A helical inner scaffold provides a structural basis for centriole cohesion

Guennec, Maeva Le; Klena, Nikolai; Gambarotto, Davide; Laporte, Marine H.; Tassin, Anne‐Marie; Hoek, Hugo van den; Erdmann, Philipp; Schaffer, Miroslava; Kováčik, Ľubomír; Borgers, Susanne; Goldie, Kenneth N.; Stahlberg, Henning; Bornens, Michel; Azimzadeh, Juliette; Engel, Benjamin D.; Hamel, Virginie; Guichard, Paul

doi:10.1126/sciadv.aaz4137

Cited by 136 publications

(249 citation statements)

References 41 publications

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“…Briefly, coverslips were incubated for 5 hours in 2X 0.7 % AA/ 1% FA mix at 37°C prior gelation in APS/ Temed /Monomer solution (19% Sodium Acrylate; 10% AA; 0,1% BIS-AA in PBS 10X) during 1 hour at 37°C. Then denaturation was performed during 1h30 at 95°C (50). Gels were expanded overnight in water and after shrinking in PBS gels were stained 3 hours at 37°C with primary antibodies against HA-tag (1:200), Ty-Tag (1:2.5), PolyE (1:500) and α-tubulin and β-tubulin (1:200).…”

Section: Methodsmentioning

confidence: 99%

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

Tosetti

Pacheco

Bertiaux

et al. 2020

Preprint

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Toxoplasma 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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Section: Methodsmentioning

confidence: 99%

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

Tosetti

Pacheco

Bertiaux

et al. 2020

Preprint

Self Cite

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“…Cryo-ET has provided insights into the structure and function of viruses [9][10][11][12][92][93][94][95][96][97], nuclear pore complexes [64,[98][99][100], ribosomes [101][102][103], coated vesicles [104][105][106][107][108][109], nucleosome arrangement in chromatin [110], cytoskeletal elements [111][112][113], bacterial secretion systems [114][115][116][117], chemotaxis [118,119], flagellar motors [120], and S-layers [121]. Obtaining information about the spatial arrangement of proteins by cryo-ET has also found use in assessing protein denaturation at the air-buffer interface for single-particle analysis [122].…”

Section: Examplesmentioning

confidence: 99%

The promise and the challenges of cryo‐electron tomography

2020

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Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

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“…Depending on their maturity stage and species, centrioles harbor appendages at their distal end and the cartwheel at their proximal end. The region between proximal and distal ends is the central core, which contains Y-shaped linkers [14,15]. Overview of the anatomy of the centrosome/cilium complex and its sub-compartments.…”

Section: Centrosomesmentioning

confidence: 99%

A Proximity Mapping Journey into the Biology of the Mammalian Centrosome/Cilium Complex

Arslanhan

Gulensoy

Fırat-Karalar

2020

Cells

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The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium and the centriolar satellites, which together regulate cell polarity, signaling, proliferation and motility in cells and thereby development and homeostasis in organisms. Accordingly, deregulation of its structure and functions is implicated in various human diseases including cancer, developmental disorders and neurodegenerative diseases. To better understand these disease connections, the molecular underpinnings of the assembly, maintenance and dynamic adaptations of the centrosome/cilium complex need to be uncovered with exquisite detail. Application of proximity-based labeling methods to the centrosome/cilium complex generated spatial and temporal interaction maps for its components and provided key insights into these questions. In this review, we first describe the structure and cell cycle-linked regulation of the centrosome/cilium complex. Next, we explain the inherent biochemical and temporal limitations in probing the structure and function of the centrosome/cilium complex and describe how proximity-based labeling approaches have addressed them. Finally, we explore current insights into the knowledge we gained from the proximity mapping studies as it pertains to centrosome and cilium biogenesis and systematic characterization of the centrosome, cilium and centriolar satellite interactomes.

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A helical inner scaffold provides a structural basis for centriole cohesion

Cited by 136 publications

References 41 publications

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

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

The promise and the challenges of cryo‐electron tomography

A Proximity Mapping Journey into the Biology of the Mammalian Centrosome/Cilium Complex

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