The knob protein KAHRP assembles into a ring-shaped structure that underpins virulence complex assembly

Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy–Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures.


Author summary
The human malaria parasite Plasmodium falciparum causes severe disease, which is initiated by the adhesion of parasite-infected RBCs to receptors on the walls of the host's capillaries. Adhesion is mediated by a structure called the knob, which acts as a scaffold for the presentation of the virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). In this work we investigate the assembly of this complex at different stages of parasite development using a multimodal imaging approach that combines dSTORM localization microscopy and scanning electron microscopy (STORM-SEM). We

Introduction
Malaria is a devastating human disease that affects more than a quarter of the world's population and kills more than 400,000 people annually [1]. The majority of these deaths are caused by infection with Plasmodium falciparum, with children from sub-Saharan Africa most at risk. P. falciparum-infected red blood cells (RBCs) sequester within the microvasculature of the host, via a process called cytoadhesion. Adhesion removes the infected RBCs from the circulation, thereby avoiding clearance in the spleen, and thus contributing to parasite virulence. Moreover, adhesion of infected RBCs to brain venule endothelial cells or to placental syncytiotrophoblasts can lead to severe complications, known as cerebral and placental malaria, respectively [2,3]. Adhesion of infected RBCs is mediated by a family of proteins called P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is encoded by the~60-member var gene family [4,5]. Once inserted into the RBC membrane, PfEMP1 can bind to endothelial cell receptors such as Endothelial Protein C Receptor (EPCR), Cluster Determinant 36 (CD36), Intercellular Adhesion Molecule-1 (ICAM), and glycosaminoglycans, driving parasite sequestration [6,7]. Central to the parasite's ability to cytoadhere is the formation of nano-scale protrusions (called knobs) at the RBC membrane that act as platforms for the presentation of PfEMP1. The knob-associated histidine-rich protein (KAHRP) is a component of these structures and is essential for their formation [8]. In the absence of knobs, infected RBCs are unable to adhere to endothelial receptors under physiologically-relevant flow conditions [8]. A recent study suggested that the knob comprises a cone-shaped coat of electron-dense KAHRP molecules, underpinned by a spiral structure (of unknown composition) and connected by multiple links to the actin-spectrin meshwork of the RBC membrane skeleton [9]. Work with recombinant proteins and with parasite mutants expressing truncated KAHRP has shown that the C-terminal region of KAHRP contains a sequence that drives high affinity binding to spectrin [10,11] and is critical for the formation of knobs with normal morphology [12].
Despite the important contribution of the virulence complex to severe disease [13], it remains unclear how knobs are assembled at the RBC membrane and how PfEMP1 is loaded into the knobs and presented at the surface. To understand the processes underpinning the assembly of the virulence complex, it is important to determine the locations of individual components with the highest possible spatial resolution, and to place this spatial information in the context of the physical cellular structures. Here we combine direct STochastic Optical Reconstruction Microscopy (dSTORM) localization microscopy with scanning electron microscopy (SEM) to image the assembly of modular units of KAHRP and the delivery of PfEMP1 to the knobs. We show that the spectrin-actin meshwork plays an important role in marshalling knob formation and that sequences within the C-terminal region of KAHRP control assembly into rings and are essential for maintaining the structure of the spiral knob scaffold. Loading of PfEMP1 into the fully formed knob structures appears to occur by lateral transfer.

Results
KAHRP forms ring-shaped complexes that associate with the RBC membrane skeleton SEM imaging of the external surface of RBCs infected with CS2 strain P. falciparum (~30 h post-invasion) reveals knob structures as surface elevations (Fig 1A, zoom, white arrow). In agreement with previous reports the knobs have a diameter of~90 nm [14]. While KAHRP is known to be a major component of the knob, its location within the structure and its mode of assembly at the RBC membrane skeleton are not clear.
In an effort to examine the physical organization of the knobs and their interaction with the host RBC membrane skeleton, we developed a correlative imaging method that allows analysis of the cytoplasmic surface of the RBC membrane. The RBC binding lectin, phytohemagglutinin, was cross-linked to glass coverslips and P. falciparum-infected RBCs (CS2 line,~90% parasitemia,~30 h post-invasion) were adhered tightly to the lectin [15]. A stream of hypotonic buffer was applied to remove the bulk of the cell, leaving a disc of remnant RBC membrane bound to the coverslip (S1A Fig).
Labelling of the remnant discs with a monoclonal anti-KAHRP antibody (mAb89) that recognizes an epitope in the C-terminal region of KAHRP [16] (Fig 1B) gives a diffuse immunofluorescence pattern in widefield deconvolution microscopy (S1B Fig), with individual knobs below the limit of resolution (~250 nm resolution in xy). This confirms that proteins that are tightly associated with the RBC membrane skeleton are retained after shearing (although it should be noted that weakly associating proteins could be lost).
We implemented the super-resolution microscopy technique, dSTORM, which offers an xy resolution limit of about 20 nm [17]. Sheared membrane samples labelled with anti-KAHRP and anti-mouse Alexa Fluor 647-conjugated secondary antibody were mounted in a reducing and O 2 scavenging buffer and imaged using Total Internal Reflection Fluorescence (TIRF) optics. Excitation with a 642 nm laser induces transfer of most of the Alexa-647 molecules to the triplet (dark) state, with only a subset of fluorophores emitting photons at any given time. Following collection of~10,000 frames, a population of horseshoe or ring-shaped fluorescently-labelled KAHRP structures were observed (Fig 1C, zoom, white arrows).
The average distribution of the ring structures is very similar to that of the knobs when viewed from the external surface. The ring structures exhibit an external diameter (half maximum intensity) of 134 ± 3 nm and an internal diameter of 69 ± 4 nm (S1C and S1D Fig). Given the reported diameter of IgG (7-10 nm; [18]) and the secondary labelling strategy used in this work, the diameter of the KAHRP ring structure is consistent with the observed physical size of the knobs. The paucity of labelling in the center of the structure suggests a ring of assembled KAHRP modules.

KAHRP puncta assemble into ring structures comprising discrete KAHRP modules
In addition to the ring-shaped structures, small puncta are observed with an apparent average diameter of 30 ± 2 nm (Fig 1C, zoom, blue arrows; S1C and S1D Fig). This size is consistent with the labeling of KAHRP with a primary and secondary IgG adding 7-10 nm per antibody. The diameters of the puncta are similar to the mean width of the ring walls (32 ± 1 nm), consistent with the ring structures being comprised of several individual KAHRP puncta (S1C and S1D Fig). Moreover, visual examination reveals 4 to 6 punctate components within many of the ring structures ( Fig 1C, white arrow; S1C and S1E Fig). Where individual fluorescently labelled KAHRP elements were resolved, the average distance (mean ± S.D.) between the centers of the puncta within the rings was estimated to be 53 ± 10 nm (233 measurements from 18 membranes) (S1E Fig shows representative images).
We used the number of single molecule localization events (i.e. fluorescence emissions with sufficient photons for detection as individual events [19]) as a surrogate measure of the number of fluorescent molecules in different structures. Analysis of the raw data showed that puncta are associated with 102 ± 42 events (mean ± S.D) with a shoulder of higher values (S1F Fig), consistent with a dominant single species with a weak tendency to self-associate. The ring structures are associated with a mean of 459 ± 153 events ( Fig 1D, S1G Fig), which equates to a mean ratio of 4.7 ± 2.1 puncta equivalents per ring structure (S1E Fig shows representative images). While further work is needed to confirm the structural basis for the fluorescence profile, the data suggest that the ring structures are comprised on average of five modular KAHRP units.
Following dSTORM imaging, the sheared membranes were fixed, dehydrated, gold-coated, and then imaged using high-resolution SEM. The spectrin-actin network appears as bright (raised) skeletal elements over dark patches of background ( Fig 1E). Close analysis of the SEM images reveals raised, dimpled disk-shaped structures that are closely integrated into the skeletal meshwork (Fig 1E, zoom, arrow). These dimpled disks have an average diameter of 85 ± 2 nm and a spacing and distribution (~8 per square micron) similar to that observed for knobs viewed from the external surface by SEM (Fig 1A, S1D Fig).
The dSTORM and SEM data sets were aligned using landmark registration (see Methods) to produce a STORM-SEM image ( Fig 1F). The KAHRP fluorescence signal lies around the raised disk-shaped structures, consistent with previous immuno-EM data showing KAHRP around the knob core [9] (Fig 1F, zoom, arrow). The flanking location of the dSTORM ring structure around the periphery of the raised disk-shaped SEM structures is consistent with the additional width arising from the primary and secondary antibodies.

Knob assembly occurs over the period of 16 h to 30 h post-invasion
SEM-based imaging of the knobs at the external surface of CS2-infected RBCs reveals an increase in density from 1.3 to 9.4 per μm 2 over the period 16-30 h post-invasion (S2A and S2B Fig). This timeframe is consistent with a previous analysis of knob formation [20], and with the reported timeframe for the delivery of PfEMP1 to the RBC surface [21]. We analyzed the organization of KAHRP-containing structures over this same period in sheared membranes. At 16 h post-invasion, a high density of puncta is evident with relatively few ring structures (Fig 2). During parasite maturation, the number of puncta decreases while the number of  rings increases, eventually reaching 7.8 ± 0.6 per μm 2 , which is roughly equal to the number of knob structures observed from the external surface (9.4 ± 0.6 per μm 2 ; S2A and S2B Fig). We note that the curved surface of whole infected RBCs may lead to a moderate overestimation of knob density at the external surface. Taken together, these data indicate that KAHRP is delivered to the membrane as modular units that are then assembled into the larger ring structures.

Knob formation requires remodeling of RBC membrane skeleton actin
Maturation of infected RBCs has previously been reported to involve remodeling of the RBC membrane skeleton and redistribution of a population of actin molecules from the membrane skeleton into filaments that connect to parasite-derived structures called Maurer's clefts [22,23]. The F-actin depolymerizing drug, cytochalasin D, was previously shown to destabilize Maurer's cleft-associated actin filaments in trophozoite-infected RBCs [22]. However, the effect of cytochalasin D on the rheological properties of infected RBCs and on the assembly of knobs has not been studied.
We examined the rheological properties of control and cytochalasin D-treated infected RBCs by assessing their ability to passage through a bed of microbeads designed to mimic the fenestrations in the splenic sinuses [24]. Tightly synchronized parasites (2 h window) were  (Fig 3A and 3B). Accordingly, the cytochalasin D-treated cells exhibit a higher density of small KAHRP puncta, showing that cytochalasin D treatment does not affect KAHRP delivery and binding to the RBC membrane (Fig 3B and 3C). STORM-SEM imaging confirms the loss of the disk-shaped physical knob structures (S3B Fig). Associated with this loss of physical knob structures, cytochalasin D-treated infected RBCs show increased ability to passage through the filter (Fig 3D).
To determine whether the effects of cytochalasin D are reversible, treated cells were washed and allowed to recover for 8 h (i.e. re-examined at 30 h post-invasion). dSTORM imaging reveals an increase in the number of ring structures (2.7 ± 0.4 per μm 2 ), although the density does not reach the level of the controls ( Fig 3E). The number of puncta remains higher than in the control ( Fig 3F). Despite the lack of ring structures, the treated cells show very low filterability following recovery, similar to that of controls at 30 h post-invasion ( Fig 3G).
To confirm that the cytochalasin D-mediated effects are not due to a loss of parasite viability or to developmental delay, we measured parasite area (in Giemsa-stained smears). Incubation with cytochalasin D over the 16-22 h post-invasion period had no effect on the parasite size (

Dissection of the regions of KAHRP needed for skeleton binding and ring complex assembly
The N-terminal region of KAHRP contains a signal sequence, a PEXEL motif (needed for export) and a histidine-rich region ( Fig 1B) [25,26]. The C-terminal half of KAHRP includes two repetitive sequence elements (the so-called 5´and 3´repeats; Fig 1B) that have been reported to be involved in binding to the membrane skeleton [12,27]. Recent work suggests that the 5' repeat region plays the most important role in binding to spectrin [10].
Building on a previous study [12], we generated transfectants expressing KAHRP truncated at amino acid 362 (K362), 405 (K405) or 530 (K530), in CS2 parasites, by 3' replacement of the kahrp locus, using the selection-linked integration approach (S5A Fig No KAHRP protein is detected by dSTORM imaging of sheared membranes prepared from K405 truncation mutants, despite the presence of physical structures (Fig 4B, left). The lack of fluorescence signal (either puncta or ring structures) may indicate loss of KAHRP during preparation of the sheared membranes. The K362 mutants exhibit neither physical knobs, nor evidence for the presence of KAHRP.
We used electron tomography to examine the aberrant knob structures-a method that has previously been shown to reveal a spiral structure within the knobs (9). While complete preservation of the supporting structure is not achieved in negatively-stained membrane preparations, we observed well-preserved spirals in 59% of the knobs (electron-dense structures) examined in wildtype CS2 parasites (Fig 4C and 4D, S8 and S9 Figs). In contrast, knob-like structures from the K530 mutants show complete spiral structures in only 11% of the knobs examined, with the majority (73%) showing fragmented and discontinuous spiral structures (Fig 4C and 4D, S8 and S9 Figs). The observed degradation of the supporting structure is consistent with the malformed knobs, as viewed by SEM of the external surface (S6 Fig). No complete spirals are observed in the K405 mutants, but 76% of the knobs exhibit fragmented spiral structures (Fig 4C and 4D, S8 and S9 Figs). We also observed electron-dense structures that did not contain spirals (empty knobs), particularly in the truncation mutants (CS2, 10%; K530, 16% and K405, 24%) (Fig 4C and 4D, S8 and S9 Figs). We also note that the maximum diameter of the spirals in the K530 (47 ± 3 nm) and K405 mutants (39 ± 2 nm) is smaller than in wildtype CS2 (54 ± 4 nm) parasites (S9B Fig). Similarly, the mean combined length of the spiral features (both fragmented and whole) in individual knobs is shorter in the K530 (233 ± 25 nm) and K405 (156 ± 12 nm) mutants than in wildtype parasites (322 ± 40 nm) (S9C Fig). The electron-dense knob coat has a slightly decreased diameter in the K530 (87 ± 3 nm) and K405 (80 ± 4 nm) mutants when compared to CS2 (89 ± 3 nm), consistent with our analysis of SEM images of the external surface (S9D, S6A and S6B Figs).

PfEMP1 is delivered to the membrane and targeted to the knobs
The mechanism for trafficking of PfEMP1 to knobs is poorly understood, in part due to a lack of antibody reagents that recognize PfEMP1 within the context of the knob structure [29]. In this work, we made use of transfected parasites (A4 background) expressing a mini- PfEMP1-GFP construct, A4var-V5-TM-ATS-GFP (referred to here as PfEMP1A-GFP) [30,31]. This minimal PfEMP1 contains the semi-conserved N-terminal region (DBL and CIDR), the transmembrane segment and the C-terminal ATS domain of the A4 var allele. It was previously shown that PfEMP1A-GFP molecules are delivered to the RBC surface [31].
We prepared sheared membranes from PfEMP1A-GFP transfectants and detected the transgene product using anti-GFP and anti-mouse Alexa Fluor-647 antibodies (Fig 5A, S10A  Fig). Bright fluorescent PfEMP1 foci were observed with a signal intensity suitable for dSTORM imaging. By contrast, antibodies directed against the acidic terminal sequence (ATS) domain of endogenous PfEMP1 did not provide sufficient signal for dSTORM imaging, suggesting that the antibody does not bind with high enough affinity to membrane-inserted PfEMP1 to permit single molecule localization microscopy. STORM-SEM imaging of PfEM-P1A-GFP sheared membranes was performed at 20, 24 and 30 h post-invasion and PfEMP1 foci and knob structures counted (Fig 5B and 5C). The number of PfEMP1A-GFP foci at the membranes increases from 20 to 24 h post-invasion, then remains constant through to 30 h post-invasion ( Fig 5B). The numbers of knobs at different stages of development in the PfEM-P1A-GFP parasites is similar to that seen for wildtype CS2 parasites (Fig 5C and S2 Fig). Examination of the STORM-SEM images reveals three different populations of PfEMP1A-GFP, which we have classified as being at the knobs (blue arrows, within 50 nm from a physical knob center), adjacent to the knob (yellow arrows, within 50-100 nm from a knob center) and greater than 100 nm from the center of a knob (white arrows, Fig 5A, S10B Fig). We applied this criterion and analyzed the sheared membranes to determine the location of PfEM-P1A-GFP at time points over the period of virulence complex assembly. At 20 h post-invasion only 34% of the PfEMP1A-GFP foci are located at knobs, increasing to 40% at 24 h post-invasion, and reaching 59% at 30 h post-invasion ( Fig 5D). Our data are consistent with PfEMP1 being delivered to the RBC membrane in regions away from knobs and then moving laterally to associate with and be assembled into knob structures. Alternatively, it is possible that knobs disassemble and reassemble around PfEMP1 foci.

Discussion
We developed and implemented a multimodal imaging technique that combines dSTORM localization microscopy and SEM. The technique allows us to map the locations of KAHRP and PfEMP1 to physical structures assembled at the RBC membrane skeleton. We show that KAHRP is delivered as punctate structures (with a diameter close to the resolution limit, i.e. 30 nm) that associate with the spectrin meshwork, from about 16 h post-invasion. As the parasite matures, larger (~130 nm diameter) ring-shaped structures appear and increase in number, while the puncta disappear.
The quantification of signal intensities from dSTORM data is challenging, particularly in the context of indirect labelling with antibodies [32]. Nonetheless, analysis of localization events for the individual fluorescent puncta reveals a narrow distribution of fluorescence intensities, suggesting a dominant single species, consistent with a modular population, potentially comprising single KAHRP proteins, KAHRP containing complexes or very tightly selfassociated KAHRP oligomers.
Our analysis of the number of localization events suggests that the ring structures comprise an average of 5 KAHRP modules, each separated by an average distance of~50 nm. Recent work showed that the KAHRP 5' repeats form an extended interaction interface with domains 10-14 of β-spectrin, which lie near to the ankyrin-binding domain [10]. One explanation for the 5-module ring structure is that the ring is underpinned by 5 spectrin tetramers, each presenting a KAHRP binding site (separated by~50 nm). This suggestion is consistent with recent estimates of the average branch length of the spectrin network in trophozoite stage-infected RBCs, i.e. 49-64 nm [15,23,33] and with a study showing that an average of 5 spectrin proteins converge per junctional complex in the RBC membrane skeleton [34]. Given its unusual amino acid composition and predicted disordered domains [10], we also considered the possibility that KAHRP adopts a highly elongated shape. Propagation of a weak self-association reaction could permit the formation of a five-membered ring, with individual elements separated by~50 nm (see Fig 6). Another possibility is that an as yet unidentified bridging protein facilitates the assembly process. It is also possible that the shearing method strips loosely associated structures and proteins (including KAHRP) from the membrane. The proposal that only about 5 KAHRP molecules contribute to the knob structure means that KAHRP is much sparser than envisaged in previous models (e.g. see [10]) and further work is needed to test this model.
SEM of the sheared membranes reveals dimpled, disk-shaped structures (~85 nm diameter) associated with the membrane skeleton. Correlative imaging shows that these disks sit neatly inside the ring of anti-KAHRP fluorescence. We suggest that the dimpled disks represent the physical base of the knob structures. A recent ultrastructural study visualized the knob complex as a spiral structure connected by multiple links to the RBC membrane skeleton and coated by an electron dense layer that is likely composed of KAHRP molecules [9]. In this work, we used high resolution images of negatively-stained membranes to confirm the presence of spiral structures, with a diameter of 54 nm surrounded by an electron dense coat with a diameter of 89 nm (for CS2 parasites). While it is possible that the knob structure may be altered during the preparation of samples for negative staining and EM, our data are consistent with KAHRP molecules surrounding a spiral scaffold that underpins the knob complex. Of interest, our data show that there is a region in the center of the knob complex that does not contain KAHRP, which may represent the region where PfEMP1 is displayed. However, it does not discount the possibility that other modules of KAHRP may be present (and not antibody accessible) or that they may have been lost in the shearing process.
The membrane skeleton of the RBC is reorganized during intraerythrocytic development of P. falciparum [15,35], potentially as a result of the mining of actin for use in filaments that connect to the Maurer's clefts [22]. Cytochalasin D, a cell-permeable mycotoxin that binds to the slow-growing ends of actin filaments with high affinity [36], has been shown to block RBC membrane skeleton remodeling [22,23]. We found that cytochalasin D treatment blocks knob formation. Treatment does not prevent binding of KAHRP nor affect the timing of KAHRP delivery to the RBC membrane skeleton but inhibits the reorganization of KAHRP modules into ring structures, and prevents the physical assembly of knobs. We show that the block is not due to a developmental arrest or a loss of parasite viability. This data strongly suggests that correct actin organization and/or skeletal reorganization are needed to support correct assembly and anchoring of the knob. Alternatively, it is possible that cytochalasin D treatment inhibits the trafficking of unknown third party proteins that are required for knob assembly or that cytochalasin D treatment impairs knob anchoring. Assembly of malaria parasite knobs The assembly of knobs stiffens the spectrin network [24,37], through composite strengthening, strain hardening, and knob density-dependent vertical-coupling mechanisms [38]. Similarly, the reorganization of the skeletal network, with an increased number of stretched spectrin tetramers and octamers converging on a reduced number of junction points, is predicted to increase the shear modulus of the membrane skeleton [38]. Cytochalasin D-treated infected RBCs show enhanced filterability through a microbead bed compared with control cells, consistent with important roles of knob assembly and skeleton reorganization in rigidifying the host RBC membrane. Upon removal of cytochalasin D, partial recovery of knob formation is observed, and the infected RBCs no longer survive filtration, indicating restoration of cellular rigidity.
Using C-terminal truncations of KAHRP (based on previous work [12]) we investigated the role of sequence information within the C-terminal region of KAHRP in driving formation of the ring structure. We found that transfectants expressing KAHRP truncated at residue 530 exhibit malformed (elongated bauble-like) physical structures. KAHRP modules are associated with these malformed knobs, but they exhibit fewer (and more fragmented) spiral structures with a smaller diameter and a reduced length. This indicates that the information for KAHRP self-association is retained in the K530 truncation but that the sequence information in the 3' repeat and C-terminal sequence is required for formation of canonical ring structures.
The 5' repeat region of KAHRP has been shown to be the major contributor to spectrin binding [10,12]. When KAHRP is truncated at residue 405 (mid-way through the 5' repeats), malformed knob-like protrusions are still observed by external SEM, and bauble-like structures are observed at the internal surface. Some anti-KAHRP signal is observed (by immunofluorescence microscopy of whole cells) in K405 mutant-infected RBCs, but no anti-KAHRP signal is observed in sheared membrane samples, prepared for dSTORM imaging. This suggests that the K405-KAHRP may be free in the cytoplasm or only weakly associated with the RBC membrane. In negatively-stained preparations of the K405-infected RBCs, no well-preserved spiral structures are observed, and the degraded and collapsed spiral structures exhibit significantly reduced diameter and length. Truncation of KAHRP at residue 362 completely ablates knob formation. These data confirm that the 5' repeat region of KAHRP is needed for binding to spectrin. It further demonstrates that KAHRP binding to spectrin is needed for the formation and stabilization of the spiral structure but that KAHRP, itself does not appear to be a physical component of the spiral. Whether the spiral protein comprises another parasitederived protein or reorganized RBC skeletal proteins remains to be determined. Collectively this data suggests a model where the C-terminal region of KAHRP is needed for the correct organization of KAHRP into rings structures, the correct assembly of the spiral structure and the acquisition of canonical knob morphology (S11 Fig). While PfEMP1 can be surface-displayed in the absence of knobs [39] and knobs can be assembled in parasites with a PfEMP1 trafficking defect [12,40], recent work suggests an interplay between the PfEMP1 variant that is expressed and the density of knobs at the RBC membrane [41]; and an early study suggested that the virulence complex might be pre-assembled at the Maurer's clefts [42]. Similarly, PfEMP1 variants show different affinities for spectrin and different members of the PHIST gene family [10,43]. Thus, the precise mechanism for assembly of the virulence complex remains unclear. In this work we observed three sub-populations it interacts with spectrin. Host cell remodeling is initiated (white arrows) via the dissociation of the actin junction points, allowing spectrin remodeling. PfEMP1 is delivered to the RBC membrane. (C) Host cell remodeling continues with spectrin octamers being formed (white arrow). KAHRP undergoes higher order assembly (with potential involvement of a bridging protein) forming a horseshoe-shaped structure that scaffolds the spiral knob core. PfEMP1 moves laterally to associate with the virulence complex. (D) KAHRP modules form the base of the knob complex. Most of the PfEMP1 is assembled adjacent to or at knob complexes. of PfEMP1 at the RBC membrane, located either directly at, adjacent to, or at some distance from knobs. The percentage of knob-associated PfEMP1 foci increases over the period of virulence complex assembly (20 h-30 h post-invasion). While caution should be exercised in extrapolating native PfEMP1 trafficking from the behavior of a tagged PfEMP1 construct, the data are consistent with gradual assembly of PfEMP1 into preformed knobs by lateral transfer.
Taken together with previous work, our data point towards a model of virulence complex assembly that can be interrogated with further experiments. We propose that the individual virulence complex components are assembled in situ at the RBC membrane skeleton rather than being delivered as a pre-assembled complex (Fig 6A-6D). We suggest that KAHRP molecules are trafficked to the RBC membrane from~16-20 h post-invasion, with the spectrinbinding domain driving association with the membrane skeleton (Fig 6B). We propose that reorganization of the spectrin-actin network, driven by loss of actin junction points, allows higher order assembly of KAHRP molecules that are bound to adjacent spectrin tetramers (potentially facilitated by a bridging protein, such as the spiral protein), leading to the assembly of rings of KAHRP modules (Fig 6C and 6D). We propose that individual KAHRP modules move laterally across the membrane skeleton prior to assembly. Alternatively, modules that fail to assemble may disassociate, leaving only the successfully built rings. We propose that the spiral scaffold is then assembled on the KAHRP ring in the region of destabilized membrane skeleton (Fig 6D). We propose that PfEMP1 is delivered to and inserted into the RBC membrane at random locations, where it interacts weakly with spectrin. We suggest that PfEMP1 is then transferred laterally into the central region of the preformed knob structures. Further experiments are needed to test various elements of this model.
Assembly of PfEMP1 into raised knob platforms likely promotes adhesion by presenting the receptor-binding domains of PfEMP1 above the RBC glycocalyx. The rigidification of the RBC membrane that results from knob assembly and skeletal reorganization also facilitates adhesion of infected RBCs by distributing the tensional forces imposed on individual receptor-bound PfEMP1 molecules across the entire knob region and through to the membrane skeleton [38]. The insights we have gained into the knob assembly process may lead to new ways to inhibit the presentation of PfEMP1 on infected RBCs, and thus new ways of interfering with this lethal pathogen.

Ethics statement
Red Blood Cells and serum were obtained from the Australian Red Cross blood service. All blood products were anonymous and individual donors could not be identified. This work was approved by the University of Melbourne Human Research Ethics Committee (Approval number 1135799).

Parasite culture
P. falciparum (wildtype CS2 [44] and A4 expressing the A4var-V5-TM-ATS-GFP construct (PfEMP1-A) [31]) was cultured in O+ RBCs at 5% haematocrit in complete culture media containing RPMI-GlutaMAX-HEPES (Invitrogen) supplemented with 5% v/v human serum (Australian Red Cross blood service), 0.25% w/v AlbuMAX II (Invitrogen), 200 μM hypoxanthine, 10 mM D-glucose (Sigma) and 20 μg/ml gentamicin (Sigma). Parasites were synchronsied to ring stage with 5% D-sorbitol [45] or enriched at mature stage using Percoll purification or magnetic separation [46,47]. Parasites were synchronsied to a 2 h post-invasion window as previously described [48]. Knob positive parasites were selected on gelatin [49]. Parasites were treated with 10 μM cytochalasin D as previously described [30]. Parasite survival assays were performed by diluting the cytochalasin D treated parasites by half by adding fresh RBCs and media to the culture following washout of the drug. The parasitemia was assessed by Giemsa smears in the next parasite cycle.

Plasmid constructs and transfection
Parasite lines expressing C-terminal truncations of KAHRP at amino acid 362, 405 and 530 were created in the CS2 parasites, as previously described for 3D7 [12]. A 3' replacement approach using the selection linked integration was used to create these truncations [28]. The targeting sequences for each of the truncations were PCR amplified from 3D7 genomic DNA using the common KtruncF (5'-gcggccgcTAAAATAATGGAAACGGATCCGGT-3') and either the K362R (5'-gtcgacTTAGGAATGGTGTTTTTCATCACC-3'), K405R (5'-gtcgacTTA TTTTACGCTTTCTGCATCTTC-3') and K530R (5'-gtcgacAGTACTAATGCCGCAACAA ATTAA-3') primers. The resulting PCR products were then digested and directionally cloned into the NotI and SalI digested pSLI-TGD plasmid. This leads to the removal of the GFP from this plasmid. The constructs were then transfected as previously described and maintained on media supplemented with WR99210 (5 nM) [50]. Selection linked integration was performed as previously described [28].

Scanning electron microscopy
Coverslips with adhered infected RBCs or sheared membranes were placed in a humid chamber and fixed with 2.5% glutaraldehyde in PBS for 1 h at room temperature before washing 3 times with PBS, and submerging in H 2 O for 5 min, then sequentially for 5 min each in 20%, 50%, 70%, 80%, 90%, 95% and 100% ethanol. Coverslips were dried using a compressed nitrogen gas nozzle and stored in a vacuum desiccator. Coverslips were gold-coated for 40 s at 25 mA using a Dynavac sputter coating instrument. The coating thickness was measured at 0.2 nm on a quartz crystal microbalance. Images were acquired using an FEI Teneo SEM using the ETD detector in Optiplan mode, at a working distance of 5 mm, a beam current of 50 pA and a 2-kV accelerating voltage.

dSTORM immunofluorescence microscopy
Immunolabeled membrane samples were loaded into a Chamlide chamber and immersed in a buffer containing 10% w/v glucose, 50 mM Tris pH 8, 10 mM NaCl, 10 mM cysteamine hydrochloride, 40 μg/mL catalase, 0.56 μg/mL glucose oxidase. Samples were imaged using the Del-taVision OMX V4 Monet Localization Microscopy system (GE Healthcare), with 642 nm laser TIRF illumination (60X oil immersion lens, Olympus, 1.49 NA) and a beam concentrator to assist fluorophores transition to the triplet (dark) state. A 405 nm laser was used to improve fluorophore cycling back to the ground state. Single events were captured over 10,000 frames. DeltaVision softWoRx (GE Healthcare) software was used to process data. Fluorescent events with a localization precision of 5-100 nm and persistence between 1-100 frames were included in the final reconstruction. Each event was fitted with a Gaussian distribution to localize the point of origin. Data was recolored in FIJI using the 'Red Hot' look-up table, where black, red, yellow and white indicate a gradient from low to high signal.

Electron tomography
TEM grids with Formvar support film were coated with carbon and glow discharged. The grids were functionalized with 0.1% poly-L-lysine solution for 5 min before incubation with 2% infected RBC cell suspension in PBS. Unattached cells were washed away from grids by dipping in PBS. The cells were lysed with hypotonic 5P8Q10 buffer (5 mM Na 2 HPO 4 / NaH 2 PO 4 , 10 mM NaCl, pH 8.0) for 1 min. Lysed cells were negatively stained with 1% aqueous uranyl acetate solution for 30 sec. Before imaging the back side of the grids were layered with 6 nm fiducial gold. Tilt series were collected with single tilt holder from -70˚to 70˚using an FEI Tecnai G2 F30 (FEI Company, Hillsboro, OR) electron microscope with a field emission gun operated at 200 kV. Tomograms were aligned and reconstructed using IMOD/Etomo (Boulder Laboratory for 3D Electron Microscopy of Cells). Samples from wildtype CS2 and the truncation mutants were prepared, stained and imaged in parallel on the same day.

Immunoblotting
Wildtype CS2 and truncation mutant trophozoite stage parasites were enriched from culture by Percoll purification and subjected to lysis with 0.03% Saponin/1XPBS as previously described [53]. The pellet fraction from saponin lysis was mixed with LDS sample buffer (Invitrogen) and DTT (Invitrogen) and separated on 4-12% Bolt Bis-Tris gels (Invitrogen), using MOPS buffer. Gels were then transferred to 0.2 μm nitrocellulose membrane using the iBlot 2 system (Invitrogen). The membranes were blocked in 3.5% skim milk in PBS for at least 1 h at room temperature prior to addition of the primary antibody. Anti-KAHRP (mAb18.2, 1:500) primary antibody was diluted in 3.5% skim milk/PBS and incubated on the membranes overnight. Anti-mouse (goat, 1:25,000, Promega) horseradish peroxidase-conjugated secondary antibodies in 3.5% skim milk/PBS were incubated for 1 h at room temperature. Membranes were washed between antibody incubations in 1XPBS/0.05% Tween20. Membranes were incubated with Clarity ECL substrate (BioRad) and imaged using the ChemiDoc MP Imaging System (BioRad).

Microbead filtration
The spleen mimic filtration method was used to assess the deformability properties of parasite infected RBC [24]. Briefly, synchronous parasites at the desired age were prepared at 6% parasitemia and 1% hematocrit in 1% AlbuMAX II in 1X PBS. The parasite material was flowed using a syringe pump over the microbead matrix at a flow rate of 60mL/min. A total of 8 mL of 1% AlbuMAX II/PBS was flowed through the bead matrix and the flow through collected and centrifuged to collect the parasites. The data presented is the percentage of parasites present in the flow through relative to starting parasitemia. Each experiment is performed in triplicate on at least 3 separate occasions, the mean and standard error are plotted, and a t-test is used to evaluate statistical significance.

dSTORM ring structure and puncta measurements
Ring structure measurements were performed on sheared membranes from parasite infected cells at 30 h post-invasion using FIJI. A line was drawn across the center of the puncta or ring structure and the profile plotted. The half maximum of each peak was extracted from the raw data and was used to generate the half maximal width of the structures. The puncta diameter and the outside and inside diameters of the rings were calculated. The width of the ring wall is also calculated. A total of 10 puncta or 5 individual rings from 5 separate membranes were analysed and the mean ± the standard error plotted.

Counts of ring structures and puncta from dSTORM data
Ring structures and puncta were counted from at least 2 separate 2 x 2 μm sections from each sheared membrane. Puncta were automatically counted with FIJI using the 'Analyse Particles' feature where puncta were defined as between 5-50 pixels in size. Automatic counts were manually curated to remove rings recognized as puncta. Ring structures were counted manually. All data is expressed as the number of structures per μm 2 . The mean ± the standard error is plotted.

Image alignment
STORM-SEM overlays were generated by transforming dSTORM images to align with their SEM equivalents. Transformations were performed with FIJI using the landmark correspondence plugin [54]

SEM knob diameter
Knob diameter was measured using FIJI. Sample brightness was plotted from a line across each knob. The diameter of each knob was recorded as the distance between the base of each side of its brightness peak. See S1C Fig for illustration.

Localization event counts
To determine the number of localization events per puncta or ring structures a custom Python script was created. Squares (200 x 200 nm) were placed around either isolated puncta or isolated ring structures on 23 cell membranes from 22 and 24 h post-invasion cultures. Using the squares coordinates, we counted the number of localization detections from the raw Tracked.txt tables corresponding to each punctum or ring structure, using a custom Python script (Juan Nunez-Iglesias, 2018, August 27; Count doughnuts (ring structures) Version 0.1.0. Zenodo. http://doi.org/10.5281/zenodo.1403937) depending on the pandas [55], matplotlib [56] and seaborn [57] libraries.

Statistical analysis
Statistical analysis and graphs were generated using Prism 5 (GraphPad). Unpaired t tests were performed where specified (see figure legends). P values of < 0.05 ( � ), < 0.01 ( �� ) and < 0.001 ( ��� ) were considered statistically significant.  Fig 5D. (B,C,D) The diameters of spirals (B), the lengths of spirals (C) and widths of the electron-dense knobs (D) were measured from electron tomograms of negative-stained preparations of schizont-infected RBCs. Measurements were taken from 10 knobs for each parasite line (unpaired t-test, �� p = 0.006, ��� p = 0.0009). Data is presented as the mean ± SEM. The data used to generate these graphs are shown in the tables on the right.