Pak1 kinase controls cell shape through ribonucleoprotein granules

Essential revisions:

1) The usage of ribonucleoprotein (RNP) granules

In this manuscript, two types of RNP granules are investigated: one is P body and the other is stress granule (SG). P bodies constitutively exist under all conditions, while SGs assemble only under stressed conditions including glucose starvation. It is true that P bodies coalesce with SGs under stresses, but as far as I am concerned, these two types of RNP granules are different. I think it would be better for the authors to use SGs in the text instead of RNP granules when they describe results under glucose deprivation (Figures 4-7). Also, in Figure 7D, RNP should be replaced with SG.

Thanks for this recommendation. We now refer to stress granules for experiments under glucose starvation. This distinction is strengthened by our new data showing colocalization of Pak1 and bona fide stress granule marker Pabp (new Figure 5 Supp 2).

2) Binding between Sts5 and 14-3-3

Previous work (Nuñez et al., 2016) showed that Orb6-dependent phosphorylation of Sts5 promotes binding between Sts5 and 14-3-3, thereby inhibiting its localization to P bodies. Is this also the case for Pak1-dependent phosphorylation of Sts5? Or in this case, does phosphorylation of serines (S261, S264) within the intrinsically disordered region (IDR) somehow (maybe by disrupting the disordered feature of the IDR?) interfere with the inclusion of Sts5 into P bodies?

Thanks for this suggestion, which led to new figures in the revised manuscript (Figures3C,D and Figure 3 Supp 1). We tested Sts5-Rad24 interactions using coimmunoprecipitation and found that S261A reduced this interaction similarly to S86A. Further, the double mutant had an additive effect and nearly abolished Sts5-Rad24 interactions. We conclude that Pak1 phosphorylates Sts5 to promote its binding to Rad24, and this interaction prevents protein clustering.

3) A marker of SGs

This is related to point (1). I think that the authors should have used an authentic marker of SGs, not Sum2 (a marker of P bodies), in experiments under glucose deprivation. At least, co-localization between Sum2 and a marker for SGs under this condition should be shown.

We appreciate this recommendation, which led to new data and a clearer picture of Pak1 localization to stress granules. As shown in the new Figure 5 Supp 2, we found that Pak1 and Sts5 both colocalize with Pabp, in addition to their colocalization with Sum2. Pabp is the major poly-A binding protein and a marker of stress granules. It is important to note that Pabp and Sum2 also colocalize with each other during glucose deprivation, consistent with previous studies showing that P bodies and stress granules are overlapping structures in yeast cells (Buchan et al., J Cell Biol., 2008; Protter and Parker, Trends Cell Biol., 2016).

4) Dissolution of Sts5 from RNP granules upon glucose re-addition: Figure 7

Tethering experiments presented in Figure 7 are of great interest. The difference with regards to the degree of dissolution of Sts5 between wild type cells and those containing CAAX-tethered Pak1 is modest, yet I think it convincing after I scrutinize all the data including those shown in Supplements. I am interested in the physiological consequences, if any, by this tethering. It looks to this referee that cell shape is substantially different between wild type and Pak1-CAAX strains (shown in the bottom row in Figure 7B). I would like to request showing quantification of cell length and width in these cells. I suspect that cells containing Pak1-CAAX display more roundish morphology upon glucose re-addition, which would represent down-regulation of Sts5 function.

The reviewer is correct that Pak1-CAAX cells have increased width when compared to wild type cells. We have quantified this effect in the new Figure 8 Supp 1B.

5) Does loss of sts5 suppresses any viability or cell separation defects of pak1 mutants, similarly to what happens in orb6 mutants?

We show that sts5∆ suppresses cell separation defects of pak1-as mutants in Figure 1E. This result supports the model that aberrant Sts5 clusters observed in pak1-as cells are toxic structures.

6) Following Pak1 kinase activity during glucose deprivation and refeeding could shed light on the dynamics of the Pka1/Pak1 regulatory pathway. Does Pka1 affects Pak1 kinase activity with or without glucose stress and during refeeding?

We agree with the reviewer, and attempted to address these questions with new experiments. Unfortunately, two technical problems prevented us from obtaining clear answers. First, our preferred method to assay kinase activity (³²P-ATP) is not currently available to us due to covid-related limitations in access. As an alternative, we attempted to test Pak1 kinase activity with SDS-PAGE band shifts but did not obtain conclusive results. Second, our attempts to isolate Pak1 from cells at timepoints during glucose starvation-refeeding did not yield clean purifications for in vitro testing. We have several ideas for how to address this important question going forward, but clearly it will require an extended period of time to establish reliable assays. To emphasize the importance of the reviewer’s suggestion, we have added the following statement into the revised manuscript: “Future studies will be directed at testing if/how PKA regulates Pak1 kinase activity during glucose starvation and refeeding.”

7) Do pak1-CAAX mutants display a delay in resuming polarized cell growth following glucose starvation, as compared with controls? This effect would further support a role for Pak1 in Sts5 dissolution and resumption of translation of specific mRNAs.

We agree with the reviewer’s suggestion but had difficulty obtaining clear results to this question. In brightfield microscopy videos, we observed a long delay in the resumption of tip growth for Pak1-CAAX versus wild type cells. However, the Pak1-CAAX cells have polarity and tip growth defects even under high glucose conditions, likely because the CAAX motif restricts Pak1 dynamics under all conditions. In our videos, it was not clear if the Pak1-CAAX cells resumed growth isotropically upon glucose addition. In addition, the delay in resuming clearly polarized growth was substantially longer than the delay in Sts5 dissolution, indicating that the growth delay likely reflects myriad changes in this strain. To avoid overinterpreting this result, we would prefer not to include it in this manuscript. We hope to clarify this issue with additional assays and controls in future work.

(8) The claim that absence of Orb6 and Pak1-dependent phosphorylation of Sts5 concentrates Sts5 predominantly to P bodies and causes downregulation of translation of target mRNA should be reconsidered. While Figure 3B shows colocalization among some Sts5(2A)+ and Sum2+ puncta, some Sts5(2A)+ Sum2- and Sts5(2A)- Sum2+ puncta are also present. The effect on translation levels should be validated by investigating at least a few other Sts5 mRNA targets (both the levels of mRNA and their protein products should be tested). The model presented in Figure 3F seems to be premature at this stage.

We extended our results beyond Ssp1 in response to this suggestion. In Figure 4 of the revised manuscript, we report that Cmk2 protein levels are also reduced in the sts5-2A mutant, similarly to Ssp1. The transcript levels of both ssp1 and cmk2 were previously shown to be upregulated in sts5∆ cells (Nunez et al., 2016), consistent with lower protein levels in the hyperactive sts5-2A mutant. In addition, we performed Nanostring experiments to measure mRNA levels of Sts5-regulated transcripts and observed reduced levels for ssp1, cmk2, psu1, and efc1 in the sts5-2A mutant. These new results are shown in Figures 4C-F as well as Figure 4 Supplement 1. We have also updated the model (now Figure 4G) to reflect reduced Cmk2 levels in sts5-2A mutant cells.

9) The difference in the rates of Sts5 puncta dissolution in presence or absence of punctate Pak1 (Figure 7C) is modest. The authors may consider investigating the physiological consequence of the observed difference in dissolution rates, which remains unclear. Further, not all Sts5+ puncta contain Pak1 (Figure 4D). Have the authors considered scoring the dissolution rates of Sts5+ Pak1+ vs. Sts5+ Pak1- puncta separately?

We agree that comparing the composition and dissolution kinetics of individual granules could reveal interesting differences. However, our analysis relied on fixed cells due to technical difficulties including photobleaching during time-lapse experiments. We attempted to address this question using several different approaches including microfluidics and chambered coverslips, but we were unable to reliably track and measure single granules. We will continue to trouble-shoot these issues going forward, but for the revised manuscript we focused on extending our fixed cell approach as in the new Figure 8D.

Reviewer #1:

[…] (1) The claim that absence of Orb6 and Pak1-dependent phosphorylation of Sts5 concentrates Sts5 predominantly to P bodies and causes downregulation of translation of target mRNA would need to be reconsidered. While Figure 3B shows colocalization among some Sts5(2A)+ and Sum2+ puncta, some Sts5(2A)+ Sum2- and Sts5(2A)- Sum2+ puncta are also present. The effect on translation levels should be validated by investigating at least a few other Sts5 mRNA targets (both the levels of mRNA and their protein products should be tested). The model presented in Figure 3F seems to be premature at this stage.

Please see our response to essential revision #8.

2) The difference in the rates of Sts5 puncta dissolution in presence or absence of punctate Pak1 (Figure 7C) is modest. The authors may consider investigating the physiological consequence of the observed difference in dissolution rates, which remains unclear. Further, not all Sts5+ puncta contain Pak1 (Figure 4D). It would be good to know whether the authors have considered scoring the dissolution rates of Sts5+ Pak1+ vs. Sts5+ Pak1- puncta separately.

Please see our response to essential revision #9.

3) The authors could consider revising the Introduction and Discussion section to make them more focused.

We have revised both sections with this goal in mind.

4) The cell outlines in Figure 3B (Sum2-tdTomato panel) are misleading.

Thanks for this comment, we have re-drawn the cell outlines as suggested.

Reviewer #2:

[…] 1. It should be noted that observations regarding Orb6 kinase phosphorylation of Serine 86, and the role of this residue in regulating Sts5 granule assembly were previously published (Chen et al., 2019). The role of Pak1 in Sts5 phosphorylation, and the specific residues phosphorylated by Pak1 kinase were also previously identified (Magliozzi et al., 2020). Therefore, the fact that Orb6 and Pak1 phosphorylate distinct residues of Sts5 or that Orb6 regulates Sts5 IDR is not a novel discovery in this paper (as mentioned repeatedly in lines 83-84 , 99-100, 158-161, 177-78, 186-87, etc.). However, the fact that Pak1 function had additive properties to Orb6 kinase in controlling the state of Sts5 granule assembly, in particular under conditions of glucose deprivation, is a novel, interesting expansion of knowledge.

We have revised the text to focus on the additive nature of this regulatory mechanism as suggested by the reviewer. It is worth noting that Figure 1 of our current manuscript is dedicated to establishing Sts5-S261 as a direct target of Pak1, which was not established in our 2020 publication and therefore represents a novel finding. Based on this novel finding which required in vitro reconstitution and phosphorylation-site mutants (Figure 1), we can now conclude that Pak1 and Orb6 phosphorylate distinct residues. We have attempted to be explicit and clear that Orb6 phosphorylation of Sts5-S86 was discovered by Chen et al., 2019, while stating that our contribution to understanding this additive mechanism relates to Sts5-S261. We hope that our wording appropriately balances credit for these discoveries, e.g. “Recent studies have shown that NDR/LATS kinase Orb6 similarly phosphorylates the Sts5 IDR to regulate its localization to RNP granules (Figure 2A) (Chen et al., 2019; Nuñez et al., 2016). Importantly, Orb6 phosphorylates S86 in the Sts5 IDR (Chen et al., 2019), while we have shown that Pak1 directly phosphorylates S261.”

2. Importantly, the authors refer to "stress granules" in several titles (line 185, line 225, line 244). These statements do not correspond to the data presented and should be corrected. Stress granules (SG granules) are separate membraneless organelles that contain specific factors, differentiating them from P-bodies. The marker Sum2, which is used in the paper, does not identify stress granules, but rather it colocalizes with Dcp1, a component of P-bodies. Therefore, data presented here supports the role of Pak1 in the control of P-bodies, not stress granules.

Thanks for this clarification. Based on this comment, we performed a series of additional colocalization experiments using a stress granule marker Pabp-mRFP. During glucose starvation, Sts5, Pak1, and Sum2 all strongly colocalized with Pabp (new Figure 5 Supp 2). Based on this result, we state in the revised text: “We refer to Pak1 and Sts5 localization to stress granules during glucose deprivation due to colocalization with Pabp, but we note that this result formally supports association with the overlapping stress granule and P body structures.”

3. The 2% glucose control cells used for experiments shown in Figure 4 (Figure 4, Supplement 1) appear very stressed. P-bodies (as visualized by Sum2) do not condense in healthy, exponentially growing cells. While this effect does not invalidate the results of the experiments shown in Figure 4, it would need to be corrected.

These cells were cultured in rich media and displayed no defects in growth rate or other proxies for cell health. Previous studies have shown that P bodies are constitutive structures that can be observed in exponentially growing fission yeast cells (Nilsson and Sunnerhagen, RNA, 2011; Wang et al., Mol Cell Biol, 2013; Wang et al., RNA, 2017) as well as budding yeast cells (Nissan and Parker, Methods in Enzymology, 2008). Consistent with these previous studies, we routinely observe P bodies in unstressed cells. These P bodies then become significantly enhanced upon stress such as glucose starvation.

4. Extending the quantification in Figure 7B, to include numbers of Sts5 granules (not only overall fluorescence intensity) would give a more precise assessment of the effects of Pak1 removal on Sts5 granule disassembly.

Thanks for this helpful suggestion. We performed this experiment and added the quantification in the new Figure 8D.

The overall results are interesting and the experiments are solidly performed and analyzed.

The paper could be strengthened by genetic epistasis experiments and by following up on the novelty of the Pka1-Pak1 regulatory axis.

Comments for the authors:

1. Does loss of sts5 suppresses any viability or cell separation defects of pak1 mutants, similarly to what happens in orb6 mutants?

Please see our response to essential revision #5.

2. Following Pak1 kinase activity during glucose deprivation and refeeding could shed light on the dynamics of the Pka1/Pak1 regulatory pathway. Does Pka1 affects Pak1 kinase activity with or without glucose stress and during refeeding?

Please see our response to essential revision #6.

3. Do pak1-CAAX mutants display a delay in resuming polarized cell growth following glucose starvation, as compared with controls? This effect would further support a role for Pak1 in Sts5 dissolution and resumption of translation of specific mRNAs.

Please see our response to essential revision #7.

4. What is the rationale of switching between Tukey's and Dunnett's methods in some of the ANOVA analyses?

We used Dunnett’s one-way ANOVA when comparing every mean measurement to a single control measurement. In contrast, we used a Tukey test to compare each mean to every other mean in the experiment. We have added this information into the Methods section of the revised manuscript.

Reviewer #3:

[…] Comments for the authors:

1) The usage of ribonucleoprotein (RNP) granules

In this manuscript, two types of RNP granules are investigated: one is P body and the other is stress granule (SG). P bodies constitutively exist under all conditions, while SGs assemble only under stressed conditions including glucose starvation. It is true that P bodies coalesce with SGs under stresses, but as far as I am concerned, these two types of RNP granules are different. I think it would be better for the authors to use SGs in the text instead of RNP granules when they describe results under glucose deprivation (Figures 4-7). Also, in Figure 7D, RNP should be replaced with SG.

Please see our response to essential revision #1.

2) Binding between Sts5 and 14-3-3

Previous work (Nuñez et al., 2016) showed that Orb6-dependent phosphorylation of Sts5 promotes binding between Sts5 and 14-3-3, thereby inhibiting its localization to P bodies. Is this also the case for Pak1-dependent phosphorylation of Sts5? Or in this case, does phosphorylation of serines (S261, S264) within the intrinsically disordered region (IDR) somehow (maybe by disrupting the disordered feature of the IDR?) interfere with the inclusion of Sts5 into P bodies?

Please see our response to essential revision #2.

3) A marker of SGs

This is related to point (1). I think that the authors should have used an authentic marker of SGs, not Sum2 (a marker of P bodies), in experiments under glucose deprivation. At least, co-localization between Sum2 and a marker for SGs under this condition should be shown.

Please see our response to essential revision #3.

4) Dissolution of Sts5 from RNP granules upon glucose re-addition: Figure 7

Tethering experiments presented in Figure 7 are of great interest. The difference with regards to the degree of dissolution of Sts5 between wild type cells and those containing CAAX-tethered Pak1 is modest, yet I think it convincing after I scrutinize all the data including those shown in Supplements. I am interested in the physiological consequences, if any, by this tethering. It looks to this referee that cell shape is substantially different between wild type and Pak1-CAAX strains (shown in the bottom row in Figure 7B). I would like to request showing quantification of cell length and width in these cells. I suspect that cells containing Pak1-CAAX display more roundish morphology upon glucose re-addition, which would represent down-regulation of Sts5 function.

Please see our response to essential revision #4.