Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Reviewer #1 (Public Review):

This paper consists in a comprehensive analysis of the malaria parasite Plasmodium falciparum during its development in erythrocytes, using expansion microscopy. The authors used general dyes to stain membranes or proteins and a set of specific markers to label diverse cellular structures of the parasite, with a particular focus on the microtubule organizing center (MTOC).

This is by nature a purely descriptive study, providing remarkable images with great details on subcellular structures such as the MTOC, the basal complex, the cytostome and rhoptries. The work is extremely well performed and the images are beautiful. It confirms a number of previous observations, but does not bring much novel biological insights. However, the study illustrates the strength of expansion microscopy, an affordable and adaptable sample preparation method that will undoubtedly become standard in the field.

While the narrative could be improved, this study provides a valuable resource that can serve as a reference dataset for analysis of P. falciparum and other apicomplexan parasites.

Reviewer #2 (Public Review):

In this work the authors describe the shape and interconnectedness of intracellular structures of malaria blood stage parasites by taking advantage of expansion microscopy. Compared to previous microscopy work with these parasites, the strength of this paper lies in the increased resolution and the fact that the NHE ester highlights protein densities. Together with the BodipyC membrane staining, this results in data that is somewhere in between EM and standard fluorescence microscopy: it has higher resolution than standard fluorescence microscopy and provides some points of reference of different cellular structures due to the NHE ester/BodipyC.

This study makes many interesting and useful observations and although it is somewhat "old school descriptory" in its presentation, researchers working in many different areas will find something of interest here. This ranges from mitosis, to organisation and distribution of major cellular structures, endocytosis and invasion, overall providing a rich and interesting resource. The results section is long but by taking the space to explain everything in detail, it has the advantage that it clearly transpires how things were done and on how many cells a conclusion is based on. Further the authors often also included a brief interpretation of their findings with a very open assessment what it does and what it does not show, highlighting interesting questions left by the data.

Overall this is a very nice and useful paper that will be of interest to many, particularly those working on nuclear division, cytokinesis, endocytosis or invasion in malaria parasites. The spatiotemporal arrangement and interconnection of subcellular structures will also give a framework for specific functional studies.

Reviewer #3 (Public Review):

Summary:

In their study the authors analyze the localization of multiple organelles and subcellular structure of blood stage malaria parasites with unprecedented detail. They use a 3D super-resolution imaging technique that has gained popularity in the protozoan field, ultrastructure expansion microscopy. Building on markers and labels established in the field they generate an appealing collection of images for all stages of the intraerythrocytic developmental stages of asexual blood stage parasites with some focus on nuclear division and cell segmentation stages.

Strengths:

The authors generated an impressive amount of imaging data that presents the most comprehensive analysis of ultrastructural organization of the parasite cell so far. This atlas can serve as a reference for researchers studying the cell biology of the intraerythrocytic development cycle. The authors achieve a nice catalogue of the reorganization of well-established markers, which together with the improved resolution allows them to highlight some novel observations and consolidate previous findings. They e.g. improve our understanding of organization, duplication and constitutive tethering of the malaria parasite centrosome to the plasma membrane. Further they provide some interesting observations on rhoptry biogenesis, cytostome morphology, and organelle fission during segmentation.

Weaknesses:

While the comprehensiveness of the study is its strength the authors do not present any novel markers, stainings, or imaging protocols. There is no fundamentally new mechanistic insight derived from this study although some earlier findings are consolidated by the higher spatial resolution.

In the following I want to comment on some major points.

Most importantly, in order to justify the authors claim to provide an "Atlas", I want to strongly suggest they share their raw 3D-imaging data (at least of the main figures) in a data repository. This would allow the readers to browse their structure of interest in 3D and significantly improve the impact of their study in the malaria cell biology field.

The organization of the manuscript can be improved. Aside some obvious modifications as citing the figures in the correct order (see also further comments and recommendations), I would maybe suggest one subsection and one figure per analyzed cellular structure/organelle (i.e. 13 sections). This would in my opinion improve readability and facilitate "browsing the atlas".

Considering the importance of reliability of the U-ExM protocol for this study the authors should provide some validation for the isotropic expansion of the sample e.g. by measuring one well defined cellular structure.

In the absence of time-resolved data and more in-depth mechanistic analysis the authors must down tone some of their conclusions specifically around mitochondrial membrane potential, supellicular microtubule depolymerization, and kinetics of the basal complex. More detailed suggestions for improvement are provided as further comments.

In conclusion the authors provide an exciting cell biological reference framework and new working hypotheses about the function of some subcellular structures, which are still largely enigmatic in the malaria parasite, and can be investigated in the future.