PtdIns(3,4)P2, Lamellipodin, and VASP coordinate actin dynamics during phagocytosis in macrophages

  1. Fernando Montaño-Rendón
  2. Glenn F.W. Walpole
  3. Matthias Krause
  4. Gerald R.V. Hammond
  5. Sergio Grinstein
  6. Gregory D. Fairn

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Abstract

Phosphoinositides are pivotal regulators of vesicular traffic and signaling during phagocytosis. Phagosome formation, the initial step of the process, is characterized by local membrane remodeling and reorganization of the actin cytoskeleton that leads to formation of the pseudopods that drive particle engulfment. Using genetically encoded fluorescent probes, we found that upon particle engagement a localized pool of PtdIns(3,4)P2 is generated by the sequential activities of class I phosphoinositide 3-kinases and phosphoinositide 5-phosphatases. Depletion of this locally generated pool of PtdIns(3,4)P2 blocks pseudopod progression and ultimately phagocytosis. We show that the PtdIns(3,4)P2 effector Lamellipodin (Lpd) is recruited to nascent phagosomes by PtdIns(3,4)P2. Furthermore, we show that silencing of Lpd inhibits phagocytosis and produces aberrant pseudopodia with disorganized actin filaments. Finally, vasodilator-stimulated phosphoprotein (VASP) was identified as a key actin-regulatory protein mediating phagosome formation downstream of Lpd. Mechanistically, our findings imply that a pathway involving PtdIns(3,4)P2, Lpd, and VASP mediates phagocytosis at the stage of particle engulfment.

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  1. Version published to 10.1083/jcb.202207042
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    Response to previous review

    Reviewer____ #1 (Evidence, reproducibility and clarity (Required)):

    The authors use newly available probes to show that the phosphoinositide PI(3,4)P2 plays a previously undescribed role in FcgammaR-mediated phagocytosis. Using RAW macrophages, they show that PI(3,4)P2 is enriched at the plasma membrane and also at phagocytic cups internalizing IgG-opsonized sheep red blood cells. Pharmacological inhibition using wortmannin, and also expression of membrane-targeted INPP4B phosphatase showed that PI(3,4)P2 production depends on PI3 kinase activity. Further experiments using selective inhibitors showed that PI(3,4)5P2 is mainly derived by dephosphorylation of PI(3,4,5)P3, likely by multiple phosphatases such as SHIP1 or OCRL. Depletion of PI(3,4)P2 at the plasma membrane by INPP4B also resulted in strongly decreased internalization of red blood cells, although their attachment to macrophages seemed unaltered, pointing to defects in particle engulfment. The authors then tested the potential role of lamellipodin, one of the few known PI(3,4)P2 specific effectors. Lamellipodin was found to be enriched at phagocytic cups, and this enrichment was shown to be dependent on the presence of PI(3,4)P2, by targeting of INPP4B to the plasma membrane. Macrophages depleted of lamellipodin by shRNA treatment showed reduced phagocytic efficiency and also aberrant phagocytic cup formation. As VASP is a known binding partner of lamellipodin and involved in actin polymerization, the authors next tested its potential involvement. Overexpression experiments showed that VASP colocalizes with lamellipodin at phagocytic cups. Sequestering of VASP at mitochondria through a respective construct containing the VASP binding site of ActA, together with a mitochondrial targeting sequence, showed that this also results in incompletely formed phagocytic cups and reduced phagocytic efficiency. Similar effects were observed upon expression of a lamellipodin construct with mutated binding sites for VASP. Collectively, the authors propose that PI(3,4)P2 is localized produced at phagocytic cups through the sequential activity of PI3 kinase and PI5 phosphatase, that it recruits lamellipodin and its binding partner VASP, and that this cascade is necessary for proper phagocytic cup formation and closure and thus phagocytic capacity of cells. This is an interesting study that uncovers a novel role for PI(3,4)P2 in phagocytic cup formation and closure. It is very well controlled, and the claims of the study are supported by the presented data. Statistical analysis is sound.

    Major comments:

    1. The localization of VASP at phagocytic cups is only shown by overexpression of constructs. Endogenous staining of VASP should support this finding.

    We agree with the reviewer that localization of the endogenous VASP would strengthen our conclusions. We have therefore performed the suggested experiments and in the revised manuscript include a new panel (E) in the revised Figure 6 showing immunostaining of endogenous VASP during phagocytosis. The result confirms the localization of the GFP-chimeric protein.

    1. It is unclear whether the roles of PI(3,4)P2, lamellipodin, and VASP are restricted to FcgammaR-mediated phagocytosis. Their potential involvement in CR3-mediated phagocytosis should be discussed or addressed in a basic set of experiments.

    In the revised manuscript we have extended our original observations to analyze also CR3-mediated phagocytosis, as recommended by the reviewer. A new supplemental figure (Supplemental Figure 10) now documents that PtdIns(3,4)P2 is also accumulated and Lamellipodin and VASP are recruited to the phagocytic cup during CR3-mediated phagocytosis. These results imply that the role of this lipid and its effectors extend to other modes of phagocytosis. These new observations are discussed on Page 14 of the revised manuscript.

    Minor comments:

    1. A very recent study (Körber and Faix, EJCB, 2022) describes the role of VASP in macroendocytosis in Dictyostelium. Specifically, VASP is found to be important for proper cup closure. The results are of direct importance to the current study and should be cited accordingly.

    Thank you for bringing this study to our attention. We now discuss the findings of Körber & Faix on Page 9 of the revised text.

    1. direct labelling of the figures would be helpful in assessing the manuscript

    To facilitate re-assessment of the paper, we have added the Figure numbers directly to the individual figures in the manuscript as suggested.

    Reviewer #1 (Significance (Required)): This study highlights the role of an underappreciated phospholipid in phagocytosis. It also describes for the first time a role for lamellipodin in formation of phagocytic cups and confirms the recent finding that also VASP is necessary for phagocytic cup closure. The paper should be of interest to researchers working on host-pathogen interaction, regulation of the actin cytoskeleton, and also to the general cell biological community

    Reviewer´s expertise: Actin regulation Microtubule-based transport Adhesion, migration, invasion Phagocytosis

    We thank the reviewers for his/her comments and suggestions that have clearly improved the manuscript.

    Reviewer____ #2 (Evidence, reproducibility and clarity (Required)):

    Review of Montano-Rendon et al: 'PI(3,4)P2, Lamellipodin and VASP coordinate cytoskeletal remodeling during phagocytic cup formation in macrophages'

    The authors employ biosensors for PI(3,4)P2 in RAW 264.7 macrophages to identify localized pools of PIP2 that were sensitive to INPP4B and wortmannin (Fig1). The biosensors for PIP2 are enriched on the forming phagocytic cup (Fig2, movies) in these macrophage cells. Inhibitors for PI3K blocked the recruitment of this biosensor to the membrane.

    Overall, the data are clear with the exceptions noted below. Krause et al (Dev Cell 2004) published a manuscript looking at PIP2, Lpd, and VASP in non-macrophage cells (fibroblasts, HeLa, etc...) where the influence of PI(3,4)P2 and these proteins was found to regulate actin and lamellipodial membrane extensions. This study also implicated Lpd protein coordinated actin networks in the docking of pathogens such as Vaccinia virus and EPEC bacteria. Given the additional reports of these proteins participating in dorsal ruffling (Michael et al Curr Bio 2010) and invasion (Carmona et al Oncogene 2016), it comes as no surprise that they participate in phagophore formation and phagocytosis. These studies are referenced, but having this in mind does diminish the novelty of implicating Lpd and VASP in the phagocytic process, though it seems to be the first time this machinery was directly implicated in macrophage cells.

    We would like to point out that docking of viruses or dorsal ruffling are very different biological processes from phagocytosis and that the common involvement of Lamellipodin in these very disparate processes does not, in our view, detract from the novelty of our studies.

    Specific Comments Although the images and movies graphically demonstrate a PI(3,4)P2 enrichment on phagocytic structures, the authors could provide some additional images that include fluorescently tagged phagocytic cargo such as the erythrocytes used. The addition of a fluorescent marker or phase image would be especially beneficial in the experiments where a lack of cPHx-biosensor recruitment is seen to the docked phagocytic cargo.

    We thank the reviewer for this suggestion. In the revised manuscript figures now include micrographs of the fluorescently labelled particles or phase-contrast images where appropriate.

    Otherwise, readers are left with the impression that perturbations such as INPP4B compromise docking and phagocytic cup formation altogether (Fig 2C)- which is perhaps the authors point? Make this clear?

    We apologize for the ambiguity of the former version of the manuscript. Initially, we noticed that particle engulfment -which is what we believe the reviewer means by “cup formation”- was the main defect in INPP4B-CaaX expressing cells. However, since the reviewer raised the possibility, we have gone back and re-analyzed the data and found that cells expressing the INPP4B-CaaX also have a small (~35%) decrease in particle engagement/binding (Reviewer uses the term “docking”). This suggests that the plasmalemmal pool of PtdIns(3,4)P2 in resting cells supports the actin dynamics at the cell surface which allows the RAW cells to survey their immediate environment and thereby contact more potential prey. This new finding is included in and discussed the revised manuscript. We thank the reviewer for prompting us to consider this alternative mechanism.

    There has already been an implication for PI3K in the phagocytic process, perhaps verifying that initial formation/membrane extension stages of phagocytosis are impacted by targeting the D-4 position of PIP2 would be of interest?

    PtdIns(4,5)P2 is well known to be essential for actin polymerization and is increased transiently at the sites of phagocytosis (Botelho et al., 2000 J Cell Biol; Scott et al., 2005 J Cell Biol; Fairn et al., 2009 J Cell Biol.). It is not clear whether the reviewer is curious about the possible consequences of converting PtdIns(4,5)P2 to PtdIns5P prior to activation of PI3K. Whether PtdIns5P itself has biological activity is a subject of debate and, to our knowledge, its existence has not been documented at sites of phagocytosis. It is also unclear whether PtdIns5P would serve as an effective substrate for PI3K and, if so whether the putative product, PtdIns(3,5)P2 that is normally found in endomembranes, would be functionally relevant.

    Depletion of PI(3,4)P2 through the expression of the INPP4B phosphatase demonstrated a reduction in phagocytic uptake of red blood cells (Fig4). The readout for this assay relied upon what appears to be differential labeling of phagocytosed red blood cells, though there are examples of cargo that is supposedly inside the macrophages labeled in green? Perhaps the authors can reconcile this and make the methods more clear for this approach?

    Thank you for this comment; we apologize if the original text was unclear in this regard. In the revised manuscript a detailed description of the staining protocol we used to distinguish inside from outside particles is now included in the Methods section. It is also worth pointing out that the green-only SRBCs in the INPP4B-CaaX panel in Figure 3 indicate targets that were fully internalized by those RAW macrophages not expressing BFP-INPP4B-CAAX (see image below)

    Fig4 demonstrates the PI(3,4)P2 dependent recruitment of Lamellipodin (Lpd) to the phagocytic cup, which is clear. Lpd is found to be necessary for effective phagocytic uptake in Fig 5. There is no blotting/qPCR data for the verification of Lpd knockdown shown?

    RAW macrophages and other macrophage cell lines are rather refractory to transfection, resulting in only a minor (10-20%) fraction of the cells expressing transfected constructs. For this reason, immunoblotting or qPCR analyses of the entire population yield misleading results, not reflective of the comparatively small transfected sub-population of cells. To overcome this limitation, we co-transfected the shRNA-containing plasmid with a smaller amount of a plasmid containing a fluorescent protein used to identify transfectants visually (a 5:1 ratio of shRNA:EGFP). By using a 5:1 ratio of the plasmids we ensured that cells expressing the fluorescent protein had a high likelihood of also expressing the shRNA. In this manner, the Lpd-depleted cells could be scored separately from the untransfected, wild-type cells following immunostaining (Supplemental Figure 5). Note that some immunostaining persisted in the Lpd-silenced cells, in all likelihood because some of the antibody binding is nonspecific, as is commonly seen in immunostaining. Nevertheless, the data indicate that substantial silencing of Lpd is achieved when transfecting the shRNA.

    The authors demonstrate a co-localization of Lpd/VASP proteins at the phagocytic cup of these macrophages in Fig 6 and sequester VASP protein to the mitochondria with some ActA derived fusion proteins to functionally block phagocytosis. The functional interaction of Lpd/VASP is further explored with experiments utilizing Ena/VASP mutants in Fig7, demonstrating a dependence on this interaction to promote phagocytic uptake.

    Reviewer #2 (Significance (Required)): see above

    Reviewer____ #3 (Evidence, reproducibility and clarity (Required)):

    In this study, Montano-Rendon and colleagues address the role of phosphatidylinositol (3,4)-bisphosphate in phagocytosis by RAW macrophages. Using small molecule inhibitors, they show that dephosphorylation of PI(3,4,5)P3 is the main source of PI(3,4)P2 in phagocytosis. Using an elegant approach based on overexpression of a PI(3,4)P2-specific phosphatase, they show that the selective depletion of PI(3,4)P2 impairs phagosome formation. Moreover, they identify two PI(3,4)P2 interacting proteins involved in phagocytosis: lamellipodin and VASP. They show that shRNA silencing of lamellipodin arrests phagocytosis, as well as mistargeting of VASP to mitochondria by a fusion protein. Overall, this is a high-quality study, well designed and written. I hence support publication, and only have a few relatively minor comments that the authors should consider as I believe it would improve the quality of the manuscript.

    The role of PI(3,4)P2 in the actin organisation in phagocytosis has been shown previously in various studies, see for example PMID: 16418223, 27806292 and review 32296634. In these studies, different mechanisms have been proposed of how PI(3,4)P2 affects the cytoskeleton and phagocytic process. It would be good to discuss how the findings with lamellipodin and VASP relate to these previously described mechanisms.

    We now include and discuss the references recommended by the reviewer to highlight that the importance of PtdIns(3,4)P2 extends to dendritic cells and HL60 neutrophils.

    In figure 6, a role for VASP in phagocytosis is shown by mistargeting it to mitochondria using a fusion protein consisting of a VASP binding region and a mitochondrial targeting motif. While this is an elegant approach, I wonder why not simply shRNA is used, similar to lamellipodin?

    We decided to use this approach because macrophages (including RAW cells) express other members of the Ena/VASP family of proteins such as EVL (Coppolino et al., 2001 *J Cell Sci) *that could potentially substitute for VASP; simultaneously silencing multiple, distinct members of the Ena/VASP family poses an experimental challenge. Moreover, in our experience introducing siRNA into RAW cells, even when using electroporation, is often insufficient to generate robust silencing of certain genes (e.g. Levin-Konigsberg, et al., 2019 Nature Cell Biology). Thus, we took advantage of the robust, more globally effective ActA-based molecular tool. To demonstrate its effectiveness, we now include a new Supplemental figure (Supplemental Figure 6, reproduced below) using immunostaining that shows how virtually all of the endogenous VASP is sequestered to the surface of mitochondria when the MITO-FP4 is expressed.

    Supplemental Figure 6. MITO-FP4 targets endogenous VASP to the Mitochondria

    In figure 3A: How was the inside-outside staining performed? I cannot find this information in the Methods.

    We apologize for the omission. The inside/outside staining protocol is now detailed in the Methods section of the manuscript.

    Figures are overall good quality. However, in figure 1,2, and 4 individual cells are shown in the graphs, whereas figures 3, 5, 6 and 7, and the supplementary figures only show averages with bar graphs. Please change these graphs to all show individual cells, as this will allow to see the variation among cells.

    Thank you for the suggestion. The graphs have been modified to violin plots to show the variation and distribution of results amongst the individual cells and experiments.

    Reviewer #3 (Significance (Required)):

    see above

  3. 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 #3

    Evidence, reproducibility and clarity

    In this study, Montano-Rendon and colleagues address the role of phosphatidylinositol (3,4)-bisphosphate in phagocytosis by RAW macrophages. Using small molecule inhibitors, they show that dephosphorylation of PI(3,4,5)P3 is the main source of PI(3,4)P2 in phagocytosis. Using an elegant approach based on overexpression of a PI(3,4)P2-specific phosphatase, they show that the selective depletion of PI(3,4)P2 impairs phagosome formation. Moreover, they identify two PI(3,4)P2 interacting proteins involved in phagocytosis: lamellipodin and VASP. They show that shRNA silencing of lamellipodin arrests phagocytosis, as well as mistargeting of VASP to mitochondria by a fusion protein. Overall, this is a high-quality study, well designed and written. I hence support publication, and only have a few relatively minor comments that the authors should consider as I believe it would improve the quality of the manuscript.

    The role of PI(3,4)P2 in the actin organisation in phagocytosis has been shown previously in various studies, see for example PMID: 16418223, 27806292 and review 32296634. In these studies, different mechanisms have been proposed of how PI(3,4)P2 affects the cytoskeleton and phagocytic process. It would be good to discuss how the findings with lamellipodin and VASP relate to these previously described mechanisms.

    In figure 6, a role for VASP in phagocytosis is shown by mistargeting it to mitochondria using a fusion protein consisting of a VASP binding region and a mitochondrial targeting motif. While this is an elegant approach, I wonder why not simply shRNA is used, similar to lamellipodin? In figure 3A: How was the inside-outside staining performed? I cannot find this information in the Methods.

    Figures are overall good quality. However, in figure 1,2, and 4 individual cells are shown in the graphs, whereas figures 3, 5, 6 and 7, and the supplementary figures only show averages with bar graphs. Please change these graphs to all show individual cells, as this will allow to see the variation among cells.

    Significance

    see above

  4. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Review of Montano-Rendon et al: 'PI(3,4)P2, Lamellipodin and VASP coordinate cytoskeletal remodeling during phagocytic cup formation in macrophages'

    The authors employ biosensors for PI(3,4)P2 in RAW 264.7 macrophages to identify localized pools of PIP2 that were sensitive to INPP4B and wortmannin (Fig1). The biosensors for PIP2 are enriched on the forming phagocytic cup (Fig2, movies) in these macrophage cells. Inhibitors for PI3K blocked the recruitment of this biosensor to the membrane.

    Overall, the data are clear with the exceptions noted below. Krause et al (Dev Cell 2004) published a manuscript looking at PIP2, Lpd, and VASP in non-macrophage cells (fibroblasts, HeLa, etc...) where the influence of PI(3,4)P2 and these proteins was found to regulate actin and lamellipodial membrane extensions. This study also implicated Lpd protein coordinated actin networks in the docking of pathogens such as Vaccinia virus and EPEC bacteria. Given the additional reports of these proteins participating in dorsal ruffling (Michael et al Curr Bio 2010) and invasion (Carmona et al Oncogene 2016), it comes as no surprise that they participate in phagophore formation and phagocytosis. These studies are referenced, but having this in mind does diminish the novelty of implicating Lpd and VASP in the phagocytic process, though it seems to be the first time this machinery was directly implicated in macrophage cells.

    Specific Comments

    Although the images and movies graphically demonstrate a PI(3,4)P2 enrichment on phagocytic structures , the authors could provide some additional images that include fluorescently tagged phagocytic cargo such as the erythrocytes used. The addition of a fluorescent marker or phase image would be especially beneficial in the experiments where a lack of cPHx-biosensor recruitment is seen to the docked phagocytic cargo. Otherwise, readers are left with the impression that perturbations such as INPP4B compromise docking and phagocytic cup formation altogether (Fig 2C)- which is perhaps the authors point? Make this clear? There has already been an implication for PI3K in the phagocytic process, perhaps verifying that initial formation/membrane extension stages of phagocytosis are impacted by targeting the D-4 position of PIP2 would be of interest?

    Depletion of PI(3,4)P2 through the expression of the INPP4B phosphatase demonstrated a reduction in phagocytic uptake of red blood cells (Fig4). The readout for this assay relied upon what appears to be differential labeling of phagocytosed red blood cells, though there are examples of cargo that is supposedly inside the macrophages labeled in green? Perhaps the authors can reconcile this and make the methods more clear for this approach?

    Fig4 demonstrates the PI(3,4)P2 dependent recruitment of Lamellipodin (Lpd) to the phagocytic cup, which is clear. Lpd is found to be necessary for effective phagocytic uptake in Fig 5. There is no blotting/qPCR data for the verification of Lpd knockdown shown? The authors demonstrate a co-localization of Lpd/VASP proteins at the phagocytic cup of these macrophages in Fig 6 and sequester VASP protein to the mitochondria with some ActA derived fusion proteins to functionally block phagocytosis. The functional interaction of Lpd/VASP is further explored with experiments utilizing Ena/VASP mutants in Fig7, demonstrating a dependence on this interaction to promote phagocytic uptake.

    Significance

    see above

  5. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The authors use newly available probes to show that the phosphoinositide PI(3,4)P2 plays a previously undescribed role in FcgammaR-mediated phagocytosis. Using RAW macrophages, they show that PI(3,4)P2 is enriched at the plasma membrane and also at phagocytic cups internalizing IgG-opsonized sheep red blood cells. Pharmacological inhibition using wortmannin, and also expression of membrane-targeted INPP4B phosphatase showed that PI(3,4)P2 production depends on PI3 kinase activity. Further experiments using selective inhibitors showed that PI(3,4)5P2 is mainly derived by dephosphorylation of PI(3,4,5)P3, likely by multiple phosphatases such as SHIP1 or OCRL. Depletion of PI(3,4)P2 at the plasma membrane by INPP4B also resulted in strongly decreased internalization of red blood cells, although their attachment to macrophages seemed unaltered, pointing to defects in particle engulfment.

    The authors then tested the potential role of lamellipodin, one of the few known PI(3,4)P2 specific effectors. Lamellipodin was found to be enriched at phagocytic cups, and this enrichment was shown to be dependent on the presence of PI(3,4)P2, by targeting of INPP4B to the plasma membrane. Macrophages depleted of lamellipodin by shRNA treatment showed reduced phagocytic efficiency and also aberrant phagocytic cup formation. As VASP is a known binding partner of lamellipodin and involved in actin polymerization, the authors next tested its potential involvement. Overexpression experiments showed that VASP colocalizes with lamellipodin at phagocytic cups. Sequestering of VASP at mitochondria through a respective construct containing the VASP binding site of ActA, together with a mitochondrial targeting sequence, showed that this also results in incompletely formed phagocytic cups and reduced phagocytic efficiency. Similar effects were observed upon expression of a lamellipodin construct with mutated binding sites for VASP.

    Collectively, the authors propose that PI(3,4)P2 is localized produced at phagocytic cups through the sequential activity of PI3 kinase and PI5 phosphatase, that it recruits lamellipodin and its binding partner VASP, and that this cascade is necessary for proper phagocytic cup formation and closure and thus phagocytic capacity of cells. This is an interesting study that uncovers a novel role for PI(3,4)P2 in phagocytic cup formation and closure. It is very well controlled, and the claims of the study are supported by the presented data. Statistical analysis is sound.

    Major comments:

    1. The localization of VASP at phagocytic cups is only shown by overexpression of constructs. Endogenous staining of VASP should support this finding.
    2. It is unclear whether the roles of PI(3,4)P2, lamellipodin, and VASP are restricted to FcgammaR-mediated phagocytosis. Their potential involvement in CR3-mediated phagocytosis should be discussed or addressed in a basic set of experiments.

    Minor comments:

    1. A very recent study (Körber and Faix, EJCB, 2022) describes the role of VASP in macroendocytosis in Dictyostelium. Specifically, VASP is found to be important for proper cup closure. The results are of direct importance to the current study and should be cited accordingly.
    2. direct labelling of the figures would be helpful in assessing the manuscript

    Significance

    This study highlights the role of an underappreciated phospholipid in phagocytosis. It also describes for the first time a role for lamellipodin in formation of phagocytic cups and confirms the recent finding that also VASP is necessary for phagocytic cup closure. The paper should be of interest to researchers working on host-pathogen interaction, regulation of the actin cytoskeleton, and also to the general cell biological community

    Reviewer´s expertise: Actin regulation Microtubule-based transport Adhesion, migration, invasion Phagocytosis

  6. Version published to 10.1101/2022.03.08.483476v2 on bioRxiv
  7. Version published to 10.1101/2022.03.08.483476v1 on bioRxiv