Correction: Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

[This corrects the article DOI: 10.1371/journal.pbio.3001020.].

Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid.

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.

Importance of aspartyl protease 5 in the establishment of the intracellular niche during acute and chronic infection of Toxoplasma gondii.

Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.

The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii.

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin-based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.

Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis.

Invasion and egress are two key steps in the lytic cycle of Apicomplexa that are governed by the sequential discharge of proteins from two apical secretory organelles called micronemes and rhoptries. In Toxoplasma gondii, the biogenesis of these specialized organelles depends on the post Golgi trafficking machinery, forming an endosomal-like compartment (ELC) resembling endomembrane systems found in eukaryotes. In this study, we have characterized four phylogenetically related Transporter Facilitator Proteins (TFPs) conserved among the apicomplexans. TFP1 localises to the micronemes and ELC, TFP2 and TFP3 to the rhoptries and TFP4 to the Golgi. TFP1 crucially contributes to parasite fitness and survival while the other members of this family are dispensable. Conditional depletion of TFP1 impairs microneme biogenesis and leads to a complete block in exocytosis, which hampers gliding motility, attachment, invasion and egress. Morphological investigations revealed that TFP1 participates in the condensation of the microneme content, suggesting the transport of a relevant molecule for maintaining the intraluminal microenvironment necessary for organelle maturation and exocytosis. In absence of TFP2, rhoptries adopt a considerable elongated shape, but the abundance, processing or secretion of the rhoptry content are not affected. These findings establish the relevance of TFPs in organelle maturation of T. gondii.

Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson's disease.

Lewy bodies (LBs) are intraneuronal inclusions consisting primarily of fibrillized human α-synuclein (hα-Syn) protein, which represent the major pathological hallmark of Parkinson's disease (PD). Although doubling hα-Syn expression provokes LB pathology in humans, hα-Syn overexpression does not trigger the formation of fibrillar LB-like inclusions in mice. We hypothesized that interactions between exogenous hα-Syn and endogenous mouse synuclein homologs could be attenuating hα-Syn fibrillization in mice, and therefore, we systematically assessed hα-Syn aggregation propensity in neurons derived from α-Syn-KO, ß-Syn-KO, γ-Syn-KO, and triple-KO mice lacking expression of all three synuclein homologs. Herein, we show that hα-Syn forms hyperphosphorylated (at S129) and ubiquitin-positive LB-like inclusions in primary neurons of α-Syn-KO, ß-Syn-KO, and triple-KO mice, as well as in transgenic α-Syn-KO mouse brains in vivo. Importantly, correlative light and electron microscopy, immunogold labeling, and thioflavin-S binding established their fibrillar ultrastructure, and fluorescence recovery after photobleaching/photoconversion experiments showed that these inclusions grow in size and incorporate soluble proteins. We further investigated whether the presence of homologous α-Syn species would interfere with the seeding and spreading of α-Syn pathology. Our results are in line with increasing evidence demonstrating that the spreading of α-Syn pathology is most prominent when the injected preformed fibrils and host-expressed α-Syn monomers are from the same species. These findings provide insights that will help advance the development of neuronal and in vivo models for understanding mechanisms underlying hα-Syn intraneuronal fibrillization and its contribution to PD pathogenesis, and for screening pharmacologic and genetic modulators of α-Syn fibrillization in neurons.

In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Increased axonal bouton dynamics in the aging mouse cortex.

Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline.

The relationship between PSD-95 clustering and spine stability in vivo.

The appearance and disappearance of dendritic spines, accompanied by synapse formation and elimination may underlie the experience-dependent reorganization of cortical circuits. The exact temporal relationship between spine and synapse formation in vivo remains unclear, as does the extent to which synapse formation enhances the stability of newly formed spines and whether transient spines produce synapses. We used in utero electroporation of DsRedExpress- and eGFP-tagged postsynaptic density protein 95 (PSD-95) to investigate the relationship between spine and PSD stability in mouse neocortical L2/3 pyramidal cells in vivo. Similar to previous studies, spines and synapses appeared and disappeared, even in naive animals. Cytosolic spine volumes and PSD-95-eGFP levels in spines covaried over time, suggesting that the strength of many individual synapses continuously changes in the adult neocortex. The minority of newly formed spines acquired PSD-95-eGFP puncta. Spines that failed to acquire a PSD rarely survived for more than a day. Although PSD-95-eGFP accumulation was associated with increased spine lifetimes, most new spines with a PSD did not convert into persistent spines. This indicates that transient spines may serve to produce short-lived synaptic contacts. Persistent spines that were destined to disappear showed, on average, reduced PSD-95-eGFP levels well before the actual pruning event. Altogether, our data indicate that the PSD size relates to spine stability in vivo.

Altered synaptic dynamics during normal brain aging.

What is the neuroanatomical basis for the decline in brain function that occurs during normal aging? Previous postmortem studies have blamed it on a reduction in spine density, though results remain controversial and spine dynamics were not assessed. We used chronic in vivo two-photon imaging of dendritic spines and axonal boutons in somatosensory cortex for up to 1 year in thy1 GFP mice to test the hypothesis that aging is associated with alterations in synaptic dynamics. We find that the density of spines and en passant boutons (EPBs) in pyramidal cells increases throughout adult life but is stable between mature (8-15 months) and old (>20 months) mice. However, new spines and EPBs are two to three times more likely to be stabilized over 30 d in old mice, although the long-term retention (over months) of stable spines is lower in old animals. In old mice, spines are smaller on average but are still able to make synaptic connections regardless of their size, as assessed by serial section electron microscopy reconstructions of previously imaged dendrites. Thus, our data suggest that age-related deficits in sensory perception are not associated with synapse loss in somatosensory cortex (as might be expected) but with alterations in the size and stability of spines and boutons observed in this brain area. The changes we describe here likely result in weaker synapses that are less capable of short-term plasticity in aged individuals, and therefore to less efficient circuits.

Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins.

In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.

Proteomic and electron microscopy survey of large assemblies in macrophage cytoplasm.

Many cellular processes are carried out by large macromolecular assemblies. We systematically analyzed large macromolecular assemblies in the cytoplasm of mouse macrophages (RAW264.7 cell line), cells with crucial roles in immunity and inflammation. Fractionation of the cytoplasmic fraction was performed using sucrose density gradient centrifugation, and individual fractions were subjected in parallel to (i) identification of constituent proteins by mass spectrometry and (ii) structural visualization by electron microscopy. Macromolecular assemblies present in the fractions were analyzed by integrating available data using bioinformatic approaches. We identified 368 unique proteins in our sample. Among these are components of some well-characterized assemblies involved in diverse cellular processes and structures including translation, proteolysis, protein folding, metabolism, and the cytoskeleton, as well as less characterized proteins that may correspond to additional components of known assemblies or other homo- or hetero-oligomeric structures. Single-particle analysis of electron micrographs of negatively stained samples allowed the identification of clearly distinguishable two-dimensional projections of discrete protein assemblies. Among these, we can identify small ribosomal subunits and preribosomal particles, the 26S proteasome complex and small ringlike structures resembling the molecular chaperone complexes. In addition, a broad range of discrete and different complexes were seen at size ranges between 11 to 38 nm in diameter. Our procedure selects the assemblies on the basis of abundance and ease of isolation, and therefore provides an immediately useful starting point for further study of structure and function of large assemblies. Our results will also contribute toward building a molecular cell atlas.

Toxoplasma gondii HOOK-FTS-HIP Complex is Critical for Secretory Organelle Discharge during Motility, Invasion, and Egress.

Members of the Apicomplexa phylum possess specialized secretory organelles that discharge, apically and in a timely regulated manner, key factors implicated in parasite motility, host cell invasion, egress and subversion of host cellular functions. The mechanisms regulating trafficking and apical docking of these secretory organelles are only partially elucidated. Here, we characterized two conserved endosomal trafficking regulators known to promote vesicle transport and/or fusion, HOOK and Fused Toes (FTS), in the context of organelle discharge in Toxoplasma gondii. TgHOOK and TgFTS form a complex with a coccidian-specific partner, named HOOK interacting partner (HIP). TgHOOK displays an apically enriched vesicular pattern and concentrates at the parasite apical tip where it colocalizes with TgFTS and TgHIP. Functional investigations revealed that TgHOOK is dispensable but fitness conferring. The protein regulates the apical positioning and secretion of micronemes and contributes to egress, motility, host cell attachment, and invasion. Conditional depletion of TgFTS or TgHIP impacted on the same processes but led to more severe phenotypes. This study provides evidence of endosomal trafficking regulators involved in the apical exocytosis of micronemes and possibly as a consequence or directly on the discharge of the rhoptries. IMPORTANCE Toxoplasma gondii affects between 30 and 80% of the human population, poses a life-threatening risk to immunocompromised individuals, and is a cause of abortion and birth defects following congenital transmission. T. gondii belongs to the phylum of Apicomplexa characterized by a set of unique apical secretory organelles called the micronemes and rhoptries. Upon host cell recognition, this obligatory intracellular parasite secretes specific effectors contained in micronemes and rhoptries to promote parasite invasion of host cells and subsequent persistence. Here, we identified novel T. gondii endosomal trafficking regulators and demonstrated that they regulate microneme organelle apical positioning and exocytosis, thereby strongly contributing to host cell invasion and parasite virulence.

Stable isotope labeling and ultra-high-resolution NanoSIMS imaging reveal alpha-synuclein-induced changes in neuronal metabolism in vivo.

In Parkinson's disease, pathogenic factors such as the intraneuronal accumulation of the protein α-synuclein affect key metabolic processes. New approaches are required to understand how metabolic dysregulations cause degeneration of vulnerable subtypes of neurons in the brain. Here, we apply correlative electron microscopy and NanoSIMS isotopic imaging to map and quantify 13C enrichments in dopaminergic neurons at the subcellular level after pulse-chase administration of 13C-labeled glucose. To model a condition leading to neurodegeneration in Parkinson's disease, human α-synuclein was unilaterally overexpressed in the substantia nigra of one brain hemisphere in rats. When comparing neurons overexpressing α-synuclein to those located in the control hemisphere, the carbon anabolism and turnover rates revealed metabolic anomalies in specific neuronal compartments and organelles. Overexpression of α-synuclein enhanced the overall carbon turnover in nigral neurons, despite a lower relative incorporation of carbon inside the nucleus. Furthermore, mitochondria and Golgi apparatus showed metabolic defects consistent with the effects of α-synuclein on inter-organellar communication. By revealing changes in the kinetics of carbon anabolism and turnover at the subcellular level, this approach can be used to explore how neurodegeneration unfolds in specific subpopulations of neurons.

A Plasmodium membrane receptor platform integrates cues for egress and invasion in blood forms and activation of transmission stages.

Critical events in the life cycle of malaria-causing parasites depend on cyclic guanosine monophosphate homeostasis by guanylyl cyclases (GCs) and phosphodiesterases, including merozoite egress or invasion of erythrocytes and gametocyte activation. These processes rely on a single GCα, but in the absence of known signaling receptors, how this pathway integrates distinct triggers is unknown. We show that temperature-dependent epistatic interactions between phosphodiesterases counterbalance GCα basal activity preventing gametocyte activation before mosquito blood feed. GCα interacts with two multipass membrane cofactors in schizonts and gametocytes: UGO (unique GC organizer) and SLF (signaling linking factor). While SLF regulates GCα basal activity, UGO is essential for GCα up-regulation in response to natural signals inducing merozoite egress and gametocyte activation. This work identifies a GC membrane receptor platform that senses signals triggering processes specific to an intracellular parasitic lifestyle, including host cell egress and invasion to ensure intraerythrocytic amplification and transmission to mosquitoes.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

A patatin-like phospholipase is important for mitochondrial function in malaria parasites.

Plasmodium parasites rely on a functional electron transport chain (ETC) within their mitochondrion for proliferation, and compounds targeting mitochondrial functions are validated antimalarials. Here, we localize Plasmodium falciparum patatin-like phospholipase 2 (PfPNPLA2, PF3D7_1358000) to the mitochondrion and reveal that disruption of the PfPNPLA2 gene impairs asexual replication. PfPNPLA2-null parasites are hypersensitive to proguanil and inhibitors of the mitochondrial ETC, including atovaquone. In addition, PfPNPLA2-deficient parasites show reduced mitochondrial respiration and reduced mitochondrial membrane potential, indicating that disruption of PfPNPLA2 leads to a defect in the parasite ETC. Lipidomic analysis of the mitochondrial phospholipid cardiolipin (CL) reveals that loss of PfPNPLA2 is associated with a moderate shift toward shorter-chained and more saturated CL species, implying a contribution of PfPNPLA2 to CL remodeling. PfPNPLA2-deficient parasites display profound defects in gametocytogenesis, underlining the importance of a functional mitochondrial ETC during both the asexual and sexual development of the parasite. IMPORTANCE For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, PfPNPLA2, as important for parasite replication and mitochondrial function in Plasmodium falciparum. Parasites lacking PfPNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs. Furthermore, PfPNPLA2-deficient parasites show differences in the composition of their cardiolipins, a unique class of phospholipids with key roles in mitochondrial functions. Finally, we demonstrate that parasites devoid of PfPNPLA2 have a defect in gametocyte maturation, underlining the importance of a functional ETC for parasite transmission to the mosquito vector.

Improving the quality of electron tomography image volumes using pre-reconstruction filtering.

Electron tomography produces highly magnified 3D image volumes useful for investigating the structure and function of cellular components. Image quality is degraded by multiple scattering events and quantum noise, which depend on the angle at which individual tilt projections are collected. We have adapted a biomedical imaging approach to improve image quality by enhancing individual tilt projections prior to volumetric reconstruction. Specifically, we have developed a family of non-linear anisotropic diffusion (NAD) filters parameterized by the tilt angle. We give a quantitative and qualitative evaluation of our pre-processing approach and the NAD filter. We show an improvement in the reconstructed volumes for tomograms generated from both plastic-embedded and cryo-stabilized samples of malaria parasite-infected erythrocytes.

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.