On 2017 Sep 23, Markus Meissner commented:

During this lengthy discussion, the Kursula group succeeded to solve part of the puzzle regarding the polymerisation mechanism of apicomplexan actin and confirmed our suspicions (see below) that sedimentation assays, while useful in other systems, lead to variable and unreliable results in case of apicomplexan actin (see: https://www.nature.com/articles/s41598-017-11330-w). Briefly, in this study polymerisation assays based on pyrene labelling were used to compare polymerisation kinetics of Plasmodium and rabbit actin and conclusively showed that: - Apicomplexan Actin polymerises in a cooperative manner with a similar critical concentration as canonical Actin - Shorter filament lengths result from higher depolymerisation rate. Since Skillmann et al., 2013 reached their conclusion exclusively based on sedimentation assays, their conclusion regarding an isodesmic polymerization mechanism of apicomplexan actin, as discussed below, should be seen with great scepticism. As discussed in this study these in vitro data also support our (Periz et al., 2017, Whitelaw et al., 2017 and Das et al., 2017) findings in vivo suggesting that a critical concentration of G-actin is required in order to form F-actin filaments. Therefore, the hypothesis of an isodesmic polymerisation mechanism can be considered as falsified.

On 2017 Jun 20, Robert Insall commented:

Professor Sibley's most extensive comments are based around a single paper (Skillman et al. 2013) that concluded the polymerization of Toxoplasma actin uses an isodesmic, rather than a nucleation-based mechanism. While this work was well-executed, and thorough, it is not on its own sufficient to support the level of absolutism that is in evidence in these comments. In particular, results from actin that has been exogenously expressed (in this case, in baculovirus) are less reliable than native apicomplexan actin. The folding of actin is infamously complex, with a full set of specialist chaperones and idiosyncratic N-terminal modifications. Even changes in the translation rate of native actin can affect its function and stability (see for example Zhang, 2010). Exogenously-expressed actin may be fully-folded, but still not representative of the physiological protein. Thus it is not yet appropriate to make dogmatic statements about the mechanism of apicomplexan actin function until native actin has been purified and its polymerization measured. When this occurs, as it surely will soon, stronger rulings may be appropriate.

On 2017 Jun 20, Markus Meissner commented:

We thank David Sibley for his last comment. As we mentioned previously, it was not the aim of this study to prove or disprove isodesmic polymerisation. We highlighted the current discussion in the field regarding isodesmic polymerisation (see previous comments). It is contra productive to turn the comments on this paper into a discussion on Skillmann et al., 2013, which is seen with great scepticism in the field. We made our views clear in previous responses and we hope that future results will help to clarify this issue. However, we find it concerning (and distracting) that– in contrast to his earlier comments, according to which our data can be consolidated with isodesmic polymerisation -David Sibley is now doubting the validity of our data, mentioning that CB might affect actin dynamics. This is certainly the case, as shown in the study and as is the case with most actin binding proteins used to measure actin dynamics in eukaryotic cells. This issue was discussed at length in the manuscript, by the reviewers comments and authors response, which can all be easily accessed: https://elifesciences.org/articles/24119 The above statement reflect the joint opinions of: Markus Meissner (University of Glasgow), Aoife Heaslip (University of Conneticut) and Robert Insall (Beatson Institute, Glasgow).

On 2017 Jun 19, L David Sibley commented:

Based on the most recent response by Dr. Meissner, it is clear that there is still some confusion about the difference between measuring the kinetics of actin polymerization in vivo vs. monitoring actin dynamics in vivo. These are fundamentally different processes, the former of which cannot be directly inferred from the later. Given this confusion, it is worth reviewing how these two processes are distinct, yet inter-related.

When referring to the mechanism of actin polymerization in vitro, nucleation is the process of forming stable trimers, which are normally limited by an intrinsic kinetic barrier imposed by unstable dimers. Due to this intrinsic instability, the nucleation step is normally revealed as a pronounced lag phase in the time course of polymerization, after which filaments undergo rapid elongation Pollard TD, 2000. TgACT1 lacks this nucleation step and instead uses a non-cooperative, isodesmic process. The Arp/23 complex facilitates formation of the trimer by acting as the barbed end, thus reducing the lag time and accelerating polymerization, typically by side branching from existing filaments. Toxoplasma has no use for such a step as it would not affect the efficiency of an isodesmic process since dimers and trimers normally form without a lag phase Skillman KM, 2011. By contrast, formins bind to barbed end of existing filaments and promote elongation, both by preventing capping protein from binding and by using profilin to gather actin monomers for addition to the barbed end. Formins may also nucleate F-actin by binding to two monomers to lower the lag phase for trimer formation, thus facilitating elongation, although this role is less well studied. Importantly, formins can act on actins that use either an intrinsic “nucleation-elongation” cooperative mechanism or an isodesmic process, such as that used by Toxoplasma. Hence, the fact that formins function in Toxoplasma has no bearing on the intrinsic polymerization mechanism of TgACT1.

Once the above definitions are clearly understood, it becomes apparent why the isodesmic process of actin nucleation used by Toxoplasma is fully compatible with both the short filament, rapid turnover dynamics that have been described previously Sahoo N, 2006, Skillman KM, 2011, Wetzel DM, 2003, and the new findings of long-stable filaments described in the present paper Periz J, 2017. These different states of actin polymerization represent dynamics that are driven by the combination of the intrinsic polymerization mechanism and various actin-binding proteins that modulate this process. However, the dynamic processes that affect the status of G and F-actin in vivo cannot be used to infer anything about the intrinsic mechanism of actin polymerization as it occurs in solution. As such, we strongly disagree that there is an issue to resolve regarding the intrinsic mechanism of actin polymerization in Toxoplasma nor do any of the studies in the present report address this point. Our data on the in vitro polymerization kinetics of TgACT1 clearly fit an isodesmic process Skillman KM, 2013 and we are unaware of any data that demonstrates otherwise. Hence we fail to see why this conclusion is controversial and find it surprising that these authors continue to question this point in their present work Periz J, 2017, previous report Whitelaw JA, 2017, and comments by Dr. Meissner. As it is not possible to predict the intrinsic mechanism of actin polymerization from the behavior observed in vivo, these comments are erroneous and misleading. On the other hand, if these authors have new data that speaks directly to the topic of the intrinsic polymerization mechanism of TgACT1, we would welcome them to provide it for discussion.

Although we disagree with the authors on the above points, we do agree that the fact that actin filaments can be visualized in Toxoplasma for the first time is interesting and certainly in contrast to previous studies. For example, previous studies failed to reveal such filaments using YFP-ACT1, despite the fact that this tagged form of actin is readily incorporated into Jasplakinolide-stabilized filaments Rosenberg P, 1989. As well, filaments have not been seen by CryoEM tomography Paredes-Santos TC, 2012 or by many studies using conventional transmission EM. This raises some concern that the use of chromobodies (Cb) that react to F-actin may stabilize filaments and thus affect dynamics. Although the authors make some attempt to monitor this in transfected cells, it is very difficult rule out that Cb are in fact enhancing filament formation. One example of this is seen in Figure 6 A, where in a transiently transfect cell, actin filaments are seen with both the Cb-staining and anti-actin, while in the non-transfected cell, it is much less clear that filaments are detected with anti-actin Periz J, 2017. Instead the pattern looks more like punctate clusters that concentrate at the posterior pole or residual body. Thus while we would agree that the Cb-stained filaments also stain with antibodies ot F-actin, it is much less clear that they exist in the absence of Cb expression. It would thus be nice to see these findings independently reproduced with another technique. It would also be appropriate to test the influence of Cb on TgACT1 in vitro to determine if it stabilizes filaments. There are published methods to express Toxoplasma actin in a functional state and so this could easily be tested Skillman KM, 2013. Given the isodesmic mechanism used by TgACT1, it is very likely that any F-actin binding protein would increase the stability of the short filaments that normally form spontaneously, thus leading to longer, more stable filaments. This effect is likely to be less pronounced when using yeast or mammalian actins as they intrinsically form stable filaments above their critical concentration. Testing the effects of Cb on TgACT1 polymerization in vitro would provide a much more sensitive readout than has been provided here, and would help address the question of whether expression of Cb alters in vivo actin dynamics.

In summary, we find the reported findings of interest, but do not agree that they change the view of how actin polymerization operates in Toxoplasma at the level of the intrinsic mechanism. They instead reveal an important aspect of in vivo dynamics and it will be import to determine what factors regulate this process in future studies.

The above statement reflect the joint opinions of: John Cooper (Washington University), Dave Sept (University of Michigan) and David Sibley (Washington University).

On 2017 Jun 16, Markus Meissner commented:

Thank you for your comment which appears to be only a slight update of the comments already made on the eLIFE website and it would be helpful for all readers who wish to follow this discussion if we could stick to the website where the discussion started (see: https://elifesciences.org/articles/24119).

Regarding the second comment of David Sibley: It is good to see that the authors of the Skillmann paper (Skilmann et al., 2013) are able to reconcile our data with their unusual, isodesmic polymerisation model, despite their initial interpretations that clearly states that “…an isodesmic mechanism results in a distribution of SMALL OLIGOMERS, which explains why TgACTI only sediments efficiently at higher g force. Our findings also explain why long TgACTI filaments have not been observed in parasites by any method, including EM, fluorescence imaging of GFP–TgACTI and Ph staining." While it appears that we will need a lengthy discussion about Skillmann et al., 2013 or even better more reliable assays to answer the question of isodesmic vs cooperative polymerisation, our study did not aim to answer this open issue that we briefly introduced in Periz et al., 2017 to give a more complete picture of the open questions regarding apicomplexan actin. As soon as more convincing evidences are available for cooperative or isodesmic polymerisation of apicomplexan actin, we will be happy to integrate it in our interpretation. Meanwhile we remain of the opinion that our in vivo data (see also Whitelaw et al., 2017) best reflects the known behaviours of canonical actin. While it seems that under the conditions used by Skillmann et al., 2013 apicomplexan actin polymerizes in an isodemic manner, in the in vivo situation F-actin behaviour appears very similar to other, well characterised model systems. However, we would like to point out that a major argument in the interpretation of Skillmann et al., 2013 for isodesmic polymerisation is that “This discovery explains previous differences from conventional actins and offers insight into the behaviour of the parasite in vivo. First, nucleation is not rate limiting, so that T.gondii does not need nucleation-promoting factors. Indeed, homologs of actin nucleating proteins, such as Arp2/3 complex have not been identified within apicomplexan genomes”. This statement is oversimplified and cannot be reconciled with the literature on eukaryotic actin. For example, Arp2/3 knockouts have been produced in various cell lines (and obviously their actin doesn’t switch to an isodesmic polymerisation process). Instead, within cells, regulated actin assembly is initiated by two major classes of actin nucleators, the Arp2/3 complex and the formins (Butler and Cooper, 2012). Therefore, we thought it is necessary to mention in Periz et al., 2017 that apicomplexans do possess nucleators, such as formins. Several studies agree, that apicomplexan formins efficiently NUCLEATE actin in vitro, both rabbit and apicomplexan actin (Skillmann et al., 2012, Daher et al., 2010 and Baum et al., 2008). In summary we agree that future experiments will be required to solve this issue and we are glad that David Sibley agrees with the primary findings of our study. We hope that future in vitro studies will help to solve the question of isodesmic vs cooperative polymerisation mechanism in the case of apicomplexan actin so that a better integration of in vivo and in vitro data will be possible.

On 2017 Jun 14, L David Sibley commented:

We feel it is worth briefly reviewing the concept of the critical concentration (Cc), and the properties of nucleation-dependent actin polymerization, since there seems to be some misconception about these terms as they are used in this paper Periz J, 2017.

Polymerization assays using muscle or yeast actin clearly show that these actins undergo nucleation dependent assembly. Nucleation is a cooperative assembly process in which monomers of actin (i.e. G-actin) form small, unstable oligomers that readily dissociate. The Cc is the concentration of free actin above which a stable nucleus is formed and the filament elongation begins, a process that is more thermodynamically favorable than the nucleation step. A key feature of this nucleation-elongation mechanism is that for total actin concentrations above the Cc, the concentration of free G-actin remains fixed at the Cc, and all of the additional actin, over and above the Cc, is polymerized into filaments (i.e. F-actin). In contrast, an isodesmic polymerization process is not cooperative, and all steps (formation of dimer, trimer, etc.,) occur with the same binding and rate constants. With isodesmic polymerization, the monomer concentration (G-actin concentration) does not display a fixed limit; instead, as total actin concentration increased, the G-actin concentration continues to increase. Another key difference with isodesmic polymerization is that polymer forms at all concentrations of total actin (i.e. there is no concept of a critical concentration, Cc, that must be exceeded in order to achieve polymer formation).

The inherent differences between nucleation-elongation and isodesmic polymerization give rise to distinct kinetic and thermodynamic signatures in experiments. Because the nucleation process is unfavorable and cooperative, the time course of nucleation-elongation polymerization shows a characteristic lag phase, with a relatively low rate of initial growth, before the favorable elongation phase occurs. In contrast, isodesmic polymerization shows no lag phase, but exhibits linear growth vs. time from the start at time zero. The thermodynamic differences are manifested in experiments examining the fractions of polymer (F-actin) and monomer (G-actin) at steady state. Since nucleation-elongation has a critical concentration (Cc), the monomer concentration plateaus at this value and remains flat as the total protein concentration is increased. Polymer concentration is zero until the total concentration exceeds the critical concentration, and above that point, all the additional protein exists as polymer. In the isodesmic model, in stark contrast, the monomer concentration continues to increase and polymer form at all concentrations of total protein. These two distinct behaviors are illustrated in Figure 1 from Miraldi ER, 2008.

Our previous study on yeast and Toxoplasma actin Skillman KM, 2013 shows sedimentation assays that are closely matched by the theoretical results discussed above. In our study yeast actin (ScACT, Figure 2c) displays the saturation behavior characteristic of a nucleation-elongation mechanism; however, for TgACT1 (Figure 2a), the monomer concentration (red) continues to increase as total actin increases. In addition, the inset to Figure 2a shows that filaments (blue) are present at the lowest concentrations of total actin, and also does not exhibit a lag Skillman KM, 2013. Based on these features, it is unequivocal that Toxoplasma actin follows an isodemic polymerization process, with no evidence of cooperativity.

Several of the comments in the response may lead the reader to confound polymerization behavior in vitro with that observed fro actin polymerization in vivo in cells. The question of whether actin polymerization occurs by a nucleation-elongation mechanism or by an isodesmic mechanism is one that can only be determined in vitro using a solution of pure actin, because this is a property of the actin molecule itself, irrespective of other components. While the in vitro polymerization behavior is relevant as the template upon which various actin-binding proteins act, the polymerization mechanism for the actin alone cannot be inferred from in vivo observations due to the presence of actin-interacting proteins.

The authors state that the presence of “nucleation centers” in the parasite is not easy to consolidate with the isodesmic model Periz J, 2017. We disagree completely and emphatically. We agree that there are “centers” of accumulation of F-actin in the cell, these foci should not be referred to as “nucleation” centers in this case, because the term “nucleation” has a specific meaning in regard to the polymerization mechanism. F-actin may accumulate in these foci over time as a result of any one or more of several dynamic processes – new filament formation, elongation of short filaments, decreased turnover, or clustering of pre-existing filaments. The result is interesting and important; however, the result cannot be used to infer a polymerization mechanism.

The authors imply that these centers of F-actin correspond with sites of action of formins Periz J, 2017, which are capable of binding to actin monomers or actin filaments and thereby promoting actin polymerization. With vertebrate or yeast actin, which has a nucleation-elongation mechanism, formins do accelerate the nucleation process, and they also promote the elongation process. In the case of the isodesmic model for actin polymerization, formins would still function to promote polymerization, by interacting with actin filaments and actin monomers. Indeed, the short filaments that formed with the isodesmic mechanism are ideal templates for elongation from the barbed end (which formins enhance). We have previously shown that when TgACT1 polymerized in the presence of formins assembles into clusters of intermediate sized filaments that resemble the in vivo centers Skillman KM, 2012. Hence, as we commented previously, the isodesmic mechanism is entirely consistent with the observed in vivo structures labeled by the chromobodies.

The authors also suggest that evidence of a nucleation-elongation mechanism, with a critical concentration, is provided by the observation that actin filaments seen by chromobodies in vivo do not form in a conditional knock down of TgACT1 Periz J, 2017. In our view, this conclusion is based on incorrectly using observations of in vivo dynamics to infer the intrinsic polymerization mechanism of pure actin protein. Higher total actin concentration leads to higher actin filament concentration under both models, with control provided by the various actin-binding proteins of the cell and their relative ability to drive filament formation and turnover in vivo. However, dependence on total actin concentration is not a reflection of the intrinsic polymerization mechanism. The polymerization mechanism of TgACT1, whether isodesmic or nucleation-elongation, is unlikely to be the critical determinant of actin dynamics in vitro; instead, actin monomers and filaments are substrates for numerous actin-binding proteins that regulation filament elongation, filament turnover, and G-actin sequestration, that is, the whole of actin cytoskeleton dynamics.

Although we agree that much more study is need to unlock the molecular basis of actin polymerization and dynamics in apicomplexans, it will be important to distinguish between properties that are intrinsic to the polymerization process as it occurs in vitro, vs. interactions with proteins that modulate actin dynamics in vivo. The challenge, as has been the case in better studied systems Pollard TD, 2000, will be to integrate both sets of findings into a cohesive model of actin regulation and function in apicomplexans.

The above statement reflect the joint opinions of: John Cooper (Washington University), Dave Sept (University of Michigan) and David Sibley (Washington University).

On 2017 Jun 14, L David Sibley commented:

We feel it is worth briefly reviewing the concept of the critical concentration (Cc), and the properties of nucleation-dependent actin polymerization, since there seems to be some misconception about these terms as they are used in this paper Periz J, 2017.

Polymerization assays using muscle or yeast actin clearly show that these actins undergo nucleation dependent assembly. Nucleation is a cooperative assembly process in which monomers of actin (i.e. G-actin) form small, unstable oligomers that readily dissociate. The Cc is the concentration of free actin above which a stable nucleus is formed and the filament elongation begins, a process that is more thermodynamically favorable than the nucleation step. A key feature of this nucleation-elongation mechanism is that for total actin concentrations above the Cc, the concentration of free G-actin remains fixed at the Cc, and all of the additional actin, over and above the Cc, is polymerized into filaments (i.e. F-actin). In contrast, an isodesmic polymerization process is not cooperative, and all steps (formation of dimer, trimer, etc.,) occur with the same binding and rate constants. With isodesmic polymerization, the monomer concentration (G-actin concentration) does not display a fixed limit; instead, as total actin concentration increased, the G-actin concentration continues to increase. Another key difference with isodesmic polymerization is that polymer forms at all concentrations of total actin (i.e. there is no concept of a critical concentration, Cc, that must be exceeded in order to achieve polymer formation).

The inherent differences between nucleation-elongation and isodesmic polymerization give rise to distinct kinetic and thermodynamic signatures in experiments. Because the nucleation process is unfavorable and cooperative, the time course of nucleation-elongation polymerization shows a characteristic lag phase, with a relatively low rate of initial growth, before the favorable elongation phase occurs. In contrast, isodesmic polymerization shows no lag phase, but exhibits linear growth vs. time from the start at time zero. The thermodynamic differences are manifested in experiments examining the fractions of polymer (F-actin) and monomer (G-actin) at steady state. Since nucleation-elongation has a critical concentration (Cc), the monomer concentration plateaus at this value and remains flat as the total protein concentration is increased. Polymer concentration is zero until the total concentration exceeds the critical concentration, and above that point, all the additional protein exists as polymer. In the isodesmic model, in stark contrast, the monomer concentration continues to increase and polymer form at all concentrations of total protein. These two distinct behaviors are illustrated in Figure 1 from Miraldi ER, 2008.

Our previous study on yeast and Toxoplasma actin Skillman KM, 2013 shows sedimentation assays that are closely matched by the theoretical results discussed above. In our study yeast actin (ScACT, Figure 2c) displays the saturation behavior characteristic of a nucleation-elongation mechanism; however, for TgACT1 (Figure 2a), the monomer concentration (red) continues to increase as total actin increases. In addition, the inset to Figure 2a shows that filaments (blue) are present at the lowest concentrations of total actin, and also does not exhibit a lag Skillman KM, 2013. Based on these features, it is unequivocal that Toxoplasma actin follows an isodemic polymerization process, with no evidence of cooperativity.

Several of the comments in the response may lead the reader to confound polymerization behavior in vitro with that observed fro actin polymerization in vivo in cells. The question of whether actin polymerization occurs by a nucleation-elongation mechanism or by an isodesmic mechanism is one that can only be determined in vitro using a solution of pure actin, because this is a property of the actin molecule itself, irrespective of other components. While the in vitro polymerization behavior is relevant as the template upon which various actin-binding proteins act, the polymerization mechanism for the actin alone cannot be inferred from in vivo observations due to the presence of actin-interacting proteins.

The authors state that the presence of “nucleation centers” in the parasite is not easy to consolidate with the isodesmic model Periz J, 2017. We disagree completely and emphatically. We agree that there are “centers” of accumulation of F-actin in the cell, these foci should not be referred to as “nucleation” centers in this case, because the term “nucleation” has a specific meaning in regard to the polymerization mechanism. F-actin may accumulate in these foci over time as a result of any one or more of several dynamic processes – new filament formation, elongation of short filaments, decreased turnover, or clustering of pre-existing filaments. The result is interesting and important; however, the result cannot be used to infer a polymerization mechanism.

The authors imply that these centers of F-actin correspond with sites of action of formins Periz J, 2017, which are capable of binding to actin monomers or actin filaments and thereby promoting actin polymerization. With vertebrate or yeast actin, which has a nucleation-elongation mechanism, formins do accelerate the nucleation process, and they also promote the elongation process. In the case of the isodesmic model for actin polymerization, formins would still function to promote polymerization, by interacting with actin filaments and actin monomers. Indeed, the short filaments that formed with the isodesmic mechanism are ideal templates for elongation from the barbed end (which formins enhance). We have previously shown that when TgACT1 polymerized in the presence of formins assembles into clusters of intermediate sized filaments that resemble the in vivo centers Skillman KM, 2012. Hence, as we commented previously, the isodesmic mechanism is entirely consistent with the observed in vivo structures labeled by the chromobodies.

The authors also suggest that evidence of a nucleation-elongation mechanism, with a critical concentration, is provided by the observation that actin filaments seen by chromobodies in vivo do not form in a conditional knock down of TgACT1 Periz J, 2017. In our view, this conclusion is based on incorrectly using observations of in vivo dynamics to infer the intrinsic polymerization mechanism of pure actin protein. Higher total actin concentration leads to higher actin filament concentration under both models, with control provided by the various actin-binding proteins of the cell and their relative ability to drive filament formation and turnover in vivo. However, dependence on total actin concentration is not a reflection of the intrinsic polymerization mechanism. The polymerization mechanism of TgACT1, whether isodesmic or nucleation-elongation, is unlikely to be the critical determinant of actin dynamics in vitro; instead, actin monomers and filaments are substrates for numerous actin-binding proteins that regulation filament elongation, filament turnover, and G-actin sequestration, that is, the whole of actin cytoskeleton dynamics.

Although we agree that much more study is need to unlock the molecular basis of actin polymerization and dynamics in apicomplexans, it will be important to distinguish between properties that are intrinsic to the polymerization process as it occurs in vitro, vs. interactions with proteins that modulate actin dynamics in vivo. The challenge, as has been the case in better studied systems Pollard TD, 2000, will be to integrate both sets of findings into a cohesive model of actin regulation and function in apicomplexans.

The above statement reflect the joint opinions of: John Cooper (Washington University), Dave Sept (University of Michigan) and David Sibley (Washington University).

On 2017 Jun 19, L David Sibley commented:

Based on the most recent response by Dr. Meissner, it is clear that there is still some confusion about the difference between measuring the kinetics of actin polymerization in vivo vs. monitoring actin dynamics in vivo. These are fundamentally different processes, the former of which cannot be directly inferred from the later. Given this confusion, it is worth reviewing how these two processes are distinct, yet inter-related.

When referring to the mechanism of actin polymerization in vitro, nucleation is the process of forming stable trimers, which are normally limited by an intrinsic kinetic barrier imposed by unstable dimers. Due to this intrinsic instability, the nucleation step is normally revealed as a pronounced lag phase in the time course of polymerization, after which filaments undergo rapid elongation Pollard TD, 2000. TgACT1 lacks this nucleation step and instead uses a non-cooperative, isodesmic process. The Arp/23 complex facilitates formation of the trimer by acting as the barbed end, thus reducing the lag time and accelerating polymerization, typically by side branching from existing filaments. Toxoplasma has no use for such a step as it would not affect the efficiency of an isodesmic process since dimers and trimers normally form without a lag phase Skillman KM, 2011. By contrast, formins bind to barbed end of existing filaments and promote elongation, both by preventing capping protein from binding and by using profilin to gather actin monomers for addition to the barbed end. Formins may also nucleate F-actin by binding to two monomers to lower the lag phase for trimer formation, thus facilitating elongation, although this role is less well studied. Importantly, formins can act on actins that use either an intrinsic “nucleation-elongation” cooperative mechanism or an isodesmic process, such as that used by Toxoplasma. Hence, the fact that formins function in Toxoplasma has no bearing on the intrinsic polymerization mechanism of TgACT1.

Once the above definitions are clearly understood, it becomes apparent why the isodesmic process of actin nucleation used by Toxoplasma is fully compatible with both the short filament, rapid turnover dynamics that have been described previously Sahoo N, 2006, Skillman KM, 2011, Wetzel DM, 2003, and the new findings of long-stable filaments described in the present paper Periz J, 2017. These different states of actin polymerization represent dynamics that are driven by the combination of the intrinsic polymerization mechanism and various actin-binding proteins that modulate this process. However, the dynamic processes that affect the status of G and F-actin in vivo cannot be used to infer anything about the intrinsic mechanism of actin polymerization as it occurs in solution. As such, we strongly disagree that there is an issue to resolve regarding the intrinsic mechanism of actin polymerization in Toxoplasma nor do any of the studies in the present report address this point. Our data on the in vitro polymerization kinetics of TgACT1 clearly fit an isodesmic process Skillman KM, 2013 and we are unaware of any data that demonstrates otherwise. Hence we fail to see why this conclusion is controversial and find it surprising that these authors continue to question this point in their present work Periz J, 2017, previous report Whitelaw JA, 2017, and comments by Dr. Meissner. As it is not possible to predict the intrinsic mechanism of actin polymerization from the behavior observed in vivo, these comments are erroneous and misleading. On the other hand, if these authors have new data that speaks directly to the topic of the intrinsic polymerization mechanism of TgACT1, we would welcome them to provide it for discussion.

Although we disagree with the authors on the above points, we do agree that the fact that actin filaments can be visualized in Toxoplasma for the first time is interesting and certainly in contrast to previous studies. For example, previous studies failed to reveal such filaments using YFP-ACT1, despite the fact that this tagged form of actin is readily incorporated into Jasplakinolide-stabilized filaments Rosenberg P, 1989. As well, filaments have not been seen by CryoEM tomography Paredes-Santos TC, 2012 or by many studies using conventional transmission EM. This raises some concern that the use of chromobodies (Cb) that react to F-actin may stabilize filaments and thus affect dynamics. Although the authors make some attempt to monitor this in transfected cells, it is very difficult rule out that Cb are in fact enhancing filament formation. One example of this is seen in Figure 6 A, where in a transiently transfect cell, actin filaments are seen with both the Cb-staining and anti-actin, while in the non-transfected cell, it is much less clear that filaments are detected with anti-actin Periz J, 2017. Instead the pattern looks more like punctate clusters that concentrate at the posterior pole or residual body. Thus while we would agree that the Cb-stained filaments also stain with antibodies ot F-actin, it is much less clear that they exist in the absence of Cb expression. It would thus be nice to see these findings independently reproduced with another technique. It would also be appropriate to test the influence of Cb on TgACT1 in vitro to determine if it stabilizes filaments. There are published methods to express Toxoplasma actin in a functional state and so this could easily be tested Skillman KM, 2013. Given the isodesmic mechanism used by TgACT1, it is very likely that any F-actin binding protein would increase the stability of the short filaments that normally form spontaneously, thus leading to longer, more stable filaments. This effect is likely to be less pronounced when using yeast or mammalian actins as they intrinsically form stable filaments above their critical concentration. Testing the effects of Cb on TgACT1 polymerization in vitro would provide a much more sensitive readout than has been provided here, and would help address the question of whether expression of Cb alters in vivo actin dynamics.

In summary, we find the reported findings of interest, but do not agree that they change the view of how actin polymerization operates in Toxoplasma at the level of the intrinsic mechanism. They instead reveal an important aspect of in vivo dynamics and it will be import to determine what factors regulate this process in future studies.

The above statement reflect the joint opinions of: John Cooper (Washington University), Dave Sept (University of Michigan) and David Sibley (Washington University).

On 2017 Jun 20, Robert Insall commented:

Professor Sibley's most extensive comments are based around a single paper (Skillman et al. 2013) that concluded the polymerization of Toxoplasma actin uses an isodesmic, rather than a nucleation-based mechanism. While this work was well-executed, and thorough, it is not on its own sufficient to support the level of absolutism that is in evidence in these comments. In particular, results from actin that has been exogenously expressed (in this case, in baculovirus) are less reliable than native apicomplexan actin. The folding of actin is infamously complex, with a full set of specialist chaperones and idiosyncratic N-terminal modifications. Even changes in the translation rate of native actin can affect its function and stability (see for example Zhang, 2010). Exogenously-expressed actin may be fully-folded, but still not representative of the physiological protein. Thus it is not yet appropriate to make dogmatic statements about the mechanism of apicomplexan actin function until native actin has been purified and its polymerization measured. When this occurs, as it surely will soon, stronger rulings may be appropriate.

On 2017 Sep 23, Markus Meissner commented:

During this lengthy discussion, the Kursula group succeeded to solve part of the puzzle regarding the polymerisation mechanism of apicomplexan actin and confirmed our suspicions (see below) that sedimentation assays, while useful in other systems, lead to variable and unreliable results in case of apicomplexan actin (see: https://www.nature.com/articles/s41598-017-11330-w). Briefly, in this study polymerisation assays based on pyrene labelling were used to compare polymerisation kinetics of Plasmodium and rabbit actin and conclusively showed that: - Apicomplexan Actin polymerises in a cooperative manner with a similar critical concentration as canonical Actin - Shorter filament lengths result from higher depolymerisation rate. Since Skillmann et al., 2013 reached their conclusion exclusively based on sedimentation assays, their conclusion regarding an isodesmic polymerization mechanism of apicomplexan actin, as discussed below, should be seen with great scepticism. As discussed in this study these in vitro data also support our (Periz et al., 2017, Whitelaw et al., 2017 and Das et al., 2017) findings in vivo suggesting that a critical concentration of G-actin is required in order to form F-actin filaments. Therefore, the hypothesis of an isodesmic polymerisation mechanism can be considered as falsified.