La plupart des cellules de mammifères ont la capacité d’assembler un ou plusieurs cils au cours du cycle cellulaire. Les cils immobiles, dont les cils primaires, participent à de nombreux processus sensoriels, alors que les cils mobiles sont essentiellement impliqués dans le déplacement cellulaire et la mise en mouvement de fluides extracellulaires. La longue liste de maladies dues à des défauts ciliaires met en exergue l’importance fonctionnelle de ces structures. Ces ciliopathies sont caractérisées par une impressionnante diversité de symptômes, et une étiologie génétique souvent complexe. La connaissance précise de la biologie des cils et flagelles s’avère donc essentielle pour la compréhension de ces maladies. Ces organites sont remarquablement conservés au cours de l’évolution eucaryote. Dans cette revue, nous illustrons l’importance de l’utilisation d’organismes modèles appropriés pour l’étude de divers aspects de la biologie des cils et flagelles : composition moléculaire, mode d’assemblage, mais aussi fonctions sensorielles et de motilité. Des études pionnières menées sur l’algue verte Chlamydomonas ont établi le lien entre les cils et certaines maladies génétiques. De plus, des organismes multicellulaires tels la souris, le poisson zèbre, le xénope, le nématode C. elegans ou la drosophile, ainsi que des protistes comme Paramecium, Tetrahymena et Trypanosoma ou Leishmania offrent chacun des atouts spécifiques pour l’étude de la biologie du cil. En particulier, des études fonctionnelles menées chez le trypanosome ont permis de caractériser la fonction de gènes impliqués dans les dyskinésies ciliaires primitives, une ciliopathie due à un défaut de mobilité des cils.
A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer's clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer's clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer's clefts was established for two of them. This suggests a clustering of genes encoding Maurer's cleft proteins. Our study sheds new light on the biological function of the Maurer's clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.
Background Information Eukaryotic cilia and flagella are sophisticated organelles composed of several hundreds of proteins that need to be incorporated at the right time and the right place during assembly. Results Two methods were used to investigate this process in the model protist Trypanosoma brucei : inducible expression of epitope‐tagged labelled proteins and fluorescence recovery after photobleaching of fluorescent fusion proteins. This revealed that skeletal components of the radial spokes (RSP3), the central pair (PF16) and the outer dynein arms (DNAI1) are incorporated at the distal end of the growing flagellum. They display low or even no visible turnover in mature flagella, a finding further confirmed by monitoring a heavy chain of the outer dynein arm. In contrast, the membrane‐associated protein arginine kinase 3 (AK3) showed rapid turnover in both growing and mature flagella, without particular polarity and independently of intraflagellar transport. Conclusion These results demonstrate different modes of incorporation for structural and membrane‐associated proteins in flagella. Significance The existence of two distinct modes for incorporation of proteins in growing flagella suggests the existence of different targeting machineries. Moreover, the absence of turnover of structural elements supports the view that the length of the mature flagellum in trypanosomes is not modified after assembly.
ABSTRACT Sporozoite forms of the malaria parasite Plasmodium are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection. IMPORTANCE Malaria is caused by Plasmodium parasites and remains one of the deadliest parasitic diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first invades the liver for an initial, obligatory and silent round of replication. The liver infection is an attractive target for antimalarial vaccine strategies, however the molecular mechanisms of parasite invasion of hepatocytes remain to be fully elucidated. Two hepatocyte surface proteins are known to be important for parasite entry into hepatocytes, the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1). These receptors constitute independent gateways depending on the Plasmodium species. Here, we identified the structural determinants of SR-B1, an important hepatocyte entry factor for human-infecting P. vivax . This study paves the way toward a better characterization of the molecular interactions underlying the crucial early stages of infection, a pre-requisite for the development of novel malaria vaccine strategies.
Cilia and flagella are complex organelles composed of up to 500 proteins. We have purified intact flagella from the model organism Trypanosoma brucei using mechanical shearing. Scanning and transmission electron microscopy confirmed the quality and the purity of flagella and biochemical analysis demonstrated a 15-fold enrichment of flagellar markers. Mass spectrometry investigation carried out on 5 separate experiments led to the identification of 387 proteins, 55 of which had never reported to be associated to the flagellum. 10 out of the 12 proteins investigated experimentally were indeed associated to the flagellum but turned out to localise to several sub-localisations with unique profiles: flagellar membrane, axoneme, paraflagellar rod (an extra-axonemal structure) and the adhesion zone. Two of them, termed FLAMM6 and FLAMM8 showed restricted distribution to the proximal part and to the far distal end of the axoneme, respectively. Dynamics analysis revealed that membrane proteins were incorporated by the proximal end and showed a rapid turnover whereas axonemal and PFR proteins were added to the distal end of elongating flagella but showed stable association to their structure. FLAMM6 was found only in the first half of the flagellum no matter its length, a process dependent on IFT. Finally, FLAMM8 was progressively incorporated to the elongating axoneme accumulating at the distal tip where it showed very slow turnover after flagellum formation was complete. These data highlight the existence of specific micro-domains within the eukaryotic flagellum, each with its own dynamics for assembly and turnover.
