A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication

Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission

Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility

Toxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro. TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in a ku80-ko strain reveals no vital function for tachyzoite propagation in vitro, which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity

The Skp1-Cullin1-FBXO1 complex is a pleiotropic regulator required for the formation of gametes and motile forms in Plasmodium berghei

Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium . Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCF FBXO1 ) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCF FBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts

Anti-CD20 therapeutic antibody rituximab modifies the functional organization of rafts/microdomains of B lymphoma cells

Incubation of Burkitt lymphoma-derived Raji cells at physiological temperature with submicromolar concentrations of humanized anti-CD20 antibody rituximab (RTX) redistributes CD20 to liquid-ordered, plasma membrane rafts. This accumulation of the CD20 tetraspan protein in rafts does not change the existing lipid and phosphoprotein composition but makes sphingolipids and the Src regulator Cbp/PAG (Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomain) transmembrane phosphoprotein more resistant to n-octyl-beta-pyranoside, a detergent that dissociates sphingolipid clusters. On the contrary, sphingolipids and Cbp/PAG are not protected by the presence of CD20 against the disruptive effects of methyl-beta-cyclodextrin, a cyclic carbohydrate that removes membrane cholesterol. After accumulation of CD20, the activity of the raft-associated Lyn kinase is down-regulated without apparent alteration of its relationship to substrates. Moreover, in rafts of lymphoblastoid cells that express lower amounts of Cbp/PAG, RTX redistributes CD20 to rafts but does not modulate the raft-associated protein tyrosine kinase activity, suggesting that the presence of Cbp/PAG protein in rafts is necessary for RTX to exert its transmembrane "signaling effects." Lastly, redistribution of CD20 in rafts renders the glycosylphosphatidyl inositol (GPI)-linked CD55 C'-defense protein hypersensitive to glycosylphosphatidyl inositol-specific phospholipases. By redistributing CD20 to rafts, RTX modifies their stability and organization and modulates the associated signaling pathways and C' defense capacity

Multiple short windows of Calcium-Dependent Protein Kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis

Malaria transmission relies on the production of gametes following ingestion by a mosquito. Here, we show that (Ca2+)-dependent protein kinase 4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by (Ca2+) to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. Altogether, this study reveals how a kinase integrates and transduces multiple signals to control key cell-cycle transitions during Plasmodium gametogenesis

Multiple short windows of Calcium-Dependent Protein Kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis

Malaria transmission relies on the production of gametes following ingestion by a mosquito. Here, we show that (Ca2+)-dependent protein kinase 4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by (Ca2+) to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. Altogether, this study reveals how a kinase integrates and transduces multiple signals to control key cell-cycle transitions during Plasmodium gametogenesis

Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein

Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (ARO) and myosin F (MyoF). Here, we show that two ARO partners, ARO-interacting protein (AIP) and adenylate cyclase β (ACβ) localize to a rhoptry subcompartment. In absence of AIP, ACβ disappears from the rhoptries. By assessing the contribution of each ARO armadillo (ARM) repeat, we provide evidence that ARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that ARO resembles the myosin-binding domain of the Caenorhabditis elegans myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative MyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Finally, Plasmodium falciparum ARO functionally complements ARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa

Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein (TgARO)

Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (TgARO) and myosin F (TgMyoF). Here, we show that two TgARO partners, ARO interacting protein (TgAIP) and adenylate cyclase β (TgACβ) localize to a rhoptry subcompartment. In absence of TgAIP, TgACβ disappears from the rhoptries. By assessing the contribution of each TgARO armadillo (ARM) repeat, we provide evidence that TgARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that TgARO resembles the myosin-binding domain of the myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative TgMyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Lastly, Plasmodium falciparum ARO functionally complements TgARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa