Energetics of the Microsporidian Polar Tube Invasion Machinery

Reviewer #1 (Public Review):

The authors used mathematical models to explore the mechanism(s) underlying the process of polar tube extrusion and the transport of the sporoplasm and nucleus through this structure. They combined this with experimental observations of the structure of the tube during extrusion using serial block face EM providing 3 dimensional data on this process. They also examined the effect of hyperosmolar media on this process to evaluate which model fit the predicted observed behavior of the polar tube in these various media solutions. Overall, this work resulted in the authors arriving at a model of this process that fit the data (model 5, E-OE-PTPV-ExP). This model is consistent with other data in the literature and provides support for the concept that the polar tube functions by eversion (unfolding like a finger of a glove) and that the expanding polar vacuole is part of this process. Finally, the authors provide important new insights into the bucking of the spore wall (and possible cavitation) as providing force for the nucleus to be transported via the polar tube. This is an important observation that has not been in previous models of this process.

Reviewer #2 (Public Review):

Microsporidia has a special invasion mechanism, which the polar tube (PT) ejects from mature spores at ultra-fast speeds, to penetrate the host and transfer the cargo to host. This work generated models for the physical basis of polar tube firing and cargo transport through the polar tube. They also use a combination of experiments and theory to elucidate possible biophysical mechanisms of microsporidia. Moreover, their approach also provided the potential applications of such biophysical approaches to other cellular architecture.

The conclusions of this paper are mostly well supported by data, but some analyses need to be clarified.

According to the model 5 (E-OE-PTPV-ExP) in P42 Fig. 6, is the posterior vacuole connected with the polar tube? If yes, how does the nucleus unconnected with the posterior vacuole enter the polar tube? In Fig. 6, would the posterior vacuole become two parts after spore germination? One part is transported via the polar tube, and the other is still in the spore. I recommend this process requires more experiments to prove.

Reviewer #3 (Public Review):

Abstract:

The paper follows a recent study by the same team (Jaroenlak et al Plos Pathogens 2020), which documented the dramatic ejection dynamics of the polar tube (PT) in microsporidia using live-imaging and scanning electron microscopy. Although several key observations were reported in this paper (the 3D architecture of the PT within the spore, the speed and extent of the ejection process, the translocation dynamics of the nucleus during germination), the precise geometry of the PT during ejection remain inaccessible to imaging, making it difficult to physically understand the phenomenon.

This paper aims to fill this gap with an indirect "data-driven" approach. By modeling the hydrodynamic dissipation for different unfolding mechanisms identified in the literature and by comparing the predictions with experiments of ejection in media of various viscosities, authors shows that data are compatible with an eversion (caterpillar-like) mechanism but not compatible with a "jack-in-the-box" scenario. In addition, the authors observe that most germinated spores exhibit an inward bulge, which they attribute to buckling due to internal negative pressure and which they suggest may be a mean of pushing the nucleus out of the PT during the final stage of ejection.

Major strengths:

Probably the most impressive aspect of the study is the experimental analysis of the ejection dynamics (velocity, ejection length) in medium of various viscosities over 3 orders of magnitudes, which, combined with a modeling of the viscous drag of the PT tube, provides very convincing evidence that the unfolding mechanism is not a global displacement of the tube but rather an apical extension mechanism, where the motion is localized at the end of the tube. The systematic classification of the different unfolding scenarios, consistent with the previous literature, and their confrontation with data in terms of energy, pressure and velocity also constitute an original approach in microbiology where in-situ and real time geometry is often difficult to access.

Major weaknesses:

1. While the experimental part of the paper is clear, I had (and still have) a hard time understanding the modeling part. Overall, the different unfolding mechanisms should be much better explained, with much more informative sketches to justify the dissipation and pressure terms, magnifying the different areas where dissipation occurs, showing the velocity field and pressure field, etc. In particular, a key parameter of eversion models is the geometry of the lubrication layers inside and outside the spore (h_sheath, h_slip). Where do the values of h_sheath and h_slip come from? What is the physical process that selects these parameters? For clarity, the figures showing the unfolding mechanics in the different scenario should be in the main text, not in the supplemental materials.

2. The authors compute and discuss in several places "the pressure" required to ejection, but no pressure is indicated in the various sketches and no general "ejection mechanism" involving this pressure is mentioned in the paper. What is this "required pressure" and to what element does it apply? I understand that the article focuses on the dissipation required to the deployment of the PT but I find it difficult to discuss the unfolding mechanism without having any idea on the driving mechanism of the movement. How could eversion be initiated and sustained?

3. Finally, the authors do not explain how pressure, which appears to be a positive, driving quantity at the beginning of the process, can become negative to induce buckling at the end of ejection. Although the hypothesis of rapid translocation induced by buckling is interesting, a much better mechanistic description of the process is needed to support it.