Distinct stages in stress granule assembly and disassembly

1) No evidence has been presented to show that there is no shell in any conditions examined. In order for the claim that stress granule assembly proceeds with the initial core formation to be valid, showing the presence of core at the early time point of assembly is not enough but showing the absence of shell in the initial stress granule is necessary. FRAP data in Figure 5 clearly indicate that most of G3BP in stress granule shows high mobility, consistent with liquid-like shells, 30 min after NaAsO2 treatment, which is an early time point of assembly by their definition. If shells are present in stress granule from the earliest time point they can study, it is hard to justify their argument that stress granule assembly is initiated by stable core formation.

Our model suggests stress granule core formation is an early event in stress granule assembly. The concern presented by the reviewer is whether we can also rule out if shell formation is also an early event, perhaps concomitant with core assembly, based on the data presented. We agree that our data does not conclusively rule out the possibility of shell formation as also being an early event. However, we point out that two perturbations expected to either decrease LLPS (1,6-Hexanediol treatment) or increase LLPS (lower temperatures), provide evidence that a 1,6-Hexanediol sensitive, low temperature induced LLPS is not a pre-requisite for stress granule formation. Specifically, the treatment thought to decrease LLPS (1,6-Hexanediol) actually triggers robust stress granule formation in yeast and mammals, and the treatment thought to increase LLPS (lower temperatures) actually inhibits stress granule formation. We agree with the reviewer that we cannot rule out that cores and shells form essentially at the same time, and we have re-written the text to reflect this possibility.

2) Based on the fact that treatment of yeast and mammalian cells with 1,6-Hexanediol lead to formation of stress granules, the authors draw a conclusion that "the assembly of a LLPS shell is not necessary for stress granule assembly" (Results section, subsection “Stress granule disassemble through multiple discrete steps”, Figures 8 and 9). However, no evidence has been provided for the material state of 1,6-Hexanediol induced stress granules. This claim would require FRAP measurements on these stress granules, to examine if they are indeed mostly stable cores lacking liquid-like shells.

Two issues are raised by the reviewer: first, whether the assemblies induced by 1,6-Hexanediol treatment are in fact bona fide stress granules; and two, whether the 1,6-Hexanediol-induced assemblies are more ‘core-like’ (although this is not a specific conclusion we intended to make), To address these issues, we examined the properties of 1,6-Hexanediol-induced assemblies to see how they compare to normal stress granules.

We have performed several additional experiments (now shown as Figure 10) that collectively argue 1,6-Hexanediol induces stress granule formation in a manner similar to other stresses. First, as previously shown, 1,6-Hexanediol induces the formation of assemblies that contain known stress granule proteins in both yeast and mammalian cells (HeLa and U-2 OS cells). Second, we now show 1,6-Hexanediol induced assemblies are sensitive to cycloheximide treatment, a drug known to block stress granule assembly, in both yeast and mammalian cells. Third, we now show 1,6-Hexanediol-induced assemblies from both yeast and mammals are stable in lysates suggesting that the material properties of these assemblies contain a relatively stable set of interactions similar to normal stress granules. Fourth, we show that eiF2α phosphorylation is induced to a similar extent by 1,6-Hexanediol compared to arsenite, consistent with 1,6-Hexanediol inducing stress granules by inhibiting translation initiation. Finally, as requested, we provide FRAP of 1,6-Hexanediol-induced granules in mammalian cells which indicates that 1,6-Hexanediol induced stress granules behave similar to normal stress granules. These observations lead us to conclude that 1,6-Hexanediol induces stress granules in yeast and mammalian cells.

These experiments can be interpreted in two manners: 1) that 1,6-Hexanediol disrupts LLPS in cells, and, since 1,6-Hexanediol induces stress granules indistinguishable from arsenite induced stress granules, therefore no LLPS is required for stress granule formation, or 2) that 1,6-Hexanediol is a very non-specific probe and interpretations of its effect should be carefully considered. We have re-written the discussion to clarify these interpretations.

Related to this, it is necessary to discuss the potential origin of discrepancies in experimental results for 1,6-Hexanediol treatments between this work and Kroschwald et al.

We have attempted in the discussion to explain the origins of discrepancy between our results and those presented by Kroschwald et al.

3) In Figure 2A, there are already foci in cell lysate at time 0. Does this indicate the presence of stable cores without any stress? How do these pre-existing foci affect the size distribution measured in Nanosight experiments (Figure 4)? How does the size distribution look in the earlier time point, 10 and 20 min? This is relevant since by 30 min after NaAsO2 treatment the growth phase of stress granule assembly is almost finished (based on Figure 1C).

The reviewer’s concern is whether our experiments sufficiently describe the early process of stress granule formation events to suggest cores as an early event and not as a result of rapid stress granule maturation. As the reviewer points out, our data reveals GFP-positive foci in lysates at time zero. This is because we always observe a few cells in even unstressed cultures that have stress granules. The key point of this experiment is that as stress granules are induced in cells, we observe an increasing amount of stress granule cores stable in lysates, even at early times. Thus, we feel confident in concluding that, at least as soon as we can detect stress granules in cells, stress granule cores are stable in lysates.

The reviewer also asks us to examine how the size of cores changes over very early times (10-20 minutes of stress) and whether cores are growing during this time window. To directly test whether core size increases during the ‘growth’ phase, we have examined the size using nanoparticle tracking analysis of stress granule cores at 15 minutes of arsenite stress. We observe a similar size distribution even at 15 minutes of arsenite stress, as compared to 30’, suggesting that granule cores even at early time points do not appreciably increase in size as compared to the later time points assayed. Together, we believe this result strengthens our conclusion that stress granule cores may represent an early stable compartment that serves as a platform for the accretion of substrate into the larger shell.

4) Figure 6C, what time point after NaAsO2 treatment is used for this western blot? How about at lower temperatures, like 27C? It looks like there is higher phosphorylation at 37C. This data is important to fully rule out the possibility of slower eIF2α phosphorylation at lower temperatures as an origin for delayed SG assembly.

The main issue being addressed here is similar to that of reviewer #1, and is whether translation repression is truly temperature independent, which is important to demonstrate in order to infer that stress granule assembly per se is what is affected by temperature. As detailed in comments to reviewer #1 above, we have addressed this by quantifying the eIF2α phosphorylation experiments and by adding polysome analysis of arsenite repression at different temperatures. Together, we believe this strengthens our claim that stress granule formation is delayed at lower temperatures despite being in conditions, sufficient substrate and lower temperatures, predicted to enhance phase separation (i.e. granule formation).

In addition, as requested by the reviewer, we provide the time point analyzed in the figure (20’) and in the figure legend.

5) A recent paper by Feric et.al. Cell 165(7)2016 describes liquid immiscibility of a as an organizing principle for the nucleolus. The model presented in that paper is one of two liquid droplets that do not mix with one another, with the core liquid prone to gelation over time- this seems to be a third model not considered in the current manuscript. Do the data rule out a model like this for stress granules?

We have now referenced this paper in our discussion. We should point out that this paper was published after we submitted our paper.

Reviewer #3:

My only concern is the description of granule disassembly, which the authors describe as "shell dissipation" and "core fracturing". This seems to be an over-interpretation of the images shown in Figure 10, given their low resolution. But I agree with the authors that these images do not appear to be consistent with a simple phase transition. It may be helpful to point out to the readers the features (filaments?) that appear at the edges of the stress granules during disassembly (Figure 10B, 60 min timepoint).

We thank the reviewer for their careful assessment of our microscopy data. We have added a sentence noting the appearance of features at the edges of stress granules during disassembly, however since we do not know their nature we have not discussed their possible roles in the process.