RESUMEN
Eukaryotic cilia and flagella are essential for cell motility and sensory functions. Their biogenesis and maintenance rely on the intraflagellar transport (IFT). Several cargo adapters have been identified to aid IFT cargo transport, but how ciliary cargos are discharged from the IFT remains largely unknown. During our explorations of small GTPases ARL13 and ARL3 in Trypanosoma brucei, we found that ODA16, a known IFT cargo adapter present exclusively in motile cilia, is a specific effector of ARL3. In the cilia, active ARL3 GTPases bind to ODA16 and dissociate ODA16 from the IFT complex. Depletion of ARL3 GTPases stabilizes ODA16 interaction with the IFT, leading to ODA16 accumulation in cilia and defects in axonemal assembly. The interactions between human ODA16 homolog HsDAW1 and ARL GTPases are conserved, and these interactions are altered in HsDAW1 disease variants. These findings revealed a conserved function of ARL GTPases in IFT transport of motile ciliary components, and a mechanism of cargo unloading from the IFT.
Asunto(s)
Factores de Ribosilacion-ADP , Cilios , Trypanosoma brucei brucei , Cilios/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Humanos , Trypanosoma brucei brucei/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Flagelos/metabolismo , Unión Proteica , Transporte Biológico , Transporte de Proteínas , Axonema/metabolismoRESUMEN
Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.
Asunto(s)
Malaria , Parásitos , Plasmodium , Toxoplasma , Animales , Plasmodium falciparum/metabolismo , Tomografía con Microscopio Electrónico , Proteínas Protozoarias/metabolismo , Parásitos/metabolismo , Interacciones Huésped-Parásitos , Toxoplasma/metabolismoRESUMEN
Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.
RESUMEN
The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: (1) Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. (2) Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.
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Herpesvirus Humano 1 , Herpesvirus Suido 1 , Animales , Transporte Axonal/fisiología , Axones/metabolismo , Herpesvirus Humano 1/fisiología , NeuronasRESUMEN
Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.
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Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Citoesqueleto/metabolismo , Tomografía con Microscopio Electrónico/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Orgánulos/metabolismoRESUMEN
Electron cryotomography is currently the only method capable of visualizing cells in three dimensions at nanometer resolutions. While modern instruments produce massive amounts of tomography data containing extremely rich structural information, data processing is very labor intensive and the results are often limited by the skills of the personnel rather than the data. We present an integrated workflow that covers the entire tomography data processing pipeline, from automated tilt series alignment to subnanometer resolution subtomogram averaging. Resolution enhancement is made possible through the use of per-particle per-tilt contrast transfer function correction and alignment. The workflow greatly reduces human bias, increases throughput and more closely approaches data-limited resolution for subtomogram averaging in both purified macromolecules and cells.
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Microscopía por Crioelectrón/métodos , Procesamiento Automatizado de Datos/métodos , Flujo de Trabajo , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Membrana Celular/virología , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/virología , Virus Chikungunya/fisiología , Liberación del Virus , Línea Celular , Membrana Celular/inmunología , Virus Chikungunya/genética , Humanos , Replicación ViralRESUMEN
In the unicellular parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, complex swimming behavior is driven by a flagellum laterally attached to the long and slender cell body. Using microfluidic assays, we demonstrated that T. brucei can penetrate through an orifice smaller than its maximum diameter. Efficient motility and penetration depend on active flagellar beating. To understand how active beating of the flagellum affects the cell body, we genetically engineered T. brucei to produce anucleate cytoplasts (zoids and minis) with different flagellar attachment configurations and different swimming behaviors. We used cryo-electron tomography (cryo-ET) to visualize zoids and minis vitrified in different motility states. We showed that flagellar wave patterns reflective of their motility states are coupled to cytoskeleton deformation. Based on these observations, we propose a mechanism for how flagellum beating can deform the cell body via a flexible connection between the flagellar axoneme and the cell body. This mechanism may be critical for T. brucei to disseminate in its host through size-limiting barriers.
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Citoesqueleto , Flagelos , Trypanosoma brucei brucei , Microscopía por Crioelectrón , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Flagelos/metabolismo , Flagelos/ultraestructura , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Cellular electron cryotomography offers researchers the ability to observe macromolecules frozen in action in situ, but a primary challenge with this technique is identifying molecular components within the crowded cellular environment. We introduce a method that uses neural networks to dramatically reduce the time and human effort required for subcellular annotation and feature extraction. Subsequent subtomogram classification and averaging yield in situ structures of molecular components of interest. The method is available in the EMAN2.2 software package.
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Criopreservación , Cianobacterias/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Redes Neurales de la Computación , Programas InformáticosRESUMEN
African trypanosomes have a single, membrane-bounded flagellum that is attached to the cell cortex by membrane adhesion proteins and an intracellular flagellum attachment zone (FAZ) complex. The coordinated assembly of flagellum and FAZ, during the cell cycle and the life cycle development, plays a pivotal role in organelle positioning, cell division and cell morphogenesis. To understand how the flagellum and FAZ assembly are coordinated, we examined the domain organization of the flagellum adhesion protein 1 (FLA1), a glycosylated, transmembrane protein essential for flagellum attachment and cell division. By immunoprecipitation of a FLA1-truncation mutant that mislocalized to the flagellum, a novel FLA1-binding protein (FLA1BP) was identified in procyclic Trypanosoma brucei. The interaction between FLA1 on the cell membrane and FLA1BP on the flagellum membrane acts like a molecular zipper, joining flagellum membrane to cell membrane and linking flagellum biogenesis to FAZ elongation. By coordinating flagellum and FAZ assembly during the cell cycle, morphology information is transmitted from the flagellum to the cell body.