Fluid mechanics of luminal transport in actively contracting endoplasmic reticulum

Reviewer #1 (Public Review):

Theoretical principles of viscous fluid mechanics are used here to assess likely mechanisms of transport in the ER. A set of candidate mechanisms is evaluated, making good use of imaging to represent ER network geometries. Evidence is provided that the contraction of peripheral sheets provides a much more credible mechanism than the contraction of individual tubules, junctions, or perinuclear sheets.

The work has been conducted carefully and comprehensively, making good use of underlying physical principles. There is a good discussion of the role of slip; sensible approximations (low volume fraction, small particle size, slender geometries, pragmatic treatment of boundary conditions) allow tractable and transparent calculations; clear physical arguments provide useful bounds; stochastic and deterministic features of the problem are well integrated.

There are just a couple of areas where more discussion might be warranted, in my view.

(1) The energetic cost of tubule contraction is estimated, but I did not see an equivalent estimate for the contraction of peripheral sheets. It might be helpful to estimate the energetic cost of viscous dissipation in generated flows at higher frequencies. The mechanism of peripheral sheet contraction is unclear: do ATP-driven mechanisms somehow interact with thermal fluctuations of membranes?

(2) Mutations are mentioned in the abstract but not (as far as I could see) later in the manuscript. It would be helpful if any consequences for pathologies could be developed in the text.

Reviewer #2 (Public Review):

Summary:

This study explores theoretically the consequences of structural fluctuations of the endoplasmic reticulum (ER) morphology called contractions on molecular transport. Most of the manuscript consists of the construction of an interesting theoretical flow field (physical model) under various hypothetical assumptions. The computational modeling is followed by some simulations

Strengths:

The authors are focusing their attention on testing the hypothesis that a local flow in the tubule could be driven by tubular pinching. We recall that trafficking in the ER is considered to be mostly driven by diffusion at least at a spatial scale that is large enough to account for averaging of any random flow occurring from multiple directions [note that this is not the case for plants].

Weaknesses:

The manuscript extensively details the construction of the theoretical model, occupying a significant portion of the manuscript. While this section contains interesting computations, its relevance and utility could be better emphasized, perhaps warranting a reorganization of the manuscript to foreground this critical aspect.

Overall, the manuscript appears highly technical with limited conclusive insights, particularly lacking predictions confirmed by experimental validation. There is an absence of substantial conclusions regarding molecular trafficking within the ER.

Author Response

Reviewer #1 (Public Review):

Theoretical principles of viscous fluid mechanics are used here to assess likely mechanisms of transport in the ER. A set of candidate mechanisms is evaluated, making good use of imaging to represent ER network geometries. Evidence is provided that the contraction of peripheral sheets provides a much more credible mechanism than the contraction of individual tubules, junctions, or perinuclear sheets.

The work has been conducted carefully and comprehensively, making good use of underlying physical principles. There is a good discussion of the role of slip; sensible approximations (low volume fraction, small particle size, slender geometries, pragmatic treatment of boundary conditions) allow tractable and transparent calculations; clear physical arguments provide useful bounds; stochastic and deterministic features of the problem are well integrated.

We thank the reviewer for their positive assessment of our work.

There are just a couple of areas where more discussion might be warranted, in my view.

(1) The energetic cost of tubule contraction is estimated, but I did not see an equivalent estimate for the contraction of peripheral sheets. It might be helpful to estimate the energetic cost of viscous dissipation in generated flows at higher frequencies.

This is a good point. We will also include an energetic cost estimate for the contractions of peripheral sheets in the revised manuscript.

The mechanism of peripheral sheet contraction is unclear: do ATP-driven mechanisms somehow interact with thermal fluctuations of membranes?

The new energetic estimates in the revision might help constrain possible hypotheses for the mechanism(s) driving peripheral sheet contraction, and suggest if a dedicated ATP-driven mechanism is required.

(2) Mutations are mentioned in the abstract but not (as far as I could see) later in the manuscript. It would be helpful if any consequences for pathologies could be developed in the text.

We are grateful for this suggestion. The need to rationalise pathology associated with the subtle effects of ER-morphogens’ mutations is indeed pointed out as one factor motivating the study of the interplay between ER structure and performance. In the revised manuscript, we plan to include a brief discussion potentially linking ER morphogenes’ malfunction to luminal transport, integrating additional freshly published data.

Reviewer #2 (Public Review):

Summary:

This study explores theoretically the consequences of structural fluctuations of the endoplasmic reticulum (ER) morphology called contractions on molecular transport. Most of the manuscript consists of the construction of an interesting theoretical flow field (physical model) under various hypothetical assumptions. The computational modeling is followed by some simulations

Strengths:

The authors are focusing their attention on testing the hypothesis that a local flow in the tubule could be driven by tubular pinching. We recall that trafficking in the ER is considered to be mostly driven by diffusion at least at a spatial scale that is large enough to account for averaging of any random flow occurring from multiple directions [note that this is not the case for plants].

We thank the reviewer. We have indeed explored here the possibilities of active transport, focusing especially on transport over the length scale of single tubules, as a result of structural fluctuations, and found tubular pinching to be ineffective compared to e.g. peripheral sheets fluctuations. In the revised version we plan to add text mentioning what is known about the ER in plants.

Weaknesses:

The manuscript extensively details the construction of the theoretical model, occupying a significant portion of the manuscript. While this section contains interesting computations, its relevance and utility could be better emphasized, perhaps warranting a reorganization of the manuscript to foreground this critical aspect.

Overall, the manuscript appears highly technical with limited conclusive insights, particularly lacking predictions confirmed by experimental validation. There is an absence of substantial conclusions regarding molecular trafficking within the ER.

We sought to balance the theoretical/computational details of our model with the biophysical conclusions drawn from its predictions. Given the model's complexity and novelty, it was essential to elucidate the theoretical underpinnings comprehensively, in order to allow others to implement it in the future with additional, or different, parameters. To maintain clarity and focus in the main text, we have judiciously relegated extensive technical details to the methods section or supplementary materials, and divided the text into stand-alone section headings allowing the reader to skip through to conclusions.

The primary focus of our manuscript is to introduce and explore, via our theoretical model, the interplay between ER structure dynamics and molecular transport. Our approach, while in silico, generates concrete predictions about the physical processes underpinning luminal motion within the ER. For instance, our findings challenge the previously postulated role of small tubular contractions in driving luminal flow, instead highlighting the potential significance of local flat ER areas—empirically documented entities—for facilitating such motion.

Furthermore, by deducing what type of transport may or may not occur within the range of possible ER structural fluctuations, our model offers detailed predictions designed to bridge the gap between theoretical insight and experimental verification. These predictions detail the spatial and temporal parameters essential for effective transport, delineating plausible values for these parameters. We hope that the model’s predictions will invite experimentalists to devise innovative methodologies to test them. We plan to introduce text edits to the revised version to clarify these.