RESUMEN
Kinesins are microtubule (MT)-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes. However, they are poorly studied in the divergent eukaryotic pathogens Plasmodium spp., the causative agents of malaria, which manifest atypical aspects of cell division and plasticity of morphology throughout the life cycle in both mammalian and mosquito hosts. Here, we describe a genome-wide screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 is essential for growth in the mammalian host while the other 8 kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in MT dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria.
Asunto(s)
Culicidae , Malaria , Parásitos , Plasmodium , Animales , Humanos , Cinesinas/genética , Estadios del Ciclo de Vida/genética , Malaria/metabolismo , Mamíferos , Microtúbulos/metabolismo , Plasmodium/genéticaRESUMEN
Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.
Asunto(s)
Axonema/ultraestructura , Gametogénesis/fisiología , Microscopía/métodos , Centro Organizador de los Microtúbulos/ultraestructura , Mitosis/fisiología , Plasmodium/fisiología , Animales , Ratones , Plasmodium/ultraestructuraRESUMEN
Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here, we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to CRL4 in other eukaryotes. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1, and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein important for gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both the schizont and gametocyte stages. WIG1 disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired the formation of the microtubule organization centers that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the formation of microgametes by regulating proteostasis of ciliary and DNA replication proteins.IMPORTANCEPlasmodium parasites undergo fascinating lifecycles with multiple developmental steps, converting into morphologically distinct forms in both their mammalian and mosquito hosts. Protein ubiquitination by ubiquitin ligases emerges as an important post-translational modification required to control multiple developmental stages in Plasmodium. Here, we identify a cullin RING E3 ubiquitin ligase (CRL) complex expressed in the replicating asexual blood stages and in the gametocyte stages that mediate transmission to the mosquito. WIG1, a putative substrate recognition protein of this ligase complex, is essential for the maturation of microgametocytes into microgametes upon ingestion by a mosquito. More specifically, WIG1 is required for proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. This requirement is linked to DNA replication and microtubule organization center formation, both critical to the development of flagellated microgametes.
RESUMEN
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 (SCFFBXO1) 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 SCFFBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.
Asunto(s)
Plasmodium berghei , Humanos , Eritrocitos/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , UbiquitinaciónRESUMEN
Mechanisms of cell division are remarkably diverse, suggesting the underlying molecular networks among eukaryotes differ extensively. The Aurora family of kinases orchestrates the process of chromosome segregation and cytokinesis during cell division through precise spatiotemporal regulation of their catalytic activities by distinct scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes that have three divergent aurora-related kinases (ARKs) and lack most canonical scaffolds/activators. The parasite uses unconventional modes of chromosome segregation during endomitosis and meiosis in sexual transmission stages within mosquito host. This includes a rapid threefold genome replication from 1N to 8N with successive cycles of closed mitosis, spindle formation and chromosome segregation within eight minutes (termed male gametogony). Kinome studies had previously suggested likely essential functions for all three Plasmodium ARKs during asexual mitotic cycles; however, little is known about their location, function, or their scaffolding molecules during unconventional sexual proliferative stages. Using a combination of super-resolution microscopy, mass spectrometry, omics and live-cell fluorescence imaging, we set out to investigate the contribution of the atypical Aurora paralog ARK2 to proliferative sexual stages using rodent malaria model Plasmodium berghei. We find that ARK2 primarily localises to the spindle apparatus associated with kinetochores during both mitosis and meiosis. Interactomics and co-localisation studies reveal a unique ARK2 scaffold at the spindle including the microtubule plus end-binding protein EB1 and lacking some other conserved molecules. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.
RESUMEN
The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.
Asunto(s)
División del Núcleo Celular , Segregación Cromosómica , Animales , Plasmodium berghei/genética , Proliferación Celular , Meiosis , Aurora Quinasas , EucariontesRESUMEN
PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.
Asunto(s)
Estadios del Ciclo de Vida/genética , Meiosis/genética , Mitosis/genética , Plasmodium/crecimiento & desarrollo , Proteína Fosfatasa 1/genética , Proteínas Protozoarias/genética , Proliferación Celular/genética , Malaria/prevención & control , Malaria/transmisión , Mosquitos Vectores/parasitología , Plasmodium/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The assembly of large multimeric complexes in the crowded cytoplasm is challenging. Here we reveal a mechanism that ensures accurate production of the yeast proteasome, involving ribosome pausing and co-translational assembly of Rpt1 and Rpt2. Interaction of nascent Rpt1 and Rpt2 then lifts ribosome pausing. We show that the N-terminal disordered domain of Rpt1 is required to ensure efficient ribosome pausing and association of nascent Rpt1 protein complexes into heavy particles, wherein the nascent protein complexes escape ribosome quality control. Immunofluorescence and in situ hybridization studies indicate that Rpt1- and Rpt2-encoding messenger RNAs co-localize in these particles that contain, and are dependent on, Not1, the scaffold of the Ccr4-Not complex. We refer to these particles as Not1-containing assemblysomes, as they are smaller than and distinct from other RNA granules such as stress granules and GW- or P-bodies. Synthesis of Rpt1 with ribosome pausing and Not1-containing assemblysome induction is conserved from yeast to human cells.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Algoritmos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Genoma Fúngico/genética , Humanos , Hibridación in Situ , Masculino , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.