures that were also recognized by an antibody against the P. falciparum ER resident protein, ERC (ER calcium binding protein) (Fig. 3C). By contrast, ERC was absent from the peripheral location to which GAP50–GFP is recruited as the gametocyte elongates. 3D- structured illumination microscopy (3D-SIM) can provide enhanced resolution of cellular structures (Schermelleh et al., 2008). Using 3D-SIM we found that the GAP50–GFP-labelled IMC remained closely associated with the intracellular reticular structure (Fig. 3D,E, arrows). A rendered model of the GAP50– GFP fluorescence illustrates the points of associations (Fig. 3F, arrows), as do translational and rotation views of the fluorescence micrographs (supplementary material Movies 1, 2). This is consistent with an early ultrastructural study (Sinden et al., 1978) that reported connectivity between the ER and the sub- pellicular membranes. Stage III gametocytes undergo a dramatic cellular Fig. 2. Components of the glideosome and IMC are present at the rearrangement, adopting a ‘hat-like’ appearance. During this gametocyte periphery. (A–D) Immunofluorescence microscopy of 3D7 stage, the GAP50–GFP is concentrated in the flattened rim region parasites labelled with antibodies against Plasmodium falciparum GAP50, of the ‘hat’, with additional looping structures around the top of Plasmodium falciparum GAP45, MTIP or MyoA (green) and the gametocyte the cell (Fig. 4Ai,ii; arrows; see supplementary material marker, Pfs16 (red). Bright field (BF) images are shown on the right. Scale Movie 2). As the IMC develops further it adopts a cupped bars: 5 mm. shape along the foot, in addition to the looping structures. Electron microscopy confirmed the presence of the IMC along and transferred for western blotting. Blots were probed with anti- the flattened surface and around the ‘pinching’ ends of the mouse GFP, GAP50 or MTIP. Full-length GAP50–GFP (64 kDa) gametocyte (Fig. 4Bi,ii). Fig. 4Bi depicts a longitudinal slice was detected in the precipitated pellet of the transgenic sample, through a region of the sub-pellicular microtubule layer, with the by both the anti-GFP and anti-GAP50 reagents, but not in the Journal of Cell Science IMC visible at the periphery. Some of the microtubules that pass 3D7 parent line (Fig. 3B). MTIP (28 kDa) and endogenous through the plane of the section are indicated with arrows. GAP50 (42 kDa) were detected in the precipitated pellets of Fig. 4Bii shows a cross-section in which the microtubules are both the transfectant and wild-type samples (Fig. 3B). This viewed end-on and the IMC is evident as a layer outside the demonstrates that both GAP50–GFP and endogenous GAP50 are microtubule ‘basket’. in complex with endogenous GAP45 and MTIP. These studies In stage IV of parasite development, the characteristic pointed validate the use of P. falciparum GAP50–GFP as a marker of the ends of the P. falciparum gametocyte become apparent. By this gametocyte IMC. stage, the GAP50–GFP extends further around the parasite, the Western blotting analyses revealed the presence of GAP45, surface of which was delineated by BODIPY-TR-ceramide GAP50 and MTIP from stage II to stage V gametocytes labelling (Fig. 4Aiii; see supplementary material Movie 2 for a (supplementary material Fig. S1B) and immunofluorescence 3D rotation). 3D-SIM imaging of GAP50–GFP revealed a microscopy confirmed that these proteins were present at the periodic banding pattern running perpendicular to the direction gametocyte periphery (supplementary material Fig. S2A–D). of gametocyte extension (Fig. 5A, arrows). The average width of Moreover, the solubility profiles for these proteins were similar the GAP50–GFP fluorescence bands was 460640 nm, with the in gametocytes and merozoites (supplementary material Fig. S1). interleaved regions of low fluorescence being 120640 nm in These data, in conjunction with the immunoprecipitation data, width (Fig. 5A; supplementary material Movie 3). show that the gametocyte sub-pellicular membrane complex is Cryo-electron tomography allows 3D visualization of very closely related to the merozoite IMC and we suggest that it specimens after plunge-freezing in cryogenic liquids to be renamed the gametocyte IMC. preserve membrane structures (Cyrklaff et al., 2007; Kudryashev et al., 2010). Tomographic analysis of a stage IV GAP50–GFP is present in the endoplasmic reticulum of gametocyte permits visualization of virtual sections near the early stage gametocytes prior to recruitment to the surface of the cell without the need for physical sectioning. parasite periphery Fig. 5Bi highlights the microtubule network running underneath P. falciparum GAP50 is recruited from the endoplasmic the cell surface and the locations of the IMC and parasite reticulum (ER) to the nascent IMC during asexual schizogony membranes within the cell. The section in Fig. 5Bii was (Yeoman et al., 2011). To determine the origin and physically located 100 nm above the section in Fig. 5Bi and reorganization of proteins during the formation of the shows regions of membrane (approximately 400 nm in width) 2056 Journal of Cell Science 125 (8)

Journal of Cell Science Fig. 3. Plasmodium falciparum GAP50–GFP is trafficked via the ER to the gametocyte IMC and is in complex with known IMC components. (A) Immunofluorescence microscopy of GAP50–GFP-expressing gametocytes labelled with antibodies against GFP (green) and Plasmodium falciparum GAP45 (red). Bright field (BF) images are on the right. (B) Proteins were detergent-solubilized from enriched, mature stage GAP50–GFP-transfected or parent 3D7 gametocytes. The extract was immunoprecipitated (IP) using rabbit anti-GAP45. Western blots (WB) of the pellet fractions were probed with mouse anti-GFP, anti- GAP50 and anti-MTIP. WT, wild-type. Molecular mass is shown in kDa. (C–E) Immunofluorescence (C) and 3D-SIM (D,E) of GAP50–GFP transfectants labelled with anti-GFP (green) and anti-ERC (red). (D) Projection of sections 10–15 (from a 20-section series) and (E) individual sections. DAPI fluorescence is shown on the right. In C, an accumulation of GAP50–GFP fluorescence can be seen along one side of the stage III parasite. (F) Rendered model of the 3D reconstruction. D, E and F illustrate the association of the GAP50–GFP-labelled reticular and peripherally located compartments. The single arrows indicate a region of close association between the ER and the IMC; double arrows show a region of IMC looping that appears to be continuous with the ER compartment. Scale bars: 3 mm.

interleaved with connecting bands (about 100 nm in width), IMC around the cell periphery, and the persistence of the stripes layered across the underlying microtubules. A higher (Fig. 5Aiii, arrows). The gametocyte presented in supplementary magnification view of the microtubules (Fig. 5C) highlights the material Movie 3 shows the transition from stage IV to stage V spacing and arrangement of the microtubule network within the with one rounded and one pointed end. In this rotation, the stripes mature stage parasite; the microtubules (25 nm diameter) are are clearly observed and there is evidence for some remnant spaced at intervals of approximately 10 nm. The spacing, shape GAP50–GFP fluorescence around the parasite nucleus. and orientation of the flattened membrane structures are reminiscent of the GAP50–GFP-labelled features (Fig. 5A; Temporal and spatial map of gametocyte IMC and supplementary material Fig. S3; Movie 5a,b). The intervening microtubules at different stages of gametocyte bands might represent areas of deposition of proteins that development stabilize the IMC and the underlying microtubule network. We investigated the assembly of microtubules during gametocyte In stage V, the ends of the gametocytes become rounded development. The microtubule-labelling reagent, Tubulin (Fig. 4Aiv, Fig. 5Aiii). This is probably due to dismantling of the Tracker (Invitrogen) started to accumulate in a peripheral supporting microtubule network as described below and in a region of a stage II gametocyte (Fig. 6Ai; supplementary previous study (Sinden et al., 1978). GAP50–GFP persists at the material Movie 4) before becoming concentrated along one periphery of the parasite still co-locating with BODIPY-TR- edge of the stage III gametocyte (Fig. 6Aii; supplementary ceramide (Fig. 4Aiv). 3D-SIM revealed the maintenance of the material Movie 4). Extension and elaboration of the microtubule The inner membrane complex of gametocytes 2057

around the top of the gametocyte (Fig. 6Bii; supplementary material Movie 5a,b). Some GAP50–GFP-labelled regions around the edge of the foot did not have associated microtubules (supplementary material Fig. S3; Movie 5a,b). Electron micrographs of equivalent stages show areas where the IMC had started to expand but without any underlying microtubules (Fig. 4Bii, red arrows). By stage IV there was a complete network of microtubules underlying the peripherally located IMC and forming a similar pattern to that for GAP50. This characteristic shape and the distribution of the microtubules can be best appreciated in the 3D-SIM reconstruction shown in supplementary material Movie 6. During stage III we saw a marked difference in the locations of GAP45 compared with GAP50–GFP and b-tubulin. This protein was restricted to the foot and was not co-located with the ER- associated population of GAP50–GFP (Fig. 6B,C; supplementary material Fig. S2B).

Gametocyte elongation is not driven by an actin–myosin motor Gametocytes do not display gliding motility and it is unlikely that the IMC and associated MyoA participate in a motor complex analogous to that operating in the zoite stages of P. falciparum. To test for a potential role of actin, we examined the effect of cytochalasin D on gametocyte morphology. Previous work has shown that merozoites treated with cytochalasin D (0.1–2 mM) are rendered immobile and unable to invade erythrocytes (Miller et al., 1979; Srinivasan et al., 2011). By contrast, we found that maintenance of gametocytes over a period of 6 days in the presence of cytochalasin D (at concentrations up to 10 mM) Fig. 4. IMC is assembled under the gametocyte plasma membrane. did not prevent the morphological changes that accompany (A) Live cell confocal imaging of stage III (Ai,Aii), stage IV (Aiii) and stage development to stage IV (data not shown). This indicates that V (Aiv) gametocytes expressing Plasmodium falciparum GAP50–GFP although MyoA interactions might play an important structural (green) co-labelled with BODIPY-TR-ceramide (red). White arrows indicate role, morphological changes are not driven by an actin–myosin

Journal of Cell Science an accumulation of GAP50–GFP along the rim of the stage III parasite (Ai) motor. and a loop around the top of the gametocyte (Aii). Bright field (BF) images are on the right. 3D rotations of these cells are shown in supplementary Gametocyte shape change is associated with altered material Movie 2. Scale bars: 3 mm. (B) Electron micrographs of stage III cellular deformability gametocytes. (Bi) Longitudinal and (Bii) cross-sections showing the RBC membrane, PVM, PPM and the double membrane of the IMC, above a row of In an effort to determine a functional role for the remarkable shape microtubules (MT). The electron-lucent area probably represents a vacuole changes of gametocytes, we used ektocytometry to measure within the gametocyte cytoplasm. Red arrows mark an area where the IMC cellular deformability at different stages of development (Fig. 7). has formed but no microtubules are present. Scale bars: 200 nm. See also The elongation index (EI) of synchronous enriched gametocytes supplementary material Fig. S2. was measured at sheer stresses of 0–20 Pa. Asexual trophozoite stage parasites showed a significant decrease in ability to elongate under flow (EI50.13) compared to uninfected RBCs (EI50.34) at network was observed in stage IV gametocytes (Fig. 6Aiii; the physiologically relevant sheer stress of 3 Pa (Fig. 7A). Stage supplementary material Movie 4), adopting a pattern analogous III gametocytes showed a similarly low deformability (EI50.12) to that of the peripheral population of GAP50–GFP (Fig. 4Aii). (Fig. 7A). By contrast, there was an increase in the EI upon No microtubule filament bundles were observed in the stage V transition to stage IV of gametocyte development (EI50.16); this gametocyte, consistent with the disassembly of the network in value was significantly higher than that for stage III (P50.0001). mature gametocytes. A further increase in the EI was observed upon transition to stage We used the enhanced resolution of 3D-SIM to investigate the V (EI50.18) (Fig. 7A). locations of the microtubules and IMC during gametocyte We made a rough quantification of the change in the shape at elongation. Antibody against b-tubulin labelled microtubules different stages of development by monitoring length and width within the ‘foot’ of the developing gametocyte (Fig. 6B, white in DIC images of live gametocytes. Mature trophozoite stage arrows) in addition to a diffuse cytoplasmic staining. b-tubulin in parasites displayed a roughly circular profile with a mean length the foot region was co-located with GAP50–GFP (Fig. 6B). The of 4.3160.05 mm and mean width of 4.060.2 mm (Fig. 7B,C). distribution of the microtubules and the IMC can best be The mean length of the gametocytes increased significantly from appreciated in supplementary material Movie 5a,b, which shows stage III (6.660.4 mm) to stage IV (10.960.4 mm) (P50.0001), a translational through and a rotation of the same parasite. with a slight relaxation upon transition to stage V (9.160.4 mm) GAP50–GFP and b-tubulin were also co-located in the loop (P50.005) (Fig. 7B,C). The width of the stage III gametocyte 2058 Journal of Cell Science 125 (8)

Fig. 5. Gametocyte IMC comprises rectangular membrane plates. (A) 3D-SIM of stage IV (Ai,ii) and stage V (Aiii) gamocytes transfected with Plasmodium falciparum GAP50–GFP showing vertical striations (white arrows). Rotations of the 3D- SIM data are presented in supplementary material Movie 3. The cells are co-stained with DAPI (blue). Aii is a zoomed view of the cell in Ai, highlighting the periodic striations. Scale bars: 3 mm. (B) Two 30 nm virtual sections from a cryo-electron tomogram of a region near the surface of a gametocyte. The section shown in Bii is 100 nm above the section in Bi. The IMC, parasite membrane (PM) and microtubules (MTs) are indicated. The IMC is segmented by vertical striations of amorphous material (Bii), marked as ‘sutures’. (C) Lying directly underneath, and running perpendicular to the rectangular sections of IMC, is a network of microtubules. Shown is a virtual section of a cryo-electron tomogram of a longitudinal section of microtubules. The same section is shown below rotated 90˚ to obtain a cross- sectional view. Microtubule singlets (s) of 25 nm in diameter and doublets (d) of 42 nm are indicated. The microtubules are separated by a gap (g) of about 10 nm. Scale bars: 100 nm.

(3.060.7 mm) was significantly less (P50.0001) than that of the complex that drives parasite motility (Beck et al., 2010; Fre´nal asexual stage parasite, with a further significant decrease in the et al., 2010; Gaskins et al., 2004). stage IV gametocyte (2.660.1 mm, P50.006), before remaining Our work confirms previous studies showing that the

Journal of Cell Science stable for the remainder of its development (stage V; gametocyte sub-pellicular membrane complex has ultrastructural 2.660.2 mm) (Fig. 7B,C). The length-to-width ratio increased similarity to the IMC of the motile stages of P. falciparum from 1.1 (trophozoite) to 2.2 (stage III) to 4.3 (stage IV), and then (Bannister et al., 2000; Sinden and Smalley, 1979); however, the decreased to 3.5 (stage V). molecular similarity between these structures has not previously been investigated. In this work, we show that key proteins of the Discussion merozoite glideosome complex (GAP50, GAP45, MyoA and In 1880, Alphonse Laveran observed gametocytes in the blood MTIP) are present in the gametocyte sub-pellicular membrane smear of an Algerian malaria patient and remarked upon the complex. We also show that exogenous GAP50–GFP forms a crescent shape of the parasite (Laveran, 1880). Over a century complex with endogenous GAP45, GAP50 and MTIP. These data later we are still battling to understand the molecular basis for indicate that the gametocyte membrane complex is closely related this unusual shape and its role in P. falciparum transmission and to the IMC of the motile stages of P. falciparum. virulence. Early ultrastructural analyses revealed that the gross An interesting issue is the roles that the IMC and the associated morphological restructuring of the gametocyte is coincident with glideosome proteins play in gametocyte assembly. Gametocytes the appearance of a tri-laminar membrane structure subtended by do not display gliding motility, suggesting that the MyoA does a layer of structural microtubules (Aikawa and Beaudoin, 1969; not actively participate in an actin–myosin motor complex. Sinden, 1982; Sinden et al., 1978). Comprising a double- Furthermore, we showed that treatment with cytochalasin D did membrane-bound compartment underneath the parasite plasma not prevent gametocyte shape change. This indicates that the membrane, this structure was named the sub-pellicular membrane MyoA interaction with glideosome proteins probably plays a complex (Sinden and Smalley, 1979). Indeed the presence of structural role rather than driving elongation per se. It seems alveolar sacks subtending the plasma membrane is a unifying likely that the microtubular cytoskeleton is attached to (and morphological feature of apicomplexa, dinoflagellates and organized by) the IMC, which in turn is linked to the PPM via ciliates. Different organism groups have adapted this membrane glideosome proteins. Thus, our data confirm and support the structure for different cellular functions, with roles ranging from original suggestion (Sinden, 1982) that gametocyte elongation is shape maintenance (free-living protozoa), to protective armor driven by the assembly of microtubules underneath the IMC. (dinoflagellates) to calcium stores (ciliates). In the motile stages The shape adopted by P. falciparum gametocytes is of apicomplexan parasites, the sub-pellicular membrane is termed reminiscent of the elongated sporozoite and ookinete. Indeed, the IMC and plays a vital role as the scaffold for the motor the morphological changes that accompany maturation from The inner membrane complex of gametocytes 2059

can play roles in both motility and cell morphology (Tremp and Dessens, 2011). Recent work from our laboratory indicates that the merozoite IMC is formed by the redistribution of a sub-compartment of the ER to nascent apical caps (Yeoman et al., 2011). Similarly, we show here that P. falciparum GAP50 is located in the ER in early stage gametocytes and that part of this population is recruited to the periphery to form the IMC. This is in agreement with early ultrastructural study of P. falciparum (Sinden et al., 1978) and with studies from other alveolates (Gould et al., 2008). Recruitment of GAP50 to the gametocyte periphery appears to be coordinated with the laying down of microtubules. Because the microtubule-supported IMC initially develops along one side of the gametocyte, stage III gametocytes have a ‘hat-like’ appearance. The IMC and the microtubule network then expand around the periphery, resulting in a cupped leaf shape. Our electron and fluorescence microscopy data suggest that the IMC extends first, followed by microtubule deposition, such that the leading edges of the nascent IMC are outside the supporting layer of microtubules. Other IMC proteins, GAP45 and MTIP appear to be recruited onto the nascent IMC. This process might be similar to the recruitment of pre-complexed GAP45–MTIP– MyoA onto membrane-embedded GAP50 during the formation of the Toxoplasma IMC (Gaskins et al., 2004). 3D-SIM imaging of the GAP50–GFP-labelled IMC revealed a remarkable architectural feature – bands of fluorescence of ,400 nm interrupted by ,100 nm regions of low fluorescence. Cryo-electron tomography revealed the ultrastructural analogue of these bands as flattened regions of membrane with intervening amorphous material. These structures are probably equivalent to the transverse ‘sutures’ encircling the gametocyte that were identified in an early freeze-fracture study (Meszoely et al., 1987). Similar patchworks of sub-pellicular membrane have been described in the IMC of the invasive stages of P. falciparum,as Journal of Cell Science well as in Toxoplasma and other apicomplexa (Bannister et al., 2000; Raibaud et al., 2001). Another early transmission electron microscopy study identified protein ‘crosslinks’ running Fig. 6. Coordinated development of the IMC and the microtubule perpendicular to the microtubule network (Kaidoh et al., 1993). network drives shape change. (A) Live cell confocal imaging of stage II–V We suggest that the GAP50–GFP-labelled IMC ‘stripes’ gametocytes (Ai–Aiv) labelled with Tubulin Tracker (TT, green) and represent IMC cisternae laid down as rectangular plates, BODIPY-TR-ceramide (red). DIC images are shown on the right. (B) Single whereas the gaps represent proteinaceous material that holds sections from a 3D-SIM reconstruction of stage III Plasmodium falciparum the cisternae and the microtubules in place. GAP50–GFP gametocytes prepared for immunofluorescence microscopy A concurrent large-scale analysis of IMC components in P. (anti-GFP, green; anti-b-tubulin, red). The white arrows mark areas of peripheral GAP50–GFP staining with associated b-tubulin. The red arrow in falciparum revealed a novel IMC protein that appears to be Bii highlights the looping extensions of the IMC with concentrated b-tubulin restricted to the genus Plasmodium (Mal13P1.228). In the early staining. A montage of projections of Bi is shown in supplementary material gametocyte, this protein is located in thin stripes along the ‘foot’, Fig. S3. Projections and 3D rotations of Bi and Bii are presented in which expand and eventually encircle the mature stage gametocyte supplementary material Movie 5a,b. (C) Stage III gametocyte labelled with (Kono et al., 2012). These structures might represent the regions of anti-GAP45 (green) and anti-b-tubulin (red). This image highlights the low fluorescence and proteinaceous crosslinks identified in our restricted location of GAP45 within the foot of the gametocyte. Scale bars: 3D-SIM and cryo-electron microscopy studies. 3 mm. See also supplementary material Movies 6,7. Consistent with previous ultrastructural studies of gametocytes (Sinden, 1982; Sinden et al., 1978), there is no morphologically stage II to stage IV of gametocytogenesis resemble (in reverse) identifiable apical polar ring in gametocytes to organize the the shape changes that accompany the transformation of peripheral microtubules [although a microtubule organizing the elongated sporozoites into liver stage trophozoites center is formed very quickly after activation (Aikawa, 1977)]. (Jayabalasingham et al., 2010) or ookinetes into oocysts (Carter This begs the question: how are the microtubules organized et al., 2007). Taken together these data suggest that the IMC onto the well-ordered cytoskeletal basket? We suggest that plays an important structural role in each of the cellular the proteinaceous ‘sutures’ between the IMC plates provide a remodelling events that drive parasite elongation. This is means of stabilizing the microtubule network. Proteins resident in consistent with recent work showing that specific IMC proteins these sutures might function in the targeting of the nascent IMC 2060 Journal of Cell Science 125 (8)

Fig. 7. Gametocyte shape changes are accompanied by changes in deformability. (A) Elongation index (EI) values for uninfected RBCs, asexual trophozoites (30–36 hours after invasion) and stage III–V gametocytes were measured using a RheoScan slit

Journal of Cell Science ektocytometer at shear stresses of 0–20 Pa. The data represent the mean6s.e.m. of triplicate measurements and are typical of experiments performed on two separate occasions. The red box highlights the measurements at 3 Pa. (B) Illustrations of stage III–V gametocytes (female). The dimensions were measured from DIC images (mean6s.e.m.). (C) Length-to- width ratio6s.e.m. for different developmental stages. a, apicoplast; Ac, acidocalcisomes; C, cytostome; DV, digestive vacuole; g, Golgi complex; IMC, inner membrane complex; k, knob; Lb, Laveran’s bib; m, mitochondrion; MC, Maurer’s cleft; Mt, microtubules; N, nucleus; Ob, osmophillic bodies; PV, parasitophorous vacuole; RBC, red blood cell; W, whorl.

to the periphery of the cell and in the stabilization of the IMC and human malaria species and gametocytes from most other species the sub-IMC microtubule network. of Plasmodium have a more rounded morphology, although Analysis of DIC images revealed a significant increase in the gametocytes of avian plasmodia are also elongate (Sinden et al., length and a reduction in the width of the gametocyte during 1978). P. falciparum is also unusual among human malaria maturation as compared with asexual stage parasites. A more parasites in that late stage asexual parasites drastically remodel detailed analysis of gametocyte shape by cryo-X-ray tomography the RBC through the export and insertion of parasite-derived gives similar results (Hanssen et al., 2011). The elongated form proteins into and under the host RBC membrane (Tilley et al., of P. falciparum gametocytes is unusual. Gametocytes from other 2011). These modifications lead to a significant increase in The inner membrane complex of gametocytes 2061

cellular rigidity, which would make mature parasite-infected parasitemia) was treated with 5% sorbitol (Lambros and Vanderberg, 1979), then separated using a Percoll density gradient (Knight and Sinden, 1982) to enrich RBCs vulnerable to recognition and clearance within the spleen the ring stage and remove any gametocytes. The parasites were cultured until they (Glenister et al., 2002; Safeukui et al., 2008). The parasite reached 8–10% trophozoites then sub-divided into 2% trophozoites (5% avoids these clearance mechanisms by cytoadhereing within hematocrit). The culture was maintained for 10 days in the presence of 62.5 mM N-acetyl glucosamine to inhibit merozoite invasion and thus asexual the microvasculature of the host. Early stage P. falciparum replication (Hadley et al., 1986). Giemsa-stained slides were used to monitor stage gametocytes also sequester; however, upon reaching sexual progression. The medium was changed daily but no fresh erythrocytes were added. maturity they release from their sites of adhesion and re-enter the At the desired stages of development, gametocytes were enriched by magnetic circulation to enable transmission to mosquitoes (Day et al., separation (Fivelman et al., 2007). For analysis of the effect of cytochalasin D (Sigma-Aldrich), the drug was added at concentrations of 0–10 mM at day one of 1998; Hayward et al., 1999). gametocyte generation and supplemented daily into the culture media. The In an effort to determine the functional consequences of the parasites were assessed by microscopy at different stages of development. gametocyte shape changes we investigated the ability of asexual trophozoites and stage III–V gametocytes to elongate in flow, a Microscopy and image analysis Live infected RBCs were prepared for fluorescence microscopy as previously surrogate measure of cellular deformability (Groner et al., 1980; described (Dixon et al., 2011). Immunofluorescence was performed on acetone- Hardeman et al., 1987). In agreement with previous reports, we fixed blood films (Spielmann et al., 2006) using the following primary antibodies found that asexual trophozoite-infected RBCs are highly in PBS containing 3% BSA: mouse anti-GFP (1:500, Roche), rabbit anti-GFP (1:500), rabbit anti-GAP45 [1:500 (Baum et al., 2006b)], rabbit anti-GAP50 [1:500 undeformable (Nash et al., 1989). This loss of deformability is (Baum et al., 2006b)], rabbit anti-Pfs16 [1:1000 (Baker et al., 1995)], mouse anti- probably due to a combination of membrane rigidification and Pfs16 [1:1000 (Dixon et al., 2009)], mouse anti-MTIP [1:500 (Green et al., 2006)], the presence of the large rigid intracellular parasite (Glenister mouse anti-MyoA (Baum et al., 2006b) followed by anti-mouse or anti-rabbit IgG et al., 2002; Nash et al., 1989). We found that stage III conjugated to Alexa Fluor 568, Alexa Fluor 647 or FITC. Slides were labelled with 1 mg/ml of DAPI prior to mounting. gametocytes have similar deformability properties to trophozoite- BODIPY labelling of parasite-infected cells was performed by adding BODIPY- infected RBCs; however, upon transition to stage IV the TR-ceramide at a final concentration of 0.7 mM to the parasite culture. Cells were gametocytes show a significant increase in the elongation index incubated overnight under standard culture conditions. Labelling with Tubulin Tracker Green was performed according to the manufacturer’s instructions, using a measured under flow conditions. This is probably due in part to final concentration of 250 nM and 30 minutes of incubation at 37˚C. the elongated shape. A further increase in the elongation index in Microscopy was performed using an Olympus IX81 wide field microscope, or stage V gametocytes might be due to the fact that the microtubule Leica TCS-SP2 or Zeiss LSM 510/FCS confocal microscopes. Images were processed using NIH ImageJ version 1.42 (www.rsbweb.nih.gov/ij). Samples were network is disassembled at this point, leaving an already prepared and 3D-SIM (Microbial Imaging Facility, University of Technology, elongated cell that probably extends further under flow pressure. Sydney) performed as previously described (Yeoman et al., 2011). Analysis of the These changes in rheological properties might help stage V dimensions of at least seven cells from DIC images was performed using ImageJ. Isosurface renderings of GAP50–GFP-expressing gametocytes were generated gametocytes survive in the circulation of the host. Indeed, stage using IMOD software (Kremer et al., 1996; Mastronarde, 1997). V gametocytes might have rheological properties more akin to P. vivax-infected RBCs (Handayani et al., 2009; Suwanarusk et al., Immunoprecipitation and solubility studies 2004). As its name suggests (vivax means lively), P. vivax is Gametocytes were harvested on days 3, 5, 7, 9 and 11, representing stages I to V. more amoeboid than P. falciparum and appears to avoid splenic Pellets of magnet-purified gametocytes were lysed in 0.015% saponin on ice for 15 minutes. The saponin pellets were solubilized in SDS loading buffer and equal clearance in spite of an inability to cytoadhere. Stage III–V amounts (10 mg protein) were separated by SDS-PAGE and transferred to Journal of Cell Science gametocytes lack knobs and are thought to adhere with lower nitrocellulose for immunoblotting. affinity than asexual stages and to prefer sites of reduced vascular For solubility studies, magnet-purified stage IV gametocytes were lysed in 0.015% saponin and the pellets incubated for 15 minutes with 1% Triton X-100 flow (Rogers et al., 1996; Smalley et al., 1981). It is possible that or RIPA buffer (1% IGEPAL CA-630), 150 mM NaCl and 0.5% sodium another consequence of the elongated shape of P. falciparum deoxycholate on ice, or with 0.1% SDS and 50 mM TRIS, pH 7.4 at room gametocytes is enhanced adhesion to lower affinity receptors by temperature or with 2% SDS at room temperature. The soluble supernatant and insoluble pellet fractions were collected. Equivalent amounts of protein were permitting flattening of the cell, which might increase the area in separated by SDS-PAGE and transferred to nitrocellulose for immunoblotting. contact with the capillary walls. For immunoprecipitation studies, purified parasites were lysed in RIPA buffer In conclusion, our data show that the falciform shape of P. for 15 minutes on ice in the presence of protease inhibitor, centrifuged, and the falciparum gametocytes is supported by an IMC with very similar supernatants collected. The precipitating agents were GFP–TRAP reagent (Chromotek) and Protein A Sepharose and anti-rabbit GAP45 (1:500) (Yeoman composition to the merozoite IMC, linked to underlying et al., 2011). The pellets were analyzed by SDS-PAGE and immunoblotting microtubules. A previously unrecognized correlate of gametocyte blotting. elongation is an enhanced cellular deformability. We anticipate that Immunoblotting was performed as previously described (Dixon et al., 2011). Briefly, all antibodies were prepared in PBS containing 3.5% skim milk, and both disruption of the IMC and its associated microtubules compromises primary and secondary incubations were performed for 1 hour. Three washes of the ability of P. falciparum to adopt the falciform shape; this might in PBS containing 0.1% Tween-20 were performed between each incubation. The turn affect its ability to survive in the circulation. This could provide following primary antibodies were used: mouse anti-GFP (1:500, Roche), rabbit anti-GFP (1:500), rabbit anti-GAP45 [1:500 (Baum et al., 2006b)], rabbit anti- a new avenue for interfering with this important human pathogen. GAP50 [1:500 (Baum et al., 2006b)], mouse anti-GAP50 [1:500 (Baum et al., 2006b)], mouse anti-Pfs16 [1:1000 (Dixon et al., 2009)], mouse anti-MTIP [1:500 Materials and Methods (Green et al., 2006)] and mouse mAb anti-AMA1 [mAb 1F9 1:500 (Coley et al., Parasite culture and gametocyte enrichment 2001)]. Goat anti-mouse or anti-rabbit IgG secondary antibodies conjugated to Parasite-infected RBCs were cultured in RPMI-HEPES supplemented with 5% horseradish peroxidase were used. Electrochemiluminescence reagents and human serum and 0.25% AlbuMAX II as previously described (Foley et al., 1994). autoradiography were used to visualize the immunoblots. Laboratory strains of 3D7 cultured for long periods form gametocytes with low efficiency, therefore we used a 3D7 isolate recovered from a patient (Lawrence Cell deformability et al., 2000) and cultured for only a limited time. A GAP50–GFP transgenic Mature stage asexual (30–36 hour trophozoite) and stage III–V gametocytes were parasite was created by transfecting a high gametocyte producing clone of 3D7 magnet-purified from culture, washed in PBS, then reconstituted with uninfected with the pGLUX–GAP50–GFP construct as previously described (Yeoman et al., RBCs to obtain a working parasitemia of 70% (Maier et al., 2008). The cell 2011). samples (5 ml) were mixed with 500 ml of polyvinylpyrrolidone (PVP) solution at Gametocytes were prepared using a modification of a published protocol a viscosity of 25 mPa second (Rheo Meditech). The elongation index was (Fivelman et al., 2007). A culture of mainly ring stage parasites (6–8% measured in a RheoScan Ektocytometer according to the manufacturer’s 2062 Journal of Cell Science 125 (8)

instructions. Measurements were acquired over the 0–20 Pa range. Values shown Baton, L. A. and Ranford-Cartwright, L. C. (2005). Spreading the seeds of million- are means6s.e.m. from a minimum of three individual measurements. The results murdering death: metamorphoses of malaria in the mosquito. Trends Parasitol. 21, shown are from a single experiment. The experiment was repeated showing 573-580. comparable results (two separate experiments). Baum, J., Papenfuss, A. T., Baum, B., Speed, T. P. and Cowman, A. F. (2006a). Regulation of apicomplexan actin-based motility. Nat. Rev. Microbiol. 4, 621-628. Baum, J., Richard, D., Healer, J., Rug, M., Krnajski, Z., Gilberger, T. W., Green, Electron microscopy and tomography J. L., Holder, A. A. and Cowman, A. F. (2006b). A conserved molecular motor Transmission electron microscopy and tomography of asexual stages were drives cell invasion and gliding motility across malaria life cycle stages and other performed as previously described (del Pilar Crespo et al., 2008; Hanssen et al., apicomplexan parasites. J. Biol. Chem. 281, 5197-5208. 2010); for merozoites, the cells were post-fixed with potassium-ferricyanide- Beck, J. R., Rodriguez-Fernandez, I. A., Cruz de Leon, J., Huynh, M. H., reduced osmium tetroxide. Gametocytes from selected stages were fixed and Carruthers, V. B., Morrissette, N. S. and Bradley, P. J. (2010). A novel family of embedded using a modification of a published protocol (Kass et al., 1971). Briefly, Toxoplasma IMC proteins displays a hierarchical organization and functions in cells were fixed in 5% glutaraldehyde in PBS, pH 7.3, overnight, then embedded coordinating parasite division. PLoS Pathog. 6, e1001094. in 3% agarose before undergoing post-fixation of lipids in osmium tetroxide (1% Bergman, L. W., Kaiser, K., Fujioka, H., Coppens, I., Daly, T. M., Fox, S., in PBS) for 1 hour followed by progressive dehydration in ethanol. Samples were Matuschewski, K., Nussenzweig, V. and Kappe, S. H. (2003). Myosin A tail further dehydrated using progressive ethanol:acetone ratios of 2:1, 1:1 and 1:2, and domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites. J. Cell Sci. 116, 39-49. finally immersed in acetone for 10 minutes. Acetone was gradually replaced with Carter, R. and Miller, L. H. (1979). Evidence for environmental modulation of liquid epoxy resin, and samples embedded and stained as described above. For gametocytogenesis in Plasmodium falciparum in continuous culture. Bull. World both asexual and gametocyte samples, 70-nm thick sections were observed at Health Organ. 57 Suppl. 1, 37-52. 120 keV using a JEOL 2010HC transmission electron microscope (EM Unit, La Carter, V., Nacer, A. M., Underhill, A., Sinden, R. E. and Hurd, H. (2007). Minimum Trobe University,). For electron tomography, sections of 200 or 300 nm were cut requirements for ookinete to oocyst transformation in Plasmodium. Int. J. Parasitol. and stained and observed at 200 keV on an FEI Tecnai G2 F30 electron 37, 1221-1232. microscope (Bio21 Institute, Melbourne). Tilt series were collected every 2˚ Coley, A. M., Campanale, N. V., Casey, J. L., Hodder, A. N., Crewther, P. E., between 270˚ and +70˚ and reconstructed to generate cell models as described Anders, R. F., Tilley, L. M. and Foley, M. (2001). Rapid and precise epitope previously (Abu Bakar et al., 2010; Yeoman et al., 2011). Tomograms were mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by generated using the IMOD software (Kremer et al., 1996; Mastronarde, 1997). combined phage display of fragments and random peptides. Protein Eng. 14, 691-698. IMOD was also used to generate segmentation models where contours were Cyrklaff, M., Kudryashev, M., Leis, A., Leonard, K., Baumeister, W., Menard, R., assigned manually. Meissner, M. and Frischknecht, F. (2007). Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites. J. Exp. Med. 204, 1281-1287. Cryo-electron tomography Day, K. P., Hayward, R. E., Smith, D. and Culvenor, J. G. (1998). CD36-dependent Gametocytes were transferred onto carbon-coated grids. After removal of excess adhesion and knob expression of the transmission stages of Plasmodium falciparum is liquid, grids were rapidly frozen by plunging into liquid ethane then transferring to stage specific. Mol. Biochem. Parasitol. 93, 167-177. liquid nitrogen. Grids were mounted on a Gatan cryo-holder and imaged using a del Pilar Crespo, M., Avery, T. D., Hanssen, E., Fox, E., Robinson, T. V., Valente, P., Tecnai G2 F30 (300 keV). Tilt series were collected every 2˚ between 270˚ and Taylor, D. K. and Tilley, L. (2008). Artemisinin and a series of novel endoperoxide +70˚. The total electron dose kept was 6000 e2/nm2. antimalarials exert early effects on digestive vacuole morphology. Antimicrob. Agents Chemother. 52, 98-109. Dixon, M. W. A., Peatey, C. L., Gardiner, D. L. and Trenholme, K. R. (2009). A Acknowledgements green fluorescent protein-based assay for determining gametocyte production in We would like to thank Alex Lowdin, Michael Jones and Michael Plasmodium falciparum. Mol. Biochem. Parasitol. 163, 123-126. Johnson for assistance with microscopy protocols. We thank Dixon, M. W., Thompson, J., Gardiner, D. L. and Trenholme, K. R. (2008). Sex in Plasmodium: a sign of commitment. 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Journal of Cell Science Eliza Hall Institute) for the anti-GAP50, GAP45 and MyoA antisera Fivelman, Q. L., McRobert, L., Sharp, S., Taylor, C. J., Saeed, M., Swales, C. A., and David Baker (London School of Hygiene and Tropical Sutherland, C. J. and Baker, D. A. (2007). Improved synchronous production of Medicine) for anti-Pfs16 antibodies. We thank Tim Gilberger and Plasmodium falciparum gametocytes in vitro. Mol. Biochem. Parasitol. 154, 119-123. Tobias Spielmann (Bernhard Nocht Institute) for useful discussions. Foley, M., Deady, L. W., Ng, K., Cowman, A. F. and Tilley, L. (1994). Photoaffinity labeling of chloroquine-binding proteins in Plasmodium falciparum. J. Biol. Chem. 269, 6955-6961. Funding Fre´nal, K., Polonais, V., Marq, J. B., Stratmann, R., Limenitakis, J. and Soldati- M.K.D. is supported by an Australian Postgraduate Award (APA). Favre, D. (2010). Functional dissection of the apicomplexan glideosome molecular L.T. is an ARC Australian Professorial Fellow. M.W.A.D. is architecture. Cell Host Microbe 8, 343-357. Gaskins, E., Gilk, S., DeVore, N., Mann, T., Ward, G. and Beckers, C. (2004). supported by an NHMRC training fellowship. C.B.W. is a Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. NHMRC Senior Research Fellow. This work was supported by J. Cell Biol. 165, 383-393. grants from the ANZ Group of Trustees, the Ramaciotti Foundation Glenister, F. K., Coppel, R. L., Cowman, A. F., Mohandas, N. and Cooke, B. M. and an ARC Discovery project. (2002). Contribution of parasite proteins to altered mechanical properties of malaria- infected red blood cells. Blood 99, 1060-1063. Gould, S. B., Tham, W. H., Cowman, A. F., McFadden, G. I. and Waller, R. F. Supplementary material available online at (2008). Alveolins, a new family of cortical proteins that define the protist http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.099002/-/DC1 infrakingdom Alveolata. Mol. Biol. Evol. 25, 1219-1230. Green, J. L., Martin, S. R., Fielden, J., Ksagoni, A., Grainger, M., Yim Lim, B. Y., Molloy, J. E. and Holder, A. A. (2006). The MTIP-myosin A complex in blood stage References malaria parasites. J. Mol. Biol. 355, 933-941. Abu Bakar, N. A., Klonis, N., Hanssen, E., Chan, C. and Tilley, L. (2010). Digestive- Groner, W., Mohandas, N. and Bessis, M. (1980). New optical technique for vacuole genesis and endocytic processes in the early intraerythrocytic stages of measuring erythrocyte deformability with the ektacytometer. Clin. Chem. 26, 1435- Plasmodium falciparum. J. Cell Sci. 123, 441-450. 1442. Aikawa, M. (1977). Variations in structure and function during the life cycle of malarial Hadley, T. J., Erkmen, Z., Kaufman, B. M., Futrovsky, S., McGuinnis, M. H., parasites. Bull. World Health Organ. 55, 139-156. Graves, P., Sadoff, J. C. and Miller, L. H. (1986). Factors influencing invasion of Aikawa, M. and Beaudoin, R. L. (1969). Effects of chloroquine on the morphology of erythrocytes by Plasmodium falciparum parasites: the effects of an N-acetyl glucosamine neoglycoprotein and an anti-glycophorin A antibody. Am. J. Trop. the erythrocytic stages of Plasmodium gallinaceum. Am. J. Trop. Med. Hyg. 18, 166- Med. Hyg., 35, 898-905. 181. Handayani, S., Chiu, D. T., Tjitra, E., Kuo, J. S., Lampah, D., Kenangalem, E., Baker, D. A., Thompson, J., Daramola, O. O., Carlton, J. M. R. and Targett, G. A. T. Renia, L., Snounou, G., Price, R. N., Anstey, N. M. et al. (2009). High (1995). Sexual-stage-specific RNA expression of a new Plasmodium falciparum gene deformability of Plasmodium vivax-infected red blood cells under microfluidic detected by in situ hybridisation. Mol. Biochem. Parasitol. 72, 193-201. conditions. J. Infect. Dis. 199, 445-450. Bannister, L. H., Hopkins, J. M., Fowler, R. E., Krishna, S. and Mitchell, G. H. Hanssen, E., Carlton, P., Deed, S., Klonis, N., Sedat, J., DeRisi, J. and Tilley, L. (2000). A brief illustrated guide to the ultrastructure of Plasmodium falciparum (2010). Whole cell imaging reveals novel modular features of the exomembrane asexual blood stages. Parasitol. Today (Regul. Ed.) 16, 427-433. system of the malaria parasite, Plasmodium falciparum. Int. J. Parasitol. 40, 123-134. The inner membrane complex of gametocytes 2063

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