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

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.

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

eLife ◽

10.7554/elife.56635 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

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.

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The apical annuli of Toxoplasma gondii are composed of coiled-coil and signaling proteins embedded in the IMC sutures

10.1101/711432 ◽

2019 ◽

Cited By ~ 1

Author(s):

Klemens Engelberg ◽

Chun-Ti Chen ◽

Tyler Bechtel ◽

Victoria Sánchez Guzmán ◽

Allison A. Drozda ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Coiled Coil ◽

Super Resolution ◽

Building Blocks ◽

Signaling Proteins ◽

Waste Products ◽

Membrane Complex ◽

Inner Membrane Complex

AbstractThe apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

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Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii

European Journal of Cell Biology ◽

10.1016/j.ejcb.2020.151149 ◽

2021 ◽

Vol 100 (2) ◽

pp. 151149

Author(s):

Rikako Konishi ◽

Yuna Kurokawa ◽

Kanna Tomioku ◽

Tatsunori Masatani ◽

Xuenan Xuan ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Inner Membrane ◽

Membrane Complex ◽

Inner Membrane Complex

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Toxoplasma gondii myosins B/C

The Journal of Cell Biology ◽

10.1083/jcb.200012116 ◽

2001 ◽

Vol 155 (4) ◽

pp. 613-624 ◽

Cited By ~ 66

Author(s):

Frédéric Delbac ◽

Astrid Sänger ◽

Eva M. Neuhaus ◽

Rolf Stratmann ◽

James W. Ajioka ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Daughter Cell ◽

Gliding Motility ◽

Apicomplexan Parasites ◽

Daughter Cells ◽

Membrane Complex ◽

Alternatively Spliced ◽

Butanedione Monoxime ◽

Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

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Super-resolution microscopy: successful applications in centrosome study and beyond

Biophysics Reports ◽

10.1007/s41048-019-00101-x ◽

2019 ◽

Vol 5 (5-6) ◽

pp. 235-243 ◽

Cited By ~ 1

Author(s):

Jingyan Fu ◽

Chuanmao Zhang

Keyword(s):

Molecular Basis ◽

Super Resolution ◽

Diffraction Limit ◽

Animal Cells ◽

Microtubule Organizing Center ◽

The Past ◽

Eukaryotic Species ◽

Organizing Center ◽

Cilia And Flagella ◽

Super Resolution Microscopy

AbstractCentrosome is the main microtubule-organizing center in most animal cells. Its core structure, centriole, also assembles cilia and flagella that have important sensing and motility functions. Centrosome has long been recognized as a highly conserved organelle in eukaryotic species. Through electron microscopy, its ultrastructure was revealed to contain a beautiful nine-symmetrical core 60 years ago, yet its molecular basis has only been unraveled in the past two decades. The emergence of super-resolution microscopy allows us to explore the insides of a centrosome, which is smaller than the diffraction limit of light. Super-resolution microscopy also enables the compartmentation of centrosome proteins into different zones and the identification of their molecular interactions and functions. This paper compiles the centrosome architecture knowledge that has been revealed in recent years and highlights the power of several super-resolution techniques.

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Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

The Journal of Cell Biology ◽

10.1083/jcb.200311137 ◽

2004 ◽

Vol 165 (3) ◽

pp. 383-393 ◽

Cited By ~ 185

Author(s):

Elizabeth Gaskins ◽

Stacey Gilk ◽

Nicolette DeVore ◽

Tara Mann ◽

Gary Ward ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Complex ◽

Integral Membrane Protein ◽

Gliding Motility ◽

Host Cells ◽

Inner Membrane ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

Inner Membrane Complex ◽

Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

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Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

PLoS Pathogens ◽

10.1371/journal.ppat.1005403 ◽

2016 ◽

Vol 12 (2) ◽

pp. e1005403 ◽

Cited By ~ 33

Author(s):

Clare R. Harding ◽

Saskia Egarter ◽

Matthew Gow ◽

Elena Jiménez-Ruiz ◽

David J. P. Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Inner Membrane ◽

Membrane Complex ◽

Associated Proteins ◽

Inner Membrane Complex

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Enrichment and biochemical characterization of Toxoplasma gondii tachyzoite plasmalemma

Parasitology ◽

10.1017/s0031182096008670 ◽

1997 ◽

Vol 114 (5) ◽

pp. 421-426 ◽

Cited By ~ 5

Author(s):

A. RABJEAU ◽

F. FOUSSARD ◽

G. MAURAS ◽

J. F. DUBREMETZ

Keyword(s):

Toxoplasma Gondii ◽

Plasma Membranes ◽

Biochemical Characterization ◽

Surface Membrane ◽

Surface Protein ◽

Protozoan Parasite ◽

Membrane Complex ◽

Inner Membrane Complex ◽

Partial Dissociation ◽

Major Surface

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0·7M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS–PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20–100 nm) which possess a low density (1·085–1·090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1·12–1·16 g/cm3).

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

Molecular Biology of the Cell ◽

10.1091/mbc.e17-01-0045 ◽

2017 ◽

Vol 28 (10) ◽

pp. 1361-1378 ◽

Cited By ~ 20

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.

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