KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum ‐infected erythrocytes

  1. Cecilia P. Sanchez
  2. Pintu Patra
  3. Shih‐Ying Scott Chang
  4. Christos Karathanasis
  5. Lukas Hanebutte
  6. Nicole Kilian
  7. Marek Cyrklaff
  8. Mike Heilemann
  9. Ulrich S. Schwarz
  10. Mikhail Kudryashev
  11. Michael Lanzer

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Abstract

The knob‐associated histidine‐rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite‐encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super‐resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP‐targeting nanoprobe binds close to the spiral scaffold. Single‐molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

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  1. Version published to 10.1111/mmi.14811
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    Reply to the reviewers

    Responses to reviewers’ comments

    We thank the reviewers for their encouraging comments and helpful suggestions.

    Reviewer #1

    (Evidence, reproducibility and clarity (Required)):

    Sanchez et al report several new findings about the adhesive protrusions on Plasmodium falciparum infected erythrocytes. Using super resolution microscopy and correlation analysis, they tracked associations between the knob protein KAHRP and erythrocyte membrane cytoskeleton proteins. They have expanded on and improved previous work on the unusual spiral structure of the knobs, which appears to be a spiral ribbon or blade and have shown a developmental pathway for the association of KAHRP with the cytoskeleton. They have localised KAHRP close to the spiral and determined its abundance in the knobs. They have also used cryo electron tomography and subtomogram averaging to get an improved 3D view of the knob structure.

    The work appears to be carefully and thoroughly done, and the paper is clearly written, though non specialists in the optical methods may find it challenging to navigate through the many super resolution images and correlation plots.

    Comment 1: The writing needs minor editing to fix a variety of small linguistic errors and typos. For example, line 97 "sideway positions" (they presumably mean lateral location), line 980 typo overlay, line 366 "then could reorganizes", line 435, "a predict volume".

    We apologize for the linguistic errors and typos. These have been corrected in the revised manuscript.

    (Significance (Required)):

    Comment 2: The study provides a distinct advance on the previous state of knowledge of the structure and biochemistry of the knobs. The knobs play a key role in virulence of P. falciparum and they are quite poorly understood. Although this paper does not represent a major breakthrough in determining the molecular structure or mechanistic role of the knobs, e.g. the biochemical identity of the spiral remains unknown, the new information is valuable and likely to be important in understanding the pathogenic actions of P falciparum.

    We thank the reviewer for appreciating the importance of our study. We believe that our first-time observations on the dynamics of KAHRP are a very important advance in the field and that revealing the mechanistic basis is a great challenge that at the current stage has to be left to future work.

    Comment 3: The interpretation shown in Figure 7 seems fine, except for the proposal that the actin cytoskeleton is reorganised. There is no evidence for that. The cryo tomograms of the cytoskeleton in Watermeyer et al addressed this point and did not find any evidence for reorganisation of the cytoskeleton other than the insertion of the knobs.

    In two previous studies we could show that actin is indeed reorganized by the parasite. It is mined from the protofilaments to generate long actin filaments that connect the knobs with the Maurer’s clefts and which are used for trafficking of cargo vesicles from the Maurer’s clefts to the erythrocyte plasma membrane (Cyklaff et al. Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes. Science. 2011 334:1283-1286; Cyrklaff et al. Oxidative insult can induce malaria-protective trait of sickle and fetal erythrocytes. Nat Commun. 2016 7:13401). Moreover, a life-cycle resolved AFM-study of the cytoplasmic side of iRBCs by the group of CT Lim has demonstrated dramatic coarsening of the spectrin network, which must be accompanied by changes to the actin component of the skeleton (Shi, Hui, et al. "Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes." PLoS One 8.4 (2013): e61170). Coarsening of the actin-spectrin network would imply a decrease of the amount of actin in the network, which is consistent with its use in the parasite-derived long actin filaments.

    **Referee Cross-commenting**

    I also agree with the other 2.

    Reviewer #2

    (Evidence, reproducibility and clarity (Required)):

    Malaria parasites replicate within circulating red blood cells (RBC). During parasite maturation, the parasite coordinates extensive modification of the host cell, including structural modifications of the RBC cytoskeleton and surface membrane. These host cell alterations play crucial roles in the pathology of malaria, including vascular adhesion by parasitised cells and avoidance of splenic clearance, and so are of great interest. This interesting manuscript describes a detailed examination of the role in these RBC modifications of a well-described parasite protein called KAHRP. Using a combination of cutting-edge super-resolution microscopy, cryo-electron tomography, immuno-EM, SEM and parasite mutagenesis, the authors provide evidence that KHARP localisation alters during parasite maturation but eventually becomes closely associated with the previously-described spiral structures that underlie infected RBC surface membrane protrusions called knobs. The authors provide improved resolution of the spiral formations, generate a quantitative estimate of the number of KAHRP molecules per knob, and provide a model for the role of KAHRP in attaching other proteins to the spirals based on their observations.

    In general, this study is thorough and well-performed, and the conclusions drawn are well-supported by the data. Although the work does not advance understanding of knob function or the parasite components that form the bulk of the spirals, it provides an interesting and useful contribution to understanding of the manner in which this important pathogen manipulates its host cell.

    We thank the reviewer for appreciating the importance of our study and in acknowledging that it is an important intermediate step towards a complete understanding of skeleton remodelling by the parasite.

    I have just a few minor suggestions that should improve the manuscript.

    Comment 1: Line 91 (Page 2 paragraph 2). It would be greatly helpful here if the authors could provide a more detailed background on the makeup of the RBC cytoskeleton, and in particular the interactions between beta-spectrin and the actin protofilaments of the junctional complexes. The authors should make it clear that the actin-binding domain of beta-spectrin comprises 2 calponin like domains, and that these are attached to the end of the tandem spectrin repeat domains that make up the bulk of the molecule.

    We thank the reviewer for this helpful suggestion and have added a new paragraph to the results section providing detailed background information on the makeup of the RBC membrane skeleton. The new text reads as follows:

    “Major components of the red blood cell membrane skeleton are spectrin and actin filaments (Fig. 1B). The spectrin filaments consist of α- and ß-spectrin, which form α2ß2 heterotetramers by head-to-head association of two αß dimers (Lux, 2016; Machnicka et al., 2014). The N-termini of the ß-spectrin subunits are positioned at the tail ends of the heterotetramer and contain two calponin homology (CH) domains for binding to actin protofilaments consisting of 6 to 8 actin monomers in each of the two strands (Lux, 2016; Machnicka et al., 2014). Protein 4.1R strengthens the spectrin actin interaction (Lux, 2016; Machnicka et al., 2014). Groups of up to six spectrin heterotetramers can attach to an actin protofilaments, resulting in a pseudohexagonal meshwork (Lux, 2016). Ankyrin binds to the C-terminal domain of ß-spectrin and connects integral membrane proteins with the actin spectrin network in an ankyrin complex (Lux, 2016; Machnicka et al., 2014).”

    Comment 2: Line 97 "These values are slightly larger than the reported physical dimension of the protofilament...". Please provide these reported dimensions here, as well as relevant references.

    The requested information is now provided. The sentence now reads as follows:

    “These values are slightly larger than the reported physical dimension of the protofilaments of ~37 nm (Lux, 2016) and might be explained by the lateral localization of the spectrin binding sites and the additional sizes of the primary and secondary antibody trees used to detect the two targets.”

    Comment 3: Line 366 "reorganize"

    The spelling mistake has been corrected.

    (Significance (Required)):

    Comment 4: This is a useful technical advance in understanding of the structure of the P. falciparum-infected red blood cell, and builds on the work of Watermeyer et al. (2016). The study should certainly be of interest to most malaria researchers, particularly those interested in the pathobiology of the organism.

    We thank the reviewer for supporting our study.

    **Referee Cross-commenting***

    I fully agree with and endorse the comments of the other 2 reviewers.

    Reviewer #3

    (Evidence, reproducibility and clarity (Required)):

    The binding of P. falciparum infected erythrocyte (iRBCs) to the endothelium is mediated by protuberances (knobs). These knobs are assembled by a multi-protein complex at the iRBC surface. It acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). The knob-associated histidine-rich protein (KAHRP) is an essential component of the knobs and therefore essential for the binding of iRBC to the endothelium under physiological conditions. This manuscript focusses on the knob architecture and KAHRP localization.*

    Comment 1: It is, at least for this reviewer - hard to assess how the "preparation of exposed membranes by hypotonic shock" and the analysis of the "inverted erythrocyte membrane ghosts" is i) reflective of the physiological architecture within the iRBC and ii) how the authors exclude remnants from Maurers clefts (MCs) in their preparation. The latter appears especially important for the interpretation of dynamic KAHRP repositioning, as MCs are mobile in early stages and non-mobile later on (e.g. McMilian et al. 2013, Grüring et al. 2011) and the authors observed at least some MAHRP1 signal (Figure S8), which is hard to interpret by the single representative image provided.

    We understand the reviewer’s concerns, but are convinced that we have done the necessary controls to evaluate our approaches. For example, we evaluated the exposed membrane approach by investigating uninfected erythrocytes and comparing the findings with literature reports (see Figure 1). A high degree of agreement was observed. We further would like to point out that the exposed membrane approach has been successfully used by several other studies referenced in the manuscript (Dearnley et al., 2016; Looker et al., 2019; Shi et al., 2013). Please also allow us to explain why we have used exposed membranes instead of whole cells. The reason is that the hemozoin produced by the parasite interferes with STED microscopy, resulting in a quick and strong build-up of resonance energy in the specimen and, eventually, in the disruption of the cell.

    With regard to the question of whether remnants of Maurer’s clefts are present in our preparations, we do not think so, at least we never observed membrane profiles reminiscent of Maurer’s clefts in SEM images of exposed membranes (see figure at the end of the response letter). Irrespectively, we will double check this result using STED imaging of exposed membranes treated with an antibody against the established Maurer’s clefts marker SBP1. These data could be added to a revised manuscript.

    Comment 2: line 173: Please provide a detailed description about parasite synchronization (also absent in the methods section).

    A detailed description including references are now added to the methods section:

    “For synchronization of cultures, schizont-infected erythrocytes were sterile purified using a strong magnet (VarioMACS, Miltenyi Biotec) (Staalsoe et al., 1999) and mixed with fresh erythrocytes to high parasitaemia. 5000 heparin units (Heparin-sodium 25000, Ratiopharm) were added and the cells were returned to culture for 4 hrs (Boyle et al., 2010). Following the treatment with heparin, cells were washed with pre-warmed supplemented RMPI 1640 medium and then returned to culture for 2 hrs to allow for re-invasion of erythrocytes. Subsequently, cells were treated with 5% sorbitol to remove late parasite stages (Lambros and Vanderberg, 1979).”

    Comment 3: line 136: Please re-check nomenclature of "PHIS1605w" (mixed nomenclature used throughout the manuscript). I suggest to use either LyMP or the up-to-date ID PF3D7_0532400.

    We apologize for the oversight and now consistently use the ID PF3D7_0532400.

    Comment 4: Please provide source and references for PfEMP1, MAHRP1 and "PHIS1605w" antibodies that are used. I cannot find them in the methods section or in Table S1.

    We apologize for the oversight and now provide the requested information in the amended Table S1.

    Comment 5: line 165: Warncke et al. (2016) appears to be misplaced as an appropriate MAHRP1 reference.

    We now cite the original MAHRP1 publication by Spycher et al. 2003.

    Comment 6: line 159: the sentence "The strong cross-correlation between KAHRP and actin is consistent with previous cryo-electron tomographic analysis showing long actin filaments connecting the knobs with Maurer's clefts in trophozoites (Cyrklaff et al., 2012; Cyrklaff et al., 2011; Cyrklaff et al., 2016)" could be moved to the discussion section.

    The sentence was indeed redundant with a section in the discussion and was removed.

    Comment 7: line 199: The text refers to Fig. 9AB - but should refer to 4AB or suppl. 11.

    We are sorry for this mistake and now refer to the correct figures in the revised manuscript.

    *Comment 8: Fig. 4: A solid average for the number of subtomograms, but please provide information about what the arrowheads (4E) indicate. *

    Thank you for this comment. The arrowheads indicate peripheral crown-like densities. We have updated the figure legend to clarify this issue.

    Comment 9: The "flexible periphery" is likely a combination of flexibility and occupancy as the average was made from subtomograms with varying number of turns in the spiral. As occupancy is likely a significant contributing factor to the average that should be discussed or at least mentioned.

    Thank you for this important comment. Indeed, a significant variation was observed between the individual knobs. The spirals have variable diameter, and the number of peripheral proteins also varied. We added measurements to the supplementary figure 11D. In addition, we update the text and extended the discussion.

    Comment 10. On that note, did the authors try and classify based on number of turns prior to averaging and if so did the authors see any differences in structures between few turn and many turn spirals?

    We attempted several classifications on the full knobs with variable masks. However due to a limited number of particles in the dataset we could not converge to stable solutions. Instead, we decided to adopt the subboxing strategy where locally ordered segments at the periphery could be analyzed. This showed several structural snapshot at the periphery of the knobs.

    Comment 11. What size mask was used? Was it a soft sphere around the core or big enough for the knobs with multiple spiral turns?

    While we attempted several alignments and classifications with variable masks, the final refinement and measurement of FSC was performed with a soft contour mask mask. We overlaid it with the structure in Figure S11F and uploaded it as a part of the EMDB deposition. We further show the masks used in this study in a new Figure S14.

    Comment 12. It might be useful for readers who are not familiar with Dynamo to provide a little bit more information about how the initial reference was produced. Additionally more information about the sub-boxing strategy ie: spacing etc. would helpful.

    Thank you very much for the suggestion. For the initial reference we manually aligned all the particles, summed them up and low-pass filtered them. We now describe it in the methods section.

    For the subboxing procedure we added more description to the main text:

    “40 segments were extracted at the radius of the 2nd and 3rd spirals followed by their classification into structural classes.”

    We further extended and simplified the description in the results section (line ~221).

    Comment 13: Fig. 5 Additional (earlier) maturation stages of the iRBC with Ni2+NAT-gold-labelling would be a nice add on - this could help confirm the model and would itself be a control for the later stage labelling.

    We thank the reviewer for this insightful suggestion. We are currently performing the proposed experiment and will include it in a revised version of the manuscript.

    Comment 14: line 637: DMSI typo and please provide the supplier for DMSI (DSM1).

    We corrected the typographic error and now provide the name of the supplier.

    Comment 15: Figure 7: Please provide what the purple arrows indicate.

    The figure legend has been updated.

    Comment 16: Fig S11D: The labels X, Y and Z are confusing, describing the slicing axis as "XZ, YZ and XY" view is more intuitive.

    Done as suggested by the reviewer.

    Comment 17: Figure S13 B: WBs are cropped. Please provide un-cropped WB.

    Uncropped Western blots will be provided in the revised manuscript.

    (Significance (Required)):

    In general, I highly appreciated the solid data and its thorough analysis of the microscopy data. The authors investigate the structural organization of knobs in iRBCs using high-resolution imaging techniques including STED and PALM super-resolution microscopy-based approaches and electron tomography. The beauty of this paper is that it does nicely re-investigate knob architecture in iRBC (e.g. Watermeyer et al., 2016, Cutts et al., 2017, Looker et al., 2019, McHugh et al., 2020) and provides some intriguing KHARP co-localization with cytoskeleton components. The downside of it is that - by nature - it is descriptive (and the data rather confirmative) and as it stands does not provide us with a deeper molecular dissection of the knob associated structure and its cellular function.

    We thank the reviewer for appreciating our study and would like to emphasize the following novelties in our study:

    • We show that the association of KAHRP with membrane skeletal components is highly dynamic and changes as the parasite matures. Our results on the dynamics of KAHRP organization reconciles conflicting reports in the literature, and establish for the first time a dynamical model for KAHRP organization.
    • We further show that KAHRP finally assembles at remnant actin-junctional complexes devoid of the actin-capping factors adducin and tropomodulin.
    • We further quantified the number of KAHRP molecules per knob and show that KAHRP is present as 60 copies per knob, a number one order of magnitude greater than previously thought.
    • Last but not least, we provide a 35 Å map of the spiral scaffold underlaying knobs and show that KAHRP associates with the spiral scaffold.
    • We conclude by providing a novel model on the biological function of KAHRP by proposing that KAHRP acts as a glue that connects spectrin and parasite-remodeled actin filaments with the knob spiral.

    **Referee Cross-commenting**

    Fully agreed.

    Boyle, M.J., Wilson, D.W., Richards, J.S., Riglar, D.T., Tetteh, K.K., Conway, D.J., Ralph, S.A., Baum, J., and Beeson, J.G. (2010). Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc Natl Acad Sci U S A* 107*, 14378-14383.

    Lambros, C., and Vanderberg, J.P. (1979). Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol* 65*, 418-420.

    Lux, S.E.t. (2016). Anatomy of the red cell membrane skeleton: unanswered questions. Blood* 127*, 187-199.

    Staalsoe, T., Giha, H.A., Dodoo, D., Theander, T.G., and Hviid, L. (1999). Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry* 35*, 329-336.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The binding of P. falciparum infected erythrocyte (iRBCs) to the endothelium is mediated by protuberances (knobs). These knobs are assembled by a multi-protein complex at the iRBC surface. It acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). The knob-associated histidine-rich protein (KAHRP) is an essential component of the knobs and therefore essential for the binding of iRBC to the endothelium under physiological conditions. This manuscript focusses on the knob architecture and KAHRP localization.

    General point:

    It is, at least for this reviewer - hard to assess how the "preparation of exposed membranes by hypotonic shock" and the analysis of the "inverted erythrocyte membrane ghosts" is i) reflective of the physiological architecture within the iRBC and ii) how the authors exclude remnants from Maurers clefts (MCs) in their preparation. The latter appears especially important for the interpretation of dynamic KAHRP repositioning, as MCs are mobile in early stages and non-mobile later on (e.g. McMilian et al. 2013, Grüring et al. 2011) and the authors observed at least some MAHRP1 signal (Figure S8), which is hard to interpret by the single representative image provided.

    Specific points:

    • line 173: Please provide a detailed description about parasite synchronization (also absent in the methods section)
    • line 136: Please re-check nomenclature of "PHIS1605w" (mixed nomenclature used throughout the manuscript). I suggest to use either LyMP or the up-to-date ID PF3D7_0532400.
    • Please provide source and references for PfEMP1, MAHRP1 and "PHIS1605w" antibodies that are used. I cannot find them in the methods section or in Table S1 -line 165: Warncke et al. (2016) appears to be misplaced as an appropriate MAHRP1 reference.
    • line 159: the sentence "The strong cross-correlation between KAHRP and actin is consistent with previous cryo-electron tomographic analysis showing long actin filaments connecting the knobs with Maurer's clefts in trophozoites (Cyrklaff et al., 2012; Cyrklaff et al., 2011; Cyrklaff et al., 2016)" could be moved to the discussion section
    • line 199: The text refers to Fig. 9AB - but should refer to 4AB or suppl. 11.
    • Fig. 4: A solid average for the number of subtomograms, but please provide information about what the arrowheads (4E) indicate. A few additional comments on this section:

    1: The "flexible periphery" is likely a combination of flexibility and occupancy as the average was made from subtomograms with varying number of turns in the spiral. As occupancy is likely a significant contributing factor to the average that should be discussed or at least mentioned.

    1. On that note, did the authors try and classify based on number of turns prior to averaging and if so did the authors see any differences in structures between few turn and many turn spirals?
    2. What size mask was used? Was it a soft sphere around the core or big enough for the knobs with multiple spiral turns?
    3. It might be useful for readers who are not familiar with Dynamo to provide a little bit more information about how the initial reference was produced. Additionally more information about the sub-boxing strategy ie: spacing etc. would helpful.
    • Fig. 5 Additional (earlier) maturation stages of the iRBC with Ni2+NAT-gold-labelling would be a nice add on - this could help confirm the model and would itself be a control for the later stage labelling.
    • line 637: DMSI typo and please provide the supplier for DMSI (DSM1).
    • Figure 7: Please provide what the purple arrows indicate.
    • Fig S11D: The labels X, Y and Z are confusing, describing the slicing axis as "XZ, YZ and XY" view is more intuitive.
    • Figure S13 B: WBs are cropped. Please provide un-cropped WB.

    Significance

    In general, I highly appreciated the solid data and its thorough analysis of the microscopy data. The authors investigate the structural organization of knobs in iRBCs using high-resolution imaging techniques including STED and PALM super-resolution microscopy-based approaches and electron tomography. The beauty of this paper is that it does nicely re-investigate knob architecture in iRBC (e.g. Watermeyer et al., 2016, Cutts et al., 2017, Looker et al., 2019, McHugh et al., 2020) and provides some intriguing KHARP co-localization with cytoskeleton components. The downside of it is that - by nature - it is descriptive (and the data rather confirmative) and as it stands does not provide us with a deeper molecular dissection of the knob associated structure and its cellular function.

    Referee Cross-commenting

    Fully agreed.

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    Referee #2

    Evidence, reproducibility and clarity

    Malaria parasites replicate within circulating red blood cells (RBC). During parasite maturation, the parasite coordinates extensive modification of the host cell, including structural modifications of the RBC cytoskeleton and surface membrane. These host cell alterations play crucial roles in the pathology of malaria, including vascular adhesion by parasitised cells and avoidance of splenic clearance, and so are of great interest. This interesting manuscript describes a detailed examination of the role in these RBC modifications of a well-described parasite protein called KAHRP. Using a combination of cutting-edge super-resolution microscopy, cryo-electron tomography, immuno-EM, SEM and parasite mutagenesis, the authors provide evidence that KHARP localisation alters during parasite maturation but eventually becomes closely associated with the previously-described spiral structures that underlie infected RBC surface membrane protrusions called knobs. The authors provide improved resolution of the spiral formations, generate a quantitative estimate of the number of KAHRP molecules per knob, and provide a model for the role of KAHRP in attaching other proteins to the spirals based on their observations. In general, this study is thorough and well-performed, and the conclusions drawn are well-supported by the data. Although the work does not advance understanding of knob function or the parasite components that form the bulk of the spirals, it provides an interesting and useful contribution to understanding of the manner in which this important pathogen manipulates its host cell.

    I have just a few minor suggestions that should improve the manuscript.

    Line 91 (Page 2 paragraph 2). It would be greatly helpful here if the authors could provide a more detailed background on the makeup of the RBC cytoskeleton, and in particular the interactions between beta-spectrin and the actin protofilaments of the junctional complexes. The authors should make it clear that the actin-binding domain of beta-spectrin comprises 2 calponin like domains, and that these are attached to the end of the tandem spectrin repeat domains that make up the bulk of the molecule.

    Line 97 "These values are slightly larger than the reported physical dimension of the protofilament...". Please provide these reported dimensions here, as well as relevant references.

    Line 366 "reorganize"

    Significance

    This is a useful technical advance in understanding of the structure of the P. falciparum-infected red blood cell, and builds on the work of Watermeyer et al. (2016). The study should certainly be of interest to most malaria researchers, particularly those interested in the pathobiology of the organism.

    Referee Cross-commenting

    I fully agree with and endorse the comments of the other 2 reviewers.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Sanchez et al report several new findings about the adhesive protrusions on Plasmodium falciparum infected erythrocytes. Using super resolution microscopy and correlation analysis, they tracked associations between the knob protein KAHRP and erythrocyte membrane cytoskeleton proteins. They have expanded on and improved previous work on the unusual spiral structure of the knobs, which appears to be a spiral ribbon or blade) and have shown a developmental pathway for the association of KAHRP with the cytoskeleton. They have localised KAHRP close to the spiral and determined its abundance in the knobs.

    They have also used cryo electron tomography and subtomogram averaging to get an improved 3D view of the knob structure.

    The work appears to be carefully and thoroughly done, and the paper is clearly written, though non specialists in the optical methods may find it challenging to navigate through the many super resolution images and correlation plots. The writing needs minor editing to fix a variety of small linguistic errors and typos. For example, line 97 "sideway positions" (they presumably mean lateral location), line 980 typo overlay, line 366 "then could reorganizes", line 435, "a predict volume".

    Significance

    The study provides a distinct advance on the previous state of knowledge of the structure and biochemistry of the knobs. The knobs play a key role in virulence of P. falciparum and they are quite poorly understood. Although this paper does not represent a major breakthrough in determining the molecular structure or mechanistic role of the knobs, e.g. the biochemical identity of the spiral remains unknown, the new information is valuable and likely to be important in understanding the pathogenic actions of P falciparum.

    The interpretation shown in Figure 7 seems fine, except for the proposal that the actin cytoskeleton is reorganised. There is no evidence for that. The cryo tomograms of the cytoskeleton in Watermeyer et al addressed this point and did not find any evidence for reorganisation of the cytoskeleton other than the insertion of the knobs.

    I am generally familiar with the area of this work, but not expert in the details of the optical methods and localisation analysis.

    Referee Cross-commenting

    I also agree with the other 2.

  6. Version published to 10.1101/2021.05.12.443751v1 on bioRxiv