The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells.

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin-like protein PPLP2 (here termed P-EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle-resident proteins is yet unknown. Here, we used high-resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P-EVs and that exocytosis of the P-EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress-related proteins, we identified novel components of OBs and P-EVs, including vesicle-trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

Variable microtubule architecture in the malaria parasite.

Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen.

A non-reactive natural product precursor of the duocarmycin family has potent and selective antimalarial activity.

We identify a selective nanomolar inhibitor of blood-stage malarial proliferation from a screen of microbial natural product extracts. The responsible compound, PDE-I2, is a precursor of the anticancer duocarmycin family that preserves the class's sequence-specific DNA binding but lacks its signature DNA alkylating cyclopropyl warhead. While less active than duocarmycin, PDE-I2 retains comparable antimalarial potency to chloroquine. Importantly, PDE-I2 is >1,000-fold less toxic to human cell lines than duocarmycin, with mitigated impacts on eukaryotic chromosome stability. PDE-I2 treatment induces severe defects in parasite nuclear segregation leading to impaired daughter cell formation during schizogony. Time-of-addition studies implicate parasite DNA metabolism as the target of PDE-I2, with defects observed in DNA replication and chromosome integrity. We find the effect of duocarmycin and PDE-I2 on parasites is phenotypically indistinguishable, indicating that the DNA binding specificity of duocarmycins is sufficient and the genotoxic cyclopropyl warhead is dispensable for the parasite-specific selectivity of this compound class.

Synthesis and Antiplasmodial Activity of Bisindolylcyclobutenediones.

Malaria is one of the most dangerous infectious diseases. Because the causative Plasmodium parasites have developed resistances against virtually all established antimalarial drugs, novel antiplasmodial agents are required. In order to target plasmodial kinases, novel N-unsubstituted bisindolylcyclobutenediones were designed as analogs to the kinase inhibitory bisindolylmaleimides. Molecular docking experiments produced favorable poses of the unsubstituted bisindolylcyclobutenedione in the ATP binding pocket of various plasmodial protein kinases. The synthesis of the title compounds was accomplished by sequential Friedel-Crafts acylation procedures. In vitro screening of the new compounds against transgenic NF54-luc P. falciparum parasites revealed a set of derivatives with submicromolar activity, of which some displayed a reasonable selectivity profile against a human cell line. Although the molecular docking studies suggested the plasmodial protein kinase PfGSK-3 as the putative biological target, the title compounds failed to inhibit the isolated enzyme in vitro. As selective submicromolar antiplasmodial agents, the N-unsubstituted bisindolylcyclobutenediones are promising starting structures in the search for antimalarial drugs, albeit for a rational development, the biological target addressed by these compounds has yet to be identified.

Common virulence gene expression in adult first-time infected malaria patients and severe cases.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family.

During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparumstevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface.IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery.

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACß) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

Structure-activity relationships in a series of antiplasmodial thieno[2,3-b]pyridines.

BACKGROUND: Malaria is one of the most prevalent tropical infectious diseases. Since recently cases of artemisinin resistance were reported, novel anti-malarial drugs are required which differ from artemisinins in structure and biological target. The plasmodial glycogen synthase kinase-3 (PfGSK-3) was suggested as a new anti-malarial drug target. 4-Phenylthieno[2,3-b]pyridines were previously identified as selective PfGSK-3 inhibitors with antiplasmodial activity. The present study aims at identifying a molecular position on this scaffold for the attachment of side chains in order to improve solubility and antiplasmodial activity. Furthermore, the role of axial chirality in the compound class for antiplasmodial activity and PfGSK-3 inhibition was investigated. METHODS: 4-Phenylthieno[2,3-b]pyridines with substituents in 4-position of the phenyl ring were docked into the ATP binding site of PfGSK-3. The compounds were synthesized employing a Thorpe reaction as final step. The enantiomers of one congener were separated by chiral HPLC. All derivatives were tested for inhibition of asexual erythrocytic stages of transgenic NF54-luc Plasmodium falciparum. Selected compounds with promising antiplasmodial activity were further evaluated for inhibition of HEK293 cells as well as inhibition of isolated PfGSK-3 and HsGSK-3. The kinetic aqueous solubility was assessed by laser nephelometry. RESULTS: The para position at the 4-phenyl ring of the title compounds was identified as a suitable point for the attachment of side chains. While alkoxy substituents in this position led to decreased antiplasmodial activity, alkylamino groups retained antiparasitic potency. The most promising of these congeners (4h) was investigated in detail. This compound is a selective PfGSK-3 inhibitor (versus the human GSK-3 orthologue), and exhibits improved antiplasmodial activity in vitro as well as better solubility in aqueous media than its unsubstituted parent structure. The derivative 4b was separated into the atropisomers, and it was shown that the (+)-enantiomer acts as eutomer. CONCLUSIONS: The attachment of alkylamino side chains leads to the improvement of antiplasmodial activity and aqueous solubility of selective PfGSK-inhibitors belonging to the class of 4-phenylthieno[2,3-b]pyridines. These molecules show axial chirality, a feature of high impact for biological activity. The findings can be exploited for the development of improved selective PfGSK-3 inhibitors.

Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites.

Central to the pathogenesis of malaria is the proliferation of Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of each of the six available phospho-sites in the CPD. We confirm that the cyclic AMP/protein kinase A (PKA) signalling pathway elicits a phospho-priming step upon serine 610 (S610), which enables subsequent phosphorylation in vitro of a conserved, downstream threonine residue (T613) by glycogen synthase kinase 3 (GSK3). Both phosphorylation steps are required for AMA1 to function efficiently during invasion. This provides the first evidence that the functions of key invasion ligands of the malaria parasite are regulated by sequential phosphorylation steps.

Pellicle formation in the malaria parasite.

The intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division.

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans.

Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse-genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition, and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.

The role of palmitoylation for protein recruitment to the inner membrane complex of the malaria parasite.

To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.

Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation.

Amino acid utilization is important for the growth of the erythrocytic stages of the human malaria parasite Plasmodium falciparum, however the molecular mechanism that permits survival of the parasite during conditions of limiting amino acid supply is poorly understood. We provide data here suggesting that an autophagy pathway functions in P. falciparum despite the absence of a typical lysosome for digestion of the autophagosomes. It involves PfATG8, which has a C-terminal glycine which is absolutely required for association of the protein with autophagosomes. Amino acid starvation provoked increased colocalization between PfATG8- and PfRAB7-labeled vesicles and acidification of the colabeled structures consistent with PfRAB7-mediated maturation of PfATG8-positive autophagosomes; this is a rapid process facilitating parasite survival. Immuno-electron microscopic analyses detected PfRAB7 and PfATG8 on double-membrane-bound vesicles and also near or within the parasite's food vacuole, consistent with autophagosomes fusing with the endosomal system before being routed to the food vacuole for digestion. In nonstarved parasites, PfATG8, but not PfRAB7, was found on the intact apicoplast membrane and on apicoplast-targeted vesicles and apicoplast remnants when the formation of the organelle was disrupted; a localization also requiring the C-terminal glycine. These findings suggest that in addition to a classical role in autophagy, which involves the PfRAB7-endosomal system and food vacuole, PfATG8 is associated with apicoplast-targeted vesicles and the mature apicoplast, and as such contributes to apicoplast formation and maintenance. Thus, PfATG8 may be unique in having such a second role in addition to the formation of autophagosomes required for classical autophagy.

The apicomplexan inner membrane complex.

Dinoflagellates, apicomplexans and ciliates are members of the monophyletic supergroup of Alveolata. The protists of this phylogenetic cluster have adapted to various ecological niches and lifestyles. Dinoflagellates and cilates can be found in any aquatic environment, whereas the phylum Apicomplexa solely comprises intracellular parasites. Despite their diversity all alveolates are united by the presence of membranous vesicles, so called alveoli, located beneath the plasma membrane. In addition to strengthening the cytoskeleton, these vesicles appear to possess taxon-specific functionality. In dinoflagellates and ciliates the alveoli predominantly play a structural role and can function as calcium stores. However, for the Apicomplexa, the alveolar vesicles -here jointly called the inner membrane complex (IMC)- are additionally involved in invasion of the host cell and are important scaffold elements during cytokinesis. Recent studies shed light on the architecture of the apicomplexan IMC and the number and diversity of its constituent proteins. This plethora of proteins and their varying evolutionary origin underlines the versatility of the IMC as a result of the adaption to a parasitic lifestyle.

PfSec13 is an unusual chromatin-associated nucleoporin of Plasmodium falciparum that is essential for parasite proliferation in human erythrocytes.

In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a sub-nuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex (NPC). However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging, we show that the dynamic localization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochormatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and chromatin immunoprecipitation analyses revealed that, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used peptide nucleic acids (PNA) to downregulate PfSec13 and show that it is essential for parasite proliferation in human erythrocytes.

The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion.

Members of the phylum Apicomplexa actively enter host cells by a process involving the discharge of the apically localized microneme and rhoptry organelles. To unravel the processes involved in rhoptry organelle biogenesis, we focused on the Toxoplasma gondii armadillo repeats only protein (TgARO), a conserved acylated protein homogenously anchored to the rhoptry membrane. Conditional disruption of TgARO results in the random cytosolic dispersion of rhoptries and a severe defect in T. gondii invasion, with no effects on intracellular growth or host cell egress. Importantly, rhoptry displacement upon ARO depletion can be functionally complemented with wild-type TgARO but not an acylation mutant. TgARO interacts with myosin F, and inhibition of actin polymerization or myosin function also results in rhoptry dispersal, indicating that the apical positioning of rhoptries is an actomyosin-based process. Thus, TgARO mediates the apical localization of rhoptries, which is specifically required for host cell invasion.

Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite.

Rhoptries are specialized secretory organelles characteristic of single cell organisms belonging to the clade Apicomplexa. These organelles play a key role in the invasion process of host cells by accumulating and subsequently secreting an unknown number of proteins mediating host cell entry. Despite their essential role, little is known about their biogenesis, components and targeting determinants. Here, we report on a conserved apicomplexan protein termed Armadillo Repeats-Only (ARO) protein that we localized to the cytosolic face of Plasmodium falciparum and Toxoplasma gondii rhoptries. We show that the first 20 N-terminal amino acids are sufficient for rhoptry membrane targeting. This protein relies on both - myristoylation and palmitoylation motifs - for membrane attachment. Although these lipid modifications are essential, they are not sufficient to direct ARO to the rhoptry membranes. Mutational analysis revealed additional residues within the first 20 amino acids of ARO that play an important role for rhoptry membrane attachment: the positively charged residues R9 and K14. Interestingly, the exchange of R9 with a negative charge entirely abolishes membrane attachment, whereas the exchange of K14 (and to a lesser extent K16) alters only its membrane specificity. Additionally, 17 proteins predicted to be myristoylated and palmitoylated in the first 20 N-terminal amino acids were identified in the genome of the malaria parasite. While most of the corresponding GFP fusion proteins were trafficked to the parasite plasma membrane, two were sorted to the apical organelles. Interestingly, these proteins have a similar motif identified for ARO.

Evolution and architecture of the inner membrane complex in asexual and sexual stages of the malaria parasite.

The inner membrane complex (IMC) is a unifying morphological feature of all alveolate organisms. It consists of flattened vesicles underlying the plasma membrane and is interconnected with the cytoskeleton. Depending on the ecological niche of the organisms, the function of the IMC ranges from a fundamental role as reinforcement system to more specialized roles in motility and cytokinesis. In this article, we present a comprehensive evolutionary analysis of IMC components, which exemplifies the adaptive nature of the IMCs' protein composition. Focusing on eight structurally distinct proteins in the most prominent "genus" of the Alveolata-the malaria parasite Plasmodium-we demonstrate that the level of conservation is reflected in phenotypic characteristics, accentuated in differential spatial-temporal patterns of these proteins in the motile stages of the parasite's life cycle. Colocalization studies with the centromere and the spindle apparatus reveal their discriminative biogenesis. We also reveal that the IMC is an essential structural compartment for the development of the sexual stages of Plasmodium, as it seems to drive the morphological changes of the parasite during the long and multistaged process of sexual differentiation. We further found a Plasmodium-specific IMC membrane matrix protein that highlights transversal structures in gametocytes, which could represent a genus-specific structural innovation required by Plasmodium. We conclude that the IMC has an additional role during sexual development supporting morphogenesis of the cell, which in addition to its functions in the asexual stages highlights the multifunctional nature of the IMC in the Plasmodium life cycle.