Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast

Leandro Lemgruber,Chaitali Dekiwadia,David T Riglar,Stuart A Ralph,Mikhail Kudryashev,Friedrich Frischknecht,Henning Stahlberg,Jake Baum

doi:10.1186/1475-2875-12-25

Abstract

BackgroundThe apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes.MethodsCryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology.ResultsCryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls.ConclusionsThe Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.

Highlights

The apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites
All extant plastids are believed to derive from a single primary endosymbiosis event, secondary endosymbiosis has occurred a number of times, giving rise to multiple, unrelated plastid-bearing groups
For P. falciparum merozoites to be subject to conventional glutaraldehyde and osmium fixation or for high pressure freezing and freeze substitution, purification was performed as described by Boyle and colleagues [27]

Summary

Methods

Ethics statement All animal experiments were performed according to the FELASA and GV-SOLAS standard guidelines. For P. falciparum merozoites to be subject to conventional glutaraldehyde and osmium fixation or for high pressure freezing and freeze substitution (see below), purification was performed as described by Boyle and colleagues [27]. High-pressure freezing and freeze substitution Purified P. falciparum merozoites from strain D10 were allowed to invade red blood cells, 10 min post invasion were high-pressure frozen using a LeicaEM highpressure freezer. Conventional transmission electron microscopy Purified P. falciparum merozoites (as described above) were fixed in 2.5% glutaraldehyde in pH 7.4 phosphate buffer on ice. Cells were washed in phosphate buffer and fixed in 1% osmium tetroxide (ProSciTech, Australia) in 0.1 M phosphate buffer for one hour on ice. Samples were washed in water, dehydrated in increasing concentrations of ethanol before embedding in LR Gold resin (ProSciTech, Australia) and polymerized using benzoyl peroxide (SPI-Chem, USA).

Results

Conclusions

Background

Results and discussion

Conclusion

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