scholarly journals Essential function of the alveolin network in the subpellicular microtubules and conoid assembly in Toxoplasma gondii

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Nicolò Tosetti ◽  
Nicolas Dos Santos Pacheco ◽  
Eloïse Bertiaux ◽  
Bohumil Maco ◽  
Lorène Bournonville ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Toxoplasma Gondii ◽  
Polymer Fibers ◽  
Microtubule Organizing Center ◽  
Polar Ring ◽  
Catastrophic Loss ◽  
Organizing Center ◽  
Infected Cells ◽  
Essential Function ◽  
Apical Complex

The coccidian subgroup of Apicomplexa possesses an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR). The APR serves as microtubule-organizing center for the 22 subpellicular microtubules (SPMTs) that are linked to a patchwork of flattened vesicles, via an intricate network composed of alveolins. Here, we capitalize on ultrastructure expansion microscopy (U-ExM) to localize the Toxoplasma gondii Apical Cap protein 9 (AC9) and its partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Parasites conditionally depleted in AC9 or AC10 replicate normally but are defective in microneme secretion and fail to invade and egress from infected cells. Electron microscopy revealed that the mature parasite mutants are conoidless, while U-ExM highlighted the disorganization of the SPMTs which likely results in the catastrophic loss of APR and conoid.

scholarly journals Two alveolin network proteins are essential for the subpellicular microtubules assembly and conoid anchoring to the apical pole of mature Toxoplasma gondii

2020 ◽  
Author(s):  
Nicolò Tosetti ◽  
Nicolas Dos Santos Pacheco ◽  
Eloïse Bertiaux ◽  
Bohumil Maco ◽  
Lorène Bournonville ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Microtubule Organizing Center ◽  
Membrane Complex ◽  
Catastrophic Loss ◽  
Organizing Center ◽  
Apical Complex ◽  
Critical Components ◽  
Inner Membrane Complex

AbstractToxoplasma gondii belongs to the coccidian sub-group of Apicomplexa that possess an apical complex harboring a conoid, made of unique tubulin polymer fibers. This enigmatic and dynamic organelle extrudes in extracellular invasive parasites and is associated to the apical polar ring (APR), a microtubule-organizing center for the 22 subpellicular microtubules (SPMTs). The SPMTs are linked to the Inner Membrane Complex (IMC), a patchwork of flattened vesicles, via an intricate network of small filaments composed of alveolins proteins. Here, we capitalize on super-resolution techniques including stimulated emission depletion (STED) microscopy and ultrastructure expansion microscopy (U-ExM) to localize the Apical Cap protein 9 (AC9) and its close partner AC10, identified by BioID, to the alveolin network and intercalated between the SPMTs. Conditional depletion of AC9 or AC10 using the Auxin-induced Degron (AiD) system uncovered a severe loss of fitness. Parasites lacking AC9 or AC10 replicate normally but are defective in microneme secretion and hence fail to invade and egress from infected cells. Remarkably, a series of crucial apical complex proteins (MyoH, AKMT, FRM1, CPH1, ICMAP1 and RNG2) are lost in the mature parasites although they are still present in the forming daughter cells. Electron microscopy on intracellular or deoxycholate-extracted parasites revealed that the mature parasite mutants are conoidless. Closer examination of the SPMTs by U-ExM highlighted the disassembly of the SPMTs in the apical cap region that is presumably at the origin of the catastrophic loss of APR and conoid. AC9 and AC10 are two critical components of the alveolin network that ensure the integrity of the whole apical complex in T. gondii and likely other coccidians.

scholarly journals Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii

2017 ◽  
Vol 28 (10) ◽  
pp. 1361-1378 ◽  
Author(s):  
Jacqueline M. Leung ◽  
Yudou He ◽  
Fangliang Zhang ◽  
Yu-Chen Hwang ◽  
Eiji Nagayasu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Integrity ◽  
Lytic Cycle ◽  
Host Cells ◽  
Structural Defects ◽  
Cortical Microtubules ◽  
Microtubule Organizing Center ◽  
Polar Ring ◽  
Human Parasite ◽  
Organizing Center

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

scholarly journals Stability and function of a putative microtubule organizing center in the human parasite Toxoplasma gondii

2017 ◽  
Author(s):  
Jacqueline M. Leung ◽  
Yudou He ◽  
Fangliang Zhang ◽  
Yu-Chen Hwang ◽  
Eiji Nagayasu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Integrity ◽  
Lytic Cycle ◽  
Host Cells ◽  
Structural Defects ◽  
Cortical Microtubules ◽  
Microtubule Organizing Center ◽  
Polar Ring ◽  
Human Parasite ◽  
Organizing Center

ABSTRACTThe organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here, we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and discovered that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites, and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and to invade host cells, as well as decreased secretion of effectors important for these processes. Together, the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

Immunoprobe Localization by Correlative Microscopy

2000 ◽  
Vol 6 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Patricia G. Calarco
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Correlative Microscopy ◽  
Microtubule Organizing Center ◽  
Large Size ◽  
Organizing Center ◽  
Gamma Tubulin ◽  
High Level ◽  
And Function

AbstractMammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

scholarly journals Simultaneous Cell-to-Cell Transmission of Human Immunodeficiency Virus to Multiple Targets through Polysynapses

2009 ◽  
Vol 83 (12) ◽  
pp. 6234-6246 ◽  
Author(s):  
Dominika Rudnicka ◽  
Jérôme Feldmann ◽  
Françoise Porrot ◽  
Steve Wietgrefe ◽  
Stéphanie Guadagnini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Supramolecular Organization ◽  
Microtubule Organizing Center ◽  
Multiple Targets ◽  
Gag Proteins ◽  
Immunodeficiency Virus ◽  
Organizing Center ◽  
Infected Cells ◽  
Virological Synapses ◽  
Viral Transfer

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) efficiently propagates through cell-to-cell contacts, which include virological synapses (VS), filopodia, and nanotubes. Here, we quantified and characterized further these diverse modes of contact in lymphocytes. We report that viral transmission mainly occurs across VS and through “polysynapses,” a rosette-like structure formed between one infected cell and multiple adjacent recipients. Polysynapses are characterized by simultaneous HIV clustering and transfer at multiple membrane regions. HIV Gag proteins often adopt a ring-like supramolecular organization at sites of intercellular contacts and colocalize with CD63 tetraspanin and raft components GM1, Thy-1, and CD59. In donor cells engaged in polysynapses, there is no preferential accumulation of Gag proteins at contact sites facing the microtubule organizing center. The LFA-1 adhesion molecule, known to facilitate viral replication, enhances formation of polysynapses. Altogether, our results reveal an underestimated mode of viral transfer through polysynapses. In HIV-infected individuals, these structures, by promoting concomitant infection of multiple targets in the vicinity of infected cells, may facilitate exponential viral growth and escape from immune responses.

scholarly journals Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex

2021 ◽  
Author(s):  
Dominique Soldati-Favre ◽  
Nicolas Dos Santos Pacheco ◽  
Nicolò Tosetti ◽  
Aarti Krishnan ◽  
Romuald Haase
Keyword(s):  
Toxoplasma Gondii ◽  
Comparative Proteomics ◽  
Host Cells ◽  
Wild Type ◽  
Proteomics Analysis ◽  
Polar Ring ◽  
Intracellular Parasites ◽  
Apical Complex ◽  
Microneme Proteins

Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles.

scholarly journals Visualization of the intracellular behavior of HIV in living cells

2002 ◽  
Vol 159 (3) ◽  
pp. 441-452 ◽  
Author(s):  
David McDonald ◽  
Marie A. Vodicka ◽  
Ginger Lucero ◽  
Tatyana M. Svitkina ◽  
Gary G. Borisy ◽  
...  
Keyword(s):  
Life Cycle ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Infected Cells ◽  
Resolution Imaging ◽  
Transcription Complexes ◽  
Hiv 1

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

Immunoprobe Localization by Correlative Microscopy

2000 ◽  
Vol 6 (3) ◽  
pp. 195-201
Author(s):  
Patricia G. Calarco
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Correlative Microscopy ◽  
Microtubule Organizing Center ◽  
Large Size ◽  
Organizing Center ◽  
Gamma Tubulin ◽  
High Level ◽  
And Function

Abstract Mammalian oocytes present challenges for optimal study by electron microscopy (EM) due to their high level of hydration, their large size, and their relatively undifferentiated cytoplasm. This is particularly true for immunoprobe localization which has led to a dependence on light microscopic (LM) techniques, such as immunofluorescence. This study presents correlative LM and EM data to describe an example of the failure of light microscopy to correctly predict the ultrastructure of one particular organelle. Immunoprobe localization of centrosome and microtubule organizing center (MTOC) antigens in the mammalian egg was made by immunofluorescence and post-embedding immuno-EM, with best EM results achieved in Lowicryl-embedded material. Centrosome and MTOC antigens were detected by 5051 and an antibody to gamma tubulin (γtubulin). Gamma tubulin is a highly conserved element of MTOCs in many species and, thus, is highly diagnostic for them; it is also considered essential for microtubule (MT) nucleation. Results indicate that prior to nuclear breakdown, 5051 antigens and γ-tubulin are found exclusively in a type of “organelle,” the multivesicular aggregate (MVA), that bears no resemblance to MTOCs at the ultrastructural level. Until recently, the MVA was considered an organelle without a known function, while standard MTOCs were presumed to be the entities that carry the proteins recognized by centrosome antibodies. LM localization of centrosomal antigens carried the presumption that standard MTOCs were the entities labeled. Whether or not other molecules are shown to co-localize to these MVA, the presence of γ-tubulin supports the contention that MVA, or their contents, serve as centrosomal precursors with a unique ultrastructure. Thus, dependence on LM techniques alone can lead to erroneous conclusions on organelle identity and function.

scholarly journals Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs.

1984 ◽  
Vol 98 (5) ◽  
pp. 1730-1745 ◽  
Author(s):  
E Karsenti ◽  
J Newport ◽  
R Hubble ◽  
M Kirschner
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Microtubule Organizing Center ◽  
Designed Experiments ◽  
Microtubule Organizing Centers ◽  
Organizing Center ◽  
Subsequent Activation

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.

Fine structure of zoosporulation in Perkinsus atlanticus (Apicomplexa: Perkinsea)

Parasitology ◽  
1990 ◽  
Vol 100 (3) ◽  
pp. 351-358 ◽  
Author(s):  
C. Azevedo ◽  
L. Corral ◽  
R. Cachola
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Incubation Period ◽  
Developmental Stages ◽  
The Other ◽  
Polar Ring ◽  
Transmission Electron ◽  
Apical Complex

SUMMARYLight and transmission electron microscopy were used to study different stages of Perkinsus atlanticus (Apicomplexa) during induced zoosporulation, with fluid thioglycollate medium and seawater. Cytokinesis and nucleokinesis of different developmental stages were studied every 12 h during the incubation period of 72 h, at which time the zoospores became free. Uninucleated and flagellated zoospores present the apical complex formed by conoid, polar ring, micronemes, rhoptries and subpellicular microtubules observed at different sections. Ultrastructural details were compared with the other two species of the genus Perkinsus.

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