Biology subjects

Brochet, M.

Publications and source records attributed to Brochet, M..

16 recordsLinked to original sources

bioRxiv2026

DCX enables branching of subpellicular microtubules in Plasmodium falciparum gametocytes and is required for mosquito colonisation

Plasmodium falciparum, the causative agent of malaria, relies on specialised tubulin-based cytoskeletal structures to support its parasitic lifestyle. These include the conoid required for parasite motility and host-cell invasion, as well as subpellicular microtubules (SPMTs) that support parasite shape and rigidity. Here, we investigate the function of the doublecortin-domain protein DCX, a microtubule-binding protein previously associated with the Plasmodium conoid. We first show that, in P. falciparum, DCX is not expressed in the merozoite stage and is not required for the invasion of human erythrocytes. By contrast, DCX is expressed in ookinetes, the motile stage responsible for infecting the mosquito vector, where it associates with conoid tubulin fibres, consistent with a role in stabilising the conoid architecture. Unexpectedly, we find that DCX is required for P. falciparum transmission to the mosquito independently of conoid function. We further link this requirement to the distinctive organisation of SPMTs in P. falciparum gametocytes, which display an unusual branching architecture comprising multiple microtubules of 15 to 18 protofilaments. Deletion of DCX leads to a reduction in SPMT branching and is associated with higher protofilament numbers, revealing a previously unrecognised role for DCX in shaping the ultrastructure of SPMTs in P. falciparum gametocytes. Altogether, our findings uncover the repurposing of DCX across distinct microtubule systems in transmission stages and identify DCX as a key factor mediating microtubule branching and stabilisation in SPMTs required for efficient mosquito transmission.

microbiology↗

bioRxiv2025

Targeting ligand binding sites in Plasmodium falciparum NCR1 enables antimalarial drug discovery

PfNCR1 is a Plasmodium falciparum cholesterol transporter at the plasma membrane-parasitophorous vacuole interface, which has recently emerged as a promising antimalarial target. Despite an immense interest in development of novel antimalarial compounds targeting PfNCR1, the molecular mechanism of PfNCR1 inhibition remains elusive. Here, we report cryo-EM structures of PfNCR1 in its apo state and bound to three inhibitors: MMV009108, MMV019662 and MMV028038. MMV009108 binds to the ''neck'' site at the ectodomain-membrane domain inter-face. MMV028038 displaces the sterol at the ectodomain ''ecto'' site. Remarkably, MMV019662 binds both sites: it associates near the bound sterol molecule at the ecto site and targets the neck site, thereby altering the sterol-sensing domain conformation. Importantly, we identify a novel antimalarial compound, G856-4236, which targets the ecto site exclusively. These four distinct modes of PfNCR1 inhibition advance our understanding of its conformational plasticity and es-tablish a framework for rational drug discovery targeting PfNCR1 and related transporters.

biochemistry↗

bioRxiv2025

A Modular Chromosomal Passenger Complex Rewires Chromosome Segregation in Plasmodium berghei

Faithful chromosome segregation relies on precise kinetochore-microtubule interactions and checkpoint surveillance, yet the molecular basis of these processes varies widely across eukaryotes and is only beginning to be defined in apicomplexan parasites. In the malaria parasite Plasmodium berghei, chromosome segregation is especially critical during transmission from host to mosquito: rapid mitoses generate male gametes, and subsequent meiosis in the zygote seeds the next generation of infection. Here, we identify Aurora-related kinase 1 (ARK1) as a central regulator of chromosome segregation in both mitotic and meiotic contexts. ARK1 localises to spindle poles, spindles, and kinetochores, and its depletion results in short and multipolar spindles, kinetochore misalignment, and failed chromosome partitioning. ARK1 forms a minimal Chromosomal Passenger Complex (CPC) with INCENP1 during male gametogenesis, but a more elaborate CPC with INCENP2, kinetochores, centromeric histones, and spindle assembly checkpoint proteins during meiosis. This stage-specific modularity allows Plasmodium to prioritise male gamete formation whilst safeguarding faithful chromosome inheritance during zygote development, ensuring parasite transmission to the mosquito. Our findings demonstrate that conserved CPC principles are rewired in Plasmodium, highlighting both the plasticity of eukaryotic checkpoint control and a potential vulnerability for blocking malaria transmission.

microbiology↗

bioRxiv2024

Erythrocyte membrane protein 3 (EMAP3) is exposed on the surface of the Plasmodium berghei infected red blood cell

The human malaria parasite Plasmodium falciparum invades red blood cells (RBC) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate uptake of nutrients and cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, thus protecting the iRBC from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to in vivo studies. P. berghei iRBC also sequesters in small blood vessels, mediated by binding to CD36. However, the parasite proteins binding to CD36 are unknown and only very few parasite proteins, including EMAP1 and EMAP2, have been identified that are present at the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not the major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen in vivo putative inhibitors of P. falciparum cytoadherence.

microbiology↗

bioRxiv2024

Plasmodium NEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp, the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within eight minutes followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified four Nek genes, of which two, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM) and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation and kinetochore attachment. These data suggest that Plasmodium berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.

cell biology↗

bioRxiv2023

A multistage Plasmodium CRL4-WIG1 ubiquitin ligase is critical for the formation of functional microtubule organisation centres in microgametocytes

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 the eukaryotic CRL4. 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 schizont and gametocyte stages. While WIG1 is not required for the proliferation of P. berghei asexual blood stages, its 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 formation of the microtubule organisation centres 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 transmission of malaria parasites by regulating proteostasis of ciliary and DNA replication proteins.

microbiology↗

bioRxiv2023

Plasmodium ARK2-EB1 axis drives the unconventional spindle dynamics, scaffold formation, and chromosome segregation of sexual transmission stages

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, and live-cell fluorescence imaging, we set out to investigate the role 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 in the vicinity of 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, lacking conserved Aurora scaffold proteins. Gene function studies indicate complementary functions of ARK2 and EB1 in driving endomitotic divisions and thereby parasite transmission. Our discovery of a novel Aurora kinase spindle scaffold underlines the emerging flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite Plasmodium.

cell biology↗

bioRxiv2022

An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin levels. Disruption of microtubule homeostasis caused polymerization of excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed spindle extension. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome.

cell biology↗

bioRxiv2022

Conoid extrusion serves as gatekeeper for entry of glideosome components into the pellicular space to control motility and invasion in Apicomplexa

Members of the Apicomplexa are defined by apical cytoskeletal structures and secretory or-ganelles, tailored for motility and invasion. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In Toxoplasma gondii, the conoid, composed of a cone of spiraling tubulin fibers and apposed preconoidal rings (PCRs), is an enigmatic, dynamic organelle of undefined function. Here we mapped five new components of the PCRs and deduce that the structure serves as a pivotal hub for actin polymerization and glideosome assembly. F-actin produced by Formin1 on the PCRs is used by Myosin H to generate the force for conoid extrusion. A set of B-box-type zinc finger domain containing proteins conserved in Apicomplexa is indispensable for PCRs formation, conoid extrusion and motility in Toxoplasma and Plasmodium. Conoid dynamics directs the flux of F-actin to the pellicular space, acting as dynamic gatekeeper to tightly control parasite motility during invasion and egress.

microbiology↗

bioRxiv2021

Organization and composition of apicomplexan kinetochores reveals plasticity in chromosome segregation across parasite modes of division

Kinetochores are multiprotein assemblies directing mitotic spindle attachment and chromosome segregation. In apicomplexan parasites, most known kinetochore components and associated regulators are apparently missing, suggesting a minimal structure with limited control over chromosome segregation. In this study, we use interactomics combined with deep homology searches to identify six divergent eukaryotic components, in addition to a set of eight apicomplexan kinetochore proteins (AKiTs) that bear no detectable sequence similarity to known proteins. The nanoscale organization of the apicomplexan kinetochore includes four subdomains, each displaying different evolutionary rates across the phylum. Functional analyses confirm AKiTs are essential for mitosis and reveal architectures parallel to biorientation at metaphase. Furthermore, we identify a homolog of MAD1 at the apicomplexan kinetochore, suggesting conserved spindle assembly checkpoint signaling. Finally, we show unexpected plasticity in kinetochore composition and segregation throughout the parasite lifecycle, indicating diverse requirements to maintain fidelity of chromosome segregation across apicomplexan modes of division.

microbiology↗

bioRxiv2021

Ultrastructure expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a microtubule organisation centre coordinating mitosis with axoneme assembly

The transmission of malaria-causing parasites to mosquitoes relies on the production of the gametocyte stages and their development into gametes upon a blood feed. 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 and its associated actin cytoskeleton, which confer cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it simultaneously assembles axonemes to form eight flagellated microgametes. Here, U-ExM combined with Pan-ExM revealed the molecular architecture of a single bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking acentriolar mitotic plaques to cytoplasmic basal bodies by proteinaceous filaments. The eight basal bodies are concomitantly de novo assembled from a deuterosome-like structure, where centrin, {gamma}-tubulin, SAS4/CPAP and SAS6 form distinct subdomains. Once assembled, the basal bodies show a fusion of the proximal and central cores where colocalised centrin and SAS6 are surrounded by a SAS4/CPAP-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of {gamma}-tubulin from the basal bodies to the acentriolar plaques. We finally show that this atypical MTOC architecture relies on two non-canonical MTOC 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 formation with mitosis.

microbiology↗

bioRxiv2021

Location and function of all Plasmodium kinesins: key roles in parasite proliferation, polarity, and transmission

Kinesins are microtubule-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 lifecycle 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 eight 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 microtubule dynamics during apical cell polarity formation, spindle assembly, and axoneme biogenesis. These findings help us to understand the importance of microtubule motors and may be exploited to discover new therapeutic interventions against malaria.

cell biology↗

bioRxiv2021

Protein Phosphatase 1 regulates atypical chromosome segregation and cell polarity during mitotic and meiotic division in Plasmodium sexual stages

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.

cell biology↗

bioRxiv2020

A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in both Plasmodium falciparum asexual blood stages and P. berghei gametocytes. Phosphoproteomic analyses in both Plasmodium species reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion of P. berghei ICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss of P. falciparum ICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

microbiology↗

bioRxiv2020

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of Plasmodium berghei during gametogony and parasite 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.

microbiology↗

bioRxiv2017

Sub-minute phosphoregulation of cell-cycle systems during Plasmodium gamete formation revealed by a high-resolution time course

Malaria parasites are protists of the genus Plasmodium, whose transmission to mosquitoes is initiated by the production of gametes. Male gametogenesis is an extremely rapid process that is tightly controlled to produce eight flagellated microgametes from a single haploid gametocyte within 10 minutes after ingestion by a mosquito. Regulation of the cell cycle is poorly understood in divergent eukaryotes like Plasmodium, where the highly synchronous response of gametocytes to defined chemical and physical stimuli from the mosquito has proved to be a powerful model to identify specific phosphorylation events critical for cell-cycle progression. To reveal the wider network of phosphorylation signalling in a systematic and unbiased manner, we have measured a high-resolution time course of the phosphoproteome of P. berghei gametocytes during the first minute of gametogenesis. The data show an extremely broad response in which distinct cell-cycle events such as initiation of DNA replication and mitosis are rapidly induced and simultaneously regulated. We identify several protein kinases and phosphatases that are likely central in the gametogenesis signalling pathway and validate our analysis by investigating the phosphoproteomes of mutants in two of them, CDPK4 and SRPK1. We show these protein kinases to have distinct influences over the phosphorylation of similar downstream targets that are consistent with their distinct cellular functions, which is revealed by a detailed phenotypic analysis of an SRPK1 mutant. Together, the results show that key cell-cycle systems in Plasmodium undergo simultaneous and rapid phosphoregulation. We demonstrate how a highly resolved time-course of dynamic phosphorylation events can generate deep insights into the unusual cell biology of a divergent eukaryote, which serves as a model for an important group of human pathogens.

microbiology↗