Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii
- The Francis Crick Institute, United Kingdom
- University of Vermont, United States
- University of Connecticut, United States
Abstract
Toxoplasma gondii contains a limited subset of actin binding proteins. Here we show that the putative actin regulator cyclase-associated protein (CAP) is present in two different isoforms and its deletion leads to significant defects in some but not all actin dependent processes. We observe defects in cell-cell communication, daughter cell orientation and the juxtanuclear accumulation of actin, but only modest defects in synchronicity of division and no defect in the replication of the apicoplast. 3D electron microscopy reveals that loss of CAP results in a defect in formation of a normal central residual body, but parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for F-actin regulation in Toxoplasma which appear to be achieved by partially overlapping functions of actin regulators.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 4, 5, 6, 8 and 10. Data Availability: Raw data for FIB SEM supporting movies will be uploaded to EMPIAR. Raw data for FIB SEM supporting movies have been deposited to EMPIAR.
Article and author information
Author details
Matthew Robert Geoffrey Russell
Funding
Francis Crick Institute (FC001189)
- Lucy Collinson
NIH Office of the Director (AI121885)
- Romain Carmeille
- Aoife Heaslip
NIH Office of the Director (AI139201)
- Robyn Kent
- Gary E Ward
Francis Crick Institute (FC001999)
- Matthew Robert Geoffrey Russell
- Christopher J Peddie
- Lucy Collinson
NIH Office of the Director (AI137767)
- Robyn Kent
- Gary E Ward
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experiments were performed in accordance with UK Home Office regulations (PPL 80/2616) and approved by the ethical review panel at the Francis Crick Institute.
Copyright
© 2019, Hunt et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Differential requirements for cyclase-associated protein (CAP) in actin-dependent processes of Toxoplasma gondii
eLife 8:e50598.
https://doi.org/10.7554/eLife.50598
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
