Plasmepsin X activates the PCRCR complex of Plasmodium falciparum by processing PfRh5 for erythrocyte invasion.

Plasmodium falciparum causes the most severe form of malaria in humans. The protozoan parasite develops within erythrocytes to mature schizonts, that contain more than 16 merozoites, which egress and invade fresh erythrocytes. The aspartic protease plasmepsin X (PMX), processes proteins and proteases essential for merozoite egress from the schizont and invasion of the host erythrocyte, including the leading vaccine candidate PfRh5. PfRh5 is anchored to the merozoite surface through a 5-membered complex (PCRCR), consisting of Plasmodium thrombospondin-related apical merozoite protein, cysteine-rich small secreted protein, Rh5-interacting protein and cysteine-rich protective antigen. Here, we show that PCRCR is processed by PMX in micronemes to remove the N-terminal prodomain of PhRh5 and this activates the function of the complex unmasking a form that can bind basigin on the erythrocyte membrane and mediate merozoite invasion. The ability to activate PCRCR at a specific time in merozoite invasion most likely masks potential deleterious effects of its function until they are required. These results provide an important understanding of the essential role of PMX and the fine regulation of PCRCR function in P. falciparum biology.

PCRCR complex is essential for invasion of human erythrocytes by Plasmodium falciparum.

The most severe form of malaria is caused by Plasmodium falciparum. These parasites invade human erythrocytes, and an essential step in this process involves the ligand PfRh5, which forms a complex with cysteine-rich protective antigen (CyRPA) and PfRh5-interacting protein (PfRipr) (RCR complex) and binds basigin on the host cell. We identified a heteromeric disulfide-linked complex consisting of P. falciparum Plasmodium thrombospondin-related apical merozoite protein (PfPTRAMP) and P. falciparum cysteine-rich small secreted protein (PfCSS) and have shown that it binds RCR to form a pentameric complex, PCRCR. Using P. falciparum lines with conditional knockouts, invasion inhibitory nanobodies to both PfPTRAMP and PfCSS, and lattice light-sheet microscopy, we show that they are essential for merozoite invasion. The PCRCR complex functions to anchor the contact between merozoite and erythrocyte membranes brought together by strong parasite deformations. We solved the structure of nanobody-PfCSS complexes to identify an inhibitory epitope. Our results define the function of the PCRCR complex and identify invasion neutralizing epitopes providing a roadmap for structure-guided development of these proteins for a blood stage malaria vaccine.

RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes.

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

4D analysis of malaria parasite invasion offers insights into erythrocyte membrane remodeling and parasitophorous vacuole formation.

Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens.

Guided STED nanoscopy enables super-resolution imaging of blood stage malaria parasites.

Malaria remains a major burden world-wide, but the disease-causing parasites from the genus Plasmodium are difficult to study in vitro. Owing to the small size of the parasites, subcellular imaging poses a major challenge and the use of super-resolution techniques has been hindered by the parasites' sensitivity to light. This is particularly apparent during the blood-stage of the Plasmodium life cycle, which presents an important target for drug research. The iron-rich food vacuole of the parasite undergoes disintegration when illuminated with high-power lasers such as those required for high resolution in Stimulated Emission Depletion (STED) microscopy. This causes major damage to the sample precluding the use of this super-resolution technique. Here we present guided STED, a novel adaptive illumination (AI) STED approach, which takes advantage of the highly-reflective nature of the iron deposit in the cell to identify the most light-sensitive parts of the sample. Specifically in these parts, the high-power STED laser is deactivated automatically to prevent local damage. Guided STED nanoscopy finally allows super-resolution imaging of the whole Plasmodium life cycle, enabling multicolour imaging of blood-stage malaria parasites with resolutions down to 35 nm without sample destruction.

Plasmepsin V cleaves malaria effector proteins in a distinct endoplasmic reticulum translocation interactome for export to the erythrocyte.

Plasmodium falciparum exports hundreds of virulence proteins within infected erythrocytes, a process that requires cleavage of a pentameric motif called Plasmodium export element or vacuolar transport signal by the endoplasmic reticulum (ER)-resident protease plasmepsin V. We identified plasmepsin V-binding proteins that form a unique interactome required for the translocation of effector cargo into the parasite ER. These interactions are functionally distinct from the Sec61-signal peptidase complex required for the translocation of proteins destined for the classical secretory pathway. This interactome does not involve the signal peptidase (SPC21) and consists of PfSec61, PfSPC25, plasmepsin V and PfSec62, which is an essential component of the post-translational ER translocon. Together, they form a distinct portal for the recognition and translocation of a large subset of Plasmodium export element effector proteins into the ER, thereby remodelling the infected erythrocyte that is required for parasite survival and pathogenesis.

The BTG4 and CAF1 complex prevents the spontaneous activation of eggs by deadenylating maternal mRNAs.

Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang et al. 2015 J. Genet. Genomics 42, 477-485. (doi:10.1016/j.jgg.2015.07.004); Combelles et al. 2011 Hum. Reprod. 26, 545-552. (doi:10.1093/humrep/deq363); Escrich et al. 2011 J. Assist. Reprod. Genet. 28, 111-117. (doi:10.1007/s10815-010-9493-5)). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes (Pfender et al. 2015 Nature 524, 239-242. (doi:10.1038/nature14568)), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4-depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 (Tang et al. 2010 Mol. Biol. Cell 21, 2589-2597. (doi:10.1091/mbc.E09-08-0708); Ohe et al. 2010 Mol. Biol. Cell 21, 905-913. (doi:10.1091/mbc.E09-11-0974)), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4-CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI-MII transition, and this wave of deadenylation is essential for the arrest in metaphase II. These findings establish a BTG4-dependent pathway for controlling poly(A) tail length during meiosis and identify an unexpected role for mRNA deadenylation in preventing the spontaneous activation of eggs.

Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

Heterochromatin reorganization during early mouse development requires a single-stranded noncoding transcript.

The equalization of pericentric heterochromatin from distinct parental origins following fertilization is essential for genome function and development. The recent implication of noncoding transcripts in this process raises questions regarding the connection between RNA and the nuclear organization of distinct chromatin environments. Our study addresses the interrelationship between replication and transcription of the two parental pericentric heterochromatin (PHC) domains and their reorganization during early embryonic development. We demonstrate that the replication of PHC is dispensable for its clustering at the late two-cell stage. In contrast, using parthenogenetic embryos, we show that pericentric transcripts are essential for this reorganization independent of the chromatin marks associated with the PHC domains. Finally, our discovery that only reverse pericentric transcripts are required for both the nuclear reorganization of PHC and development beyond the two-cell stage challenges current views on heterochromatin organization.

Phosphorylation of histone H3 serine 10 in early mouse embryos: active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses.

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B,  are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.