Living cells can grow to adopt a range of different shapes. For example, fission yeast cells maintain their rod-like shape by growing from their ends and then splitting in the middle to produce two new cells of an equal size. Like many other cells, fission yeast often responds to shortages of nutrients or other environmental stressors by growing more slowly or stopping its growth altogether. One way that this stress response is achieved is by preventing certain growth-promoting proteins from being made by storing or degrading the RNA molecules that are needed to make these proteins.

Fission yeast uses an enzyme called Orb6 to control its growth and its overall shape. This enzyme is a kinase, meaning that it adds phosphate groups on to other proteins. Orb6 controls cell growth in part by defining the scope of action of an important growth control factor, Cdc42. This is done by preventing the localization of a molecule that activates Cdc42 at the wrong places of the cell membrane, such as, for example, the cell sides.

Now, Nuñez et al. show that the Orb6 enzyme also controls growth via a completely separate mechanism. Orb6 prevents an RNA-binding protein called Sts5 from being recruited into clusters of RNA molecules and proteins called granules, and directs Sts5 to interact with another protein called Rad24 instead.

Since the RNA molecules in the granules tend to end up being degraded, preventing Sts5 from being recruited to the granules protects the RNA molecules that bind to Sts5. Many Sts5-bound RNAs encode proteins required for cell growth, and in this manner Orb6 promotes the production of these Sts5-controlled proteins to encourage normal cell growth.

In the future, Nuñez et al. would like to determine how Orb6 recognizes and responds to environmental signals to control cell growth. Further studies could also explore how Sts5 and Orb6 kinase affect the assembly of RNA-protein particles that have been implicated in diseases in humans.

Many cellular processes, such as cell morphogenesis, migration, and asymmetric cell division, require eukaryotic cells to alter polarity and growth patterns (Lalli, 2014; Tahirovic and Bradke, 2009; Woodham and Machesky, 2014; Knoblich, 2008). Understanding the conserved mechanisms by which cells tune polarized cell growth has implications for studies of neuronal cell morphogenesis, neurodegenerative diseases, stem cell differentiation, and cancer (Yoshimura et al., 2006; Tahirovic and Bradke, 2009; Yamashita et al., 2010; Tanos and Rodriguez-Boulan, 2008). The fission yeast Schizosaccharomyces pombe is an excellent model system to study cell morphogenesis and growth because cells have a defined cylindrical shape that enables straightforward evaluation of changes in growth and polarity. Under exponential growth conditions, fission yeast cells display a paradigmatic pattern of cell growth, growing in a monopolar fashion during early interphase and activating bipolar growth at the new cell tip once a minimal cell length has been achieved (Mitchison and Nurse, 1985). Further, S. pombe displays a distinct morphological response to nutrient deprivation, which causes cells to divide at a shorter cell length and grow in a monopolar fashion (Su et al., 1996; Yanagida, 2009; Yanagida et al., 2011).

The NDR (Nuclear Dbf2-Related) kinase family with roles in cell morphogenesis, cell growth and proliferation, mitosis, and development, is highly conserved in cells ranging from yeast to mammalian neurons (Verde et al., 1995; Verde et al., 1998; Zinn, 2004; Hergovich et al., 2006). In humans, this subset of the AGC kinase group comprises NDR1 and NDR2 and the closely related kinases LATS1 (large tumor suppressor 1) and LATS2 (Hergovich et al., 2006), which function downstream of the MST/Hippo kinases (Meng et al., 2016). While LATS1 and LATS2 kinases are central to the Hippo pathway that plays a role in organ size and tumor suppression, dysregulation of NDR kinases has been implicated in cancers such as progressive ductal cell carcinoma, melanoma, non–small-cell lung cancer, and T-cell lymphoma (Adeyinka et al., 2002; Millward et al., 1998; Hauschild et al., 1999; Ross et al., 2000; Cornils et al., 2010). In addition to their link to cancer, NDR kinases function also in neuronal growth and differentiation, dendritic branching, and dendritic tiling, and have been implicated in memory and fear conditioning (Emoto et al., 2004; Zallen et al., 2000; Koike-Kumagai et al., 2009; Stork et al., 2004). Recent work has shown that mammalian NDR1 and NDR2 promote polarity in neurons upstream of the polarity protein Par3 (Yang et al., 2014). However, the mechanisms by which NDR kinases control cell growth and polarity are not fully understood. The fission yeast NDR kinase Orb6 is a central component of the conserved morphogenesis (MOR) regulatory network (Hergovich et al., 2006). We previously showed that NDR kinase Orb6 has a role in the establishment of cell polarity and the control of polarized cell growth (Verde et al., 1995; Verde et al., 1998). Orb6 kinase regulates cell polarity, in part, by spatially controlling conserved GTPase Cdc42 (Das et al., 2009), via inhibitory phosphorylation of Cdc42 guanine exchange factor (GEF) Gef1 (Das et al., 2015).

Here, we describe a novel role for Orb6 kinase, genetically separable from its control of the Cdc42 pathway, in promoting polarized cell growth by inhibiting translational repression. Translational repression, carried out in part by the assembly of cytoplasmic granules of ribonucleoprotein particles (RNPs), is a quick and reversible cellular strategy for inhibiting cell growth in response to stress, such as nutritional deprivation, oxidative stress, or osmotic stress (Coller and Parker, 2005; Decker and Parker, 2012; Kedersha et al., 2005; Jud et al., 2008). P-bodies, stress granules, and other RNPs such as neuronal transport granules and germ granules play important roles in mRNA regulation with implications for human diseases such as ALS, frontotemporal lobar degeneration, and viral infection (Ramaswami et al., 2013; Chahar et al., 2013). P-bodies in particular contain mRNA decay machinery and serve as sites of storage or degradation for mRNAs during times of cellular stress (Decker and Parker, 2012). In this work, we describe a novel mechanism whereby NDR kinase Orb6 negatively regulates the recruitment of mRNA-binding protein Sts5 into RNP particles and Sts5 localization to P-bodies at least in part by promoting Sts5 interaction with 14-3-3 protein Rad24. This mechanism of control prevents the degradation of mRNAs encoding proteins important for polarized cell growth and cell morphogenesis during exponential cell growth, and promotes morphological adaptation during nutritional stress.

In this article, we define a novel mechanism that spatially regulates polarized cell growth and cell morphology in fission yeast during exponential cell proliferation and in response to environmental stressors, such as increased temperature or cell density. Under exponential growth conditions, fission yeast grow in a monopolar fashion during early interphase and activate growth at the new cell tip once a minimal cell length has been achieved. Different control mechanisms cooperate in the activation of the second tip, a process known as NETO (New End Take Off), including the microtubule-dependent Tea1 complex (Martin and Arkowitz, 2014; Sawin and Nurse, 1998; Martin et al., 2005; Tatebe et al., 2005), the availability of Cdc42 regulators (Coll et al., 2003; Tatebe et al., 2008; Das et al., 2012), cell transcription (Vjestica et al., 2013), and a diverse array of signaling kinases (Koyano et al., 2010; Matsusaka et al., 1995; Rupes et al., 1999; Koyano et al., 2015; Grallert et al., 2013). We have previously shown that NDR kinase Orb6 promotes cell polarity and regulates bipolar growth by spatially restricting the activation of Cdc42 GTPase, a key morphology control factor (Das et al., 2009). We recently showed that Orb6 negatively regulates Cdc42 activation by promoting the association of Cdc42 Guanine Exchange Factor (GEF) Gef1 with 14-3-3 protein Rad24, and thus limiting Gef1 activity at the membrane (Das et al., 2015). This function has the effect of spatially regulating Cdc42 activation, thus promoting the emergence of cell polarity.

In this article, we describe a genetically separable role for Orb6 kinase in the control of polarized cell growth and cell separation. We report that Orb6 kinase regulates the association of mRNA-binding protein Sts5, another Orb6 substrate target, with 14-3-3 protein Rad24. This association prevents the recruitment of Sts5 into cytoplasmic puncta (see hypothetical model in Figure 10A). Orb6 localization varies during the cell cycle, increasing at the cell tips during interphase and at the cell septum during cell division (Verde et al., 1998; Wiley et al., 2003). In small cells that have not yet undergone NETO, or in the monopolar tea1Δ mutant cells, Orb6 is enriched at the one growing cell tip. Consistent with a role for Orb6 in inhibiting Sts5 assembly into puncta, we have observed that Sts5-3xGFP puncta are spatially anti-correlated with Orb6 kinase activity and are preferentially localized near non-growing cell tips, which are depleted of Orb6 kinase (Figure 10B). Because Orb6 kinase is enriched at growing cell tips, this method of Sts5 regulation might promote the availability of Sts5-targeted mRNAs for translation near sites of polarized growth, while partially constraining growth at the non-growing, newer cell tip. Consistent with this idea, an increased percentage of sts5Δ cells exhibit a bipolar pattern of growth, as compared to control cells of similar length.

Figure 10

Download asset Open asset

By microarray and qPCR analysis we found that several transcripts, previously implicated in bipolar growth activation, accumulate in sts5Δ cells. These transcripts encode the putative CAMK kinase Cmk2, CAMKK kinase Ssp1, LAMMER kinase Lkh1, pseudokinase Tea5/Ppk2, and PDK1 kinase Ksg1, which promote bipolar growth activation in S. pombe (Koyano et al., 2010), as well as the Ras1 GEF Efc25, which affects Cdc42 activity (Papadaki et al., 2002). In higher eukaryotes, the homologues of these Sts5-regulated transcripts are also implicated in cell growth and morphology. CAMK signaling regulates cytoskeletal organization, plays a role in neuronal development and dendritic spine morphology, and has been shown to be increased in mouse models of cardiac hypertrophy (Penzes et al., 2008; Passier et al., 2000). The Drosophila LAMMER kinase Doa inhibits proliferation of germ cells (Zhao et al., 2013). Also, Ras signaling has conserved roles in cytoskeletal organization with implications for cancer development (Shields et al., 2000). Activation of PI3K signaling via PDK1 kinase has been implicated in cancer, and PDK1 has been found to regulate cell growth, proliferation, and migration (Li et al., 2010; Mora et al., 2004).

Consistent with Orb6 kinase inhibiting the extent of Sts5 recruitment during exponential growth, Sts5-containing cytoplasmic puncta assemble during the later stages of mitosis when Orb6 kinase activity is blocked by the SIN pathway (Figure 10B) (Vaggi et al., 2012; Gupta et al., 2013, Gupta et al., 2014). Sts5 recruitment during cytokinesis may function to prevent inappropriate translation of proteins involved in septum degradation while the septum is still forming. Indeed, sts5∆ cells lyse at the cell septum, in particular upon stress. Once cytokinesis is complete and Orb6 kinase is once again active, Sts5 localization is again diffuse in the cytoplasm, perhaps to allow expression of hydrolases required for cell separation. Consistent with this idea, orb6 mutants delay cell separation. We did not find obvious induction of P-body formation during mitosis or interphase, likely preventing the degradation of Sts5-regulated transcripts that will eventually be needed for cell separation and polarized cell growth. This result suggests that P-body independent, Sts5-containing RNPs are formed during mitosis and that additional stress signals are required for Sts5 to seed P-body formation under nutritional limitation conditions.

Sts5 bears closest homology to RNA exonucleases such as fission yeast Dis3L2 (Malecki et al., 2013). In humans, hDis3L2 is of particular interest because it has been implicated in the congenital Perlman syndrome, which confers fetal overgrowth and susceptibility to Wilms tumor (Astuti et al., 2012). Whereas Sts5 lacks crucial amino acids required for exonuclease activity (Figure 1C) (Malecki et al., 2013; Uesono et al., 1997), our work shows that Sts5 promotes mRNA degradation likely by promoting the interaction of Sts5-associated transcripts with P-body components. Similar to Sts5, Dis3L2 also localizes to P-bodies (Malecki et al., 2013), as well as the S. cerevisiae homologue of Sts5, Ssd1 (Jansen et al., 2009; Kurischko et al., 2011). These findings suggest that, differently from the related exonuclease Dis3 (Robinson et al., 2015), this group of RNA-binding proteins employs a mechanism of mRNA degradation that does not involve interaction with the exosome. However, it is likely that Sts5 and Dis3L2 have different roles in P-body assembly and/or recruitment of mRNAs to P-bodies. Indeed, deletion of the sts5 homologue dis3L2 does not suppress the growth defect observed upon Orb6-as2 kinase inhibition (Figure 1—figure supplement 2B). In S. cerevisiae and C. albicans, Sts5 homologue Ssd1, with roles in cell wall hydrolysis (Jansen et al., 2009; Wanless et al., 2014), fertility (Bourens et al., 2009), and transcription (Lee et al., 2015), has been proposed to promote localized mRNA translation because of its enrichment at the bud site (Kurischko et al., 2011; Lee et al., 2015). We have not observed enrichment of Sts5 at the sites of polarized growth in S. pombe cells, which may employ a different strategy for ensuring adequate mRNA localization in daughter cells.

Our work indicates that Sts5 may function as a seed for P-body formation under stress and upon Orb6 kinase inhibition. P-bodies and similar RNPs have been shown to have liquid properties and their formation has been described as condensation by a phase-separation mechanism once P-body components reach critical concentrations (Kroschwald et al., 2015; Lin et al., 2015; Elbaum-Garfinkle et al., 2015; Hyman et al., 2014; Brangwynne et al., 2009; Lee et al., 2013; Brangwynne, 2013; Becker and Gitler, 2015). It has been proposed that mRNA-binding proteins may play a role in the aggregation of mRNPs into larger structures, especially proteins containing intrinsically disordered domains, which have the potential to promote phase separation by forming multiple weak protein-protein interactions (Lin et al., 2015; Patel et al., 2015; Elbaum-Garfinkle et al., 2015; Wang et al., 2014; Kato et al., 2012; Han et al., 2012; Malinovska et al., 2013; Toretsky and Wright, 2014; Kroschwald et al., 2015). Thus, Sts5 may have the ability to function in mRNP granule formation through interactions with other P-body proteins, and one function of Orb6 phosphorylation may be to limit Sts5 recuitment to prevent inappropriate seeding of P-bodies. Consistent with this idea, Sts5 displays a predicted disordered domain at the N-terminus, which is phosphorylated in vivo (Kettenbach et al., 2015; Carpy et al., 2014; Koch et al., 2011; Wilson-Grady et al., 2008). Future experiments will address how phosphorylation of this domain modulates the function of Sts5 and affects its properties in vitro.

We found that Sts5 has a role in the morphological response to nutritional stress. Wild type S. pombe cells mount characteristic morphological responses to changing environmental conditions: increased temperature (Mitchison and Nurse, 1985), decreased nutrient availability (Costello et al., 1986; Su et al., 1996; Yanagida, 2009; Yanagida et al., 2011), and hyper-osmotic stress decrease the incidence of bipolar growth activation and alter overall cell dimensions (Rupes et al., 1999; Robertson and Hagan, 2008). The mechanisms that modulate cell morphogenesis and polarized cell growth in response to varying growth and environmental conditions are still poorly understood. We find that sts5Δ mutants delay the adaptation to starvation conditions, maintaining bipolar growth and a longer cell length as cell density increases and the medium becomes depleted of nutrients. This effect appears to be further exacerbated at higher temperatures (36°C), where sts5Δ cells become enlarged and bloated, with increased protein levels of the CAMKK Ssp1. Our findings indicate that sts5Δ mutants have defects in adapting to nutrient deprivation or temperature increase, and fail to manifest the appropriate morphological response to varying extracellular conditions. Thus Sts5 may integrate diverse nutritional and environmental signals to coordinate changes to the pattern of cell growth.

In summary, our results support a role for NDR kinases in the spatial control of polarized cell growth, during cell proliferation and in response to the nutritional environment by mediating the translational availability of specific mRNAs. Future research will seek to identify nutrient-sensitive signaling pathways upstream of Orb6 and define the specific roles of Orb6 kinase and Sts5 in the control of P-body assembly. Due to the conservation of these factors, this work has the potential to open new avenues of research linking nutrient-sensitive signaling and P-body regulation with implications for studies of cancer and neurodegenerative diseases.

1. 1. Adeyinka A
   2. Emberley E
   3. Niu Y
   4. Snell L
   5. Murphy LC
   6. Sowter H
   7. Wykoff CC
   8. Harris AL
   9. Watson PH

   (2002)

   Analysis of gene expression in ductal carcinoma in situ of the breast

   Clinical Cancer Research 8:3788–3795.

   • Google Scholar
2. 1. Alcaide-Gavilán M
   2. Lahoz A
   3. Daga RR
   4. Jimenez J

   (2014) Feedback regulation of SIN by Etd1 and Rho1 in fission yeast

   Genetics 196:455–470.

   https://doi.org/10.1534/genetics.113.155218

   • Google Scholar
3. 1. Astuti D
   2. Morris MR
   3. Cooper WN
   4. Staals RH
   5. Wake NC
   6. Fews GA
   7. Gill H
   8. Gentle D
   9. Shuib S
   10. Ricketts CJ
   11. Cole T
   12. van Essen AJ
   13. van Lingen RA
   14. Neri G
   15. Opitz JM
   16. Rump P
   17. Stolte-Dijkstra I
   18. Müller F
   19. Pruijn GJ
   20. Latif F
   21. Maher ER

   (2012) Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility

   Nature Genetics 44:277–284.

   https://doi.org/10.1038/ng.1071

   • Google Scholar
4. 1. Becker LA
   2. Gitler AD

   (2015) It's all starting to come together

   eLife 4:e09853.

   https://doi.org/10.7554/eLife.09853

   • Google Scholar
5. 1. Bolte S
   2. Cordelières FP

   (2006) A guided tour into subcellular colocalization analysis in light microscopy

   Journal of Microscopy 224:213–232.

   https://doi.org/10.1111/j.1365-2818.2006.01706.x

   • Google Scholar
6. 1. Bourens M
   2. Panozzo C
   3. Nowacka A
   4. Imbeaud S
   5. Mucchielli MH
   6. Herbert CJ

   (2009) Mutations in the Saccharomyces cerevisiae kinase Cbk1p lead to a fertility defect that can be suppressed by the absence of Brr1p or Mpt5p (Puf5p), proteins involved in RNA metabolism

   Genetics 183:161–234.

   https://doi.org/10.1534/genetics.109.105130

   • Google Scholar
7. 1. Brangwynne CP
   2. Eckmann CR
   3. Courson DS
   4. Rybarska A
   5. Hoege C
   6. Gharakhani J
   7. Jülicher F
   8. Hyman AA

   (2009) Germline P granules are liquid droplets that localize by controlled dissolution/condensation

   Science 324:1729–1732.

   https://doi.org/10.1126/science.1172046

   • Google Scholar
8. 1. Brangwynne CP

   (2013) Phase transitions and size scaling of membrane-less organelles

   Journal of Cell Biology 203:875–881.

   https://doi.org/10.1083/jcb.201308087

   • Google Scholar
9. 1. Brengues M
   2. Parker R

   (2007) Accumulation of polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-bodies in Saccharomyces cerevisiae

   Molecular Biology of the Cell 18:2592–3194.

   https://doi.org/10.1091/mbc.E06-12-1149

   • Google Scholar
10. 1. Carpy A
    2. Krug K
    3. Graf S
    4. Koch A
    5. Popic S
    6. Hauf S
    7. Macek B

    (2014) Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast)

    Molecular & Cellular Proteomics 13:1925–1961.

    https://doi.org/10.1074/mcp.M113.035824

    • Google Scholar
11. 1. Chahar HS
    2. Chen S
    3. Manjunath N

    (2013) P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication

    Virology 436:1–7.

    https://doi.org/10.1016/j.virol.2012.09.041

    • Google Scholar
12. 1. Coll PM
    2. Trillo Y
    3. Ametzazurra A
    4. Perez P

    (2003) Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

    Molecular Biology of the Cell 14:313–323.

    https://doi.org/10.1091/mbc.E02-07-0400

    • Google Scholar
13. 1. Coller J
    2. Parker R

    (2005) General translational repression by activators of mRNA decapping

    Cell 122:875–961.

    https://doi.org/10.1016/j.cell.2005.07.012

    • Google Scholar
14. 1. Cornils H
    2. Stegert MR
    3. Hergovich A
    4. Hynx D
    5. Schmitz D
    6. Dirnhofer S
    7. Hemmings BA

    (2010) Ablation of the kinase NDR1 predisposes mice to the development of T cell lymphoma

    Science Signaling 3:ra47.

    https://doi.org/10.1126/scisignal.2000681

    • Google Scholar
15. 1. Costello G
    2. Rodgers L
    3. Beach D

    (1986) Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2

    Current Genetics 11:119–125.

    https://doi.org/10.1007/BF00378203

    • Google Scholar
16. 1. Das M
    2. Drake T
    3. Wiley DJ
    4. Buchwald P
    5. Vavylonis D
    6. Verde F

    (2012) Oscillatory dynamics of Cdc42 GTPase in the control of polarized growth

    Science 337:239–243.

    https://doi.org/10.1126/science.1218377

    • Google Scholar
17. 1. Das M
    2. Nuñez I
    3. Rodriguez M
    4. Wiley DJ
    5. Rodriguez J
    6. Sarkeshik A
    7. Yates JR
    8. Buchwald P
    9. Verde F

    (2015) Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    Molecular Biology of the Cell 26:3520–3534.

    https://doi.org/10.1091/mbc.E15-02-0095

    • Google Scholar
18. 1. Das M
    2. Wiley DJ
    3. Chen X
    4. Shah K
    5. Verde F

    (2009) The conserved NDR kinase Orb6 controls polarized cell growth by spatial regulation of the small GTPase Cdc42

    Current Biology 19:1314–1319.

    https://doi.org/10.1016/j.cub.2009.06.057

    • Google Scholar
19. 1. Decker CJ
    2. Parker R

    (2012) P-bodies and stress granules: possible roles in the control of translation and mRNA degradation

    Cold Spring Harbor Perspectives in Biology 4:a012286.

    https://doi.org/10.1101/cshperspect.a012286

    • Google Scholar
20. 1. Decker CJ
    2. Teixeira D
    3. Parker R

    (2007) Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

    The Journal of Cell Biology 179:437–486.

    https://doi.org/10.1083/jcb.200704147

    • Google Scholar
21. 1. Elbaum-Garfinkle S
    2. Kim Y
    3. Szczepaniak K
    4. Chen CC
    5. Eckmann CR
    6. Myong S
    7. Brangwynne CP

    (2015) The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

    PNAS 112:7189–7283.

    https://doi.org/10.1073/pnas.1504822112

    • Google Scholar
22. 1. Emoto K
    2. He Y
    3. Ye B
    4. Grueber WB
    5. Adler PN
    6. Jan LY
    7. Jan YN

    (2004) Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons

    Cell 119:245–256.

    https://doi.org/10.1016/j.cell.2004.09.036

    • Google Scholar
23. 1. Eulalio A
    2. Behm-Ansmant I
    3. Schweizer D
    4. Izaurralde E

    (2007) P-body formation is a consequence, not the cause, of RNA-mediated gene silencing

    Molecular and Cellular Biology 27:3970–4051.

    https://doi.org/10.1128/MCB.00128-07

    • Google Scholar
24. 1. Faehnle CR
    2. Walleshauser J
    3. Joshua-Tor L

    (2014) Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway

    Nature 514:252–256.

    https://doi.org/10.1038/nature13553

    • Google Scholar
25. 1. Fromm SA
    2. Truffault V
    3. Kamenz J
    4. Braun JE
    5. Hoffmann NA
    6. Izaurralde E
    7. Sprangers R

    (2012) The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex

    The EMBO Journal 31:279–369.

    https://doi.org/10.1038/emboj.2011.408

    • Google Scholar
26. 1. García-Cortés JC
    2. McCollum D

    (2009) Proper timing of cytokinesis is regulated by Schizosaccharomyces pombe Etd1

    The Journal of Cell Biology 186:739–753.

    https://doi.org/10.1083/jcb.200902116

    • Google Scholar
27. 1. Grallert A
    2. Patel A
    3. Tallada VA
    4. Chan KY
    5. Bagley S
    6. Krapp A
    7. Simanis V
    8. Hagan IM

    (2013) Centrosomal MPF triggers the mitotic and morphogenetic switches of fission yeast

    Nature Cell Biology 15:88–95.

    https://doi.org/10.1038/ncb2633

    • Google Scholar
28. 1. Gupta S
    2. Govindaraghavan M
    3. McCollum D

    (2014) Cross talk between NDR kinase pathways coordinates cytokinesis with cell separation in Schizosaccharomyces pombe

    Eukaryotic Cell 13:1104–1112.

    https://doi.org/10.1128/EC.00129-14

    • Google Scholar
29. 1. Gupta S
    2. Mana-Capelli S
    3. McLean JR
    4. Chen CT
    5. Ray S
    6. Gould KL
    7. McCollum D

    (2013) Identification of SIN pathway targets reveals mechanisms of crosstalk between NDR kinase pathways

    Current Biology 23:333–338.

    https://doi.org/10.1016/j.cub.2013.01.014

    • Google Scholar
30. 1. Gógl G
    2. Schneider KD
    3. Yeh BJ
    4. Alam N
    5. Nguyen Ba AN
    6. Moses AM
    7. Hetényi C
    8. Reményi A
    9. Weiss EL

    (2015) The structure of an NDR/LATS kinase-mob complex reveals a novel kinase-coactivator system and substrate docking mechanism

    PLoS Biology 13:e1002146.

    https://doi.org/10.1371/journal.pbio.1002146

    • Google Scholar
31. 1. Han TW
    2. Kato M
    3. Xie S
    4. Wu LC
    5. Mirzaei H
    6. Pei J
    7. Chen M
    8. Xie Y
    9. Allen J
    10. Xiao G
    11. McKnight SL

    (2012) Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies

    Cell 149:768–847.

    https://doi.org/10.1016/j.cell.2012.04.016

    • Google Scholar
32. 1. Hao Y
    2. Chun A
    3. Cheung K
    4. Rashidi B
    5. Yang X

    (2008) Tumor suppressor LATS1 is a negative regulator of oncogene YAP

    Journal of Biological Chemistry 283:5496–5509.

    https://doi.org/10.1074/jbc.M709037200

    • Google Scholar
33. 1. Hauschild A
    2. Engel G
    3. Brenner W
    4. Gläser R
    5. Mönig H
    6. Henze E
    7. Christophers E

    (1999) S100B protein detection in serum is a significant prognostic factor in metastatic melanoma

    Oncology 56:338–344.

    https://doi.org/10.1159/000011989

    • Google Scholar
34. 1. Heinrich S
    2. Geissen EM
    3. Kamenz J
    4. Trautmann S
    5. Widmer C
    6. Drewe P
    7. Knop M
    8. Radde N
    9. Hasenauer J
    10. Hauf S

    (2013) Determinants of robustness in spindle assembly checkpoint signalling

    Nature Cell Biology 15:1328–1367.

    https://doi.org/10.1038/ncb2864

    • Google Scholar
35. 1. Hergovich A
    2. Stegert MR
    3. Schmitz D
    4. Hemmings BA

    (2006) NDR kinases regulate essential cell processes from yeast to humans

    Nature Reviews Molecular Cell Biology 7:253–264.

    https://doi.org/10.1038/nrm1891

    • Google Scholar
36. 1. Hoffman GR
    2. Cerione RA

    (2000) Flipping the switch: the structural basis for signaling through the CRIB motif

    Cell 102:403–406.

    https://doi.org/10.1016/S0092-8674(00)00045-3

    • Google Scholar
37. 1. Hogan DJ
    2. Riordan DP
    3. Gerber AP
    4. Herschlag D
    5. Brown PO

    (2008) Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system

    PLoS Biology 6:e255.

    https://doi.org/10.1371/journal.pbio.0060255

    • Google Scholar
38. 1. Hou MC
    2. Wiley DJ
    3. Verde F
    4. McCollum D

    (2003) Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression

    Journal of Cell Science 116:125–135.

    https://doi.org/10.1242/jcs.00206

    • Google Scholar
39. 1. Hyman AA
    2. Weber CA
    3. Jülicher F

    (2014) Liquid-liquid phase separation in biology

    Annual Review of Cell and Developmental Biology 30:39–58.

    https://doi.org/10.1146/annurev-cellbio-100913-013325

    • Google Scholar
40. 1. Jansen JM
    2. Wanless AG
    3. Seidel CW
    4. Weiss EL

    (2009) Cbk1 regulation of the RNA-binding protein Ssd1 integrates cell fate with translational control

    Current Biology 19:2114–2120.

    https://doi.org/10.1016/j.cub.2009.10.071

    • Google Scholar
41. 1. Jud MC
    2. Czerwinski MJ
    3. Wood MP
    4. Young RA
    5. Gallo CM
    6. Bickel JS
    7. Petty EL
    8. Mason JM
    9. Little BA
    10. Padilla PA
    11. Schisa JA

    (2008) Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

    Developmental Biology 318:38–51.

    https://doi.org/10.1016/j.ydbio.2008.02.059

    • Google Scholar
42. 1. Kanai M
    2. Kume K
    3. Miyahara K
    4. Sakai K
    5. Nakamura K
    6. Leonhard K
    7. Wiley DJ
    8. Verde F
    9. Toda T
    10. Hirata D

    (2005) Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

    The EMBO Journal 24:3012–3025.

    https://doi.org/10.1038/sj.emboj.7600782

    • Google Scholar
43. 1. Kato M
    2. Han TW
    3. Xie S
    4. Shi K
    5. Du X
    6. Wu LC
    7. Mirzaei H
    8. Goldsmith EJ
    9. Longgood J
    10. Pei J
    11. Grishin NV
    12. Frantz DE
    13. Schneider JW
    14. Chen S
    15. Li L
    16. Sawaya MR
    17. Eisenberg D
    18. Tycko R
    19. McKnight SL

    (2012) Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels

    Cell 149:753–820.

    https://doi.org/10.1016/j.cell.2012.04.017

    • Google Scholar
44. 1. Kedersha N
    2. Stoecklin G
    3. Ayodele M
    4. Yacono P
    5. Lykke-Andersen J
    6. Fritzler MJ
    7. Scheuner D
    8. Kaufman RJ
    9. Golan DE
    10. Anderson P

    (2005) Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

    Journal of Cell Biology 169:871–884.

    https://doi.org/10.1083/jcb.200502088

    • Google Scholar
45. 1. Kettenbach AN
    2. Deng L
    3. Wu Y
    4. Baldissard S
    5. Adamo ME
    6. Gerber SA
    7. Moseley JB

    (2015) Quantitative phosphoproteomics reveals pathways for coordination of cell growth and division by the conserved fission yeast kinase pom1

    Molecular & Cellular Proteomics 14:1275–1362.

    https://doi.org/10.1074/mcp.M114.045245

    • Google Scholar
46. 1. Knoblich JA

    (2008) Mechanisms of asymmetric stem cell division

    Cell 132:583–680.

    https://doi.org/10.1016/j.cell.2008.02.007

    • Google Scholar
47. 1. Koch A
    2. Krug K
    3. Pengelley S
    4. Macek B
    5. Hauf S

    (2011) Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast

    Science Signaling 4:rs6.

    https://doi.org/10.1126/scisignal.2001588

    • Google Scholar
48. 1. Koike-Kumagai M
    2. Yasunaga K
    3. Morikawa R
    4. Kanamori T
    5. Emoto K

    (2009) The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

    The EMBO Journal 28:3879–3892.

    https://doi.org/10.1038/emboj.2009.312

    • Google Scholar
49. 1. Koyano T
    2. Barnouin K
    3. Snijders AP
    4. Kume K
    5. Hirata D
    6. Toda T

    (2015) Casein kinase 1γ acts as a molecular switch for cell polarization through phosphorylation of the polarity factor Tea1 in fission yeast

    Genes to Cells 20:1046–1104.

    https://doi.org/10.1111/gtc.12309

    • Google Scholar
50. 1. Koyano T
    2. Kume K
    3. Konishi M
    4. Toda T
    5. Hirata D

    (2010) Search for kinases related to transition of growth polarity in fission yeast

    Bioscience, Biotechnology, and Biochemistry 74:1129–1133.

    https://doi.org/10.1271/bbb.100223

    • Google Scholar
51. 1. Kroschwald S
    2. Maharana S
    3. Mateju D
    4. Malinovska L
    5. Nüske E
    6. Poser I
    7. Richter D
    8. Alberti S
    9. Nuske E

    (2015) Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules

    eLife 4:e06807.

    https://doi.org/10.7554/eLife.06807

    • Google Scholar
52. 1. Kurischko C
    2. Kim HK
    3. Kuravi VK
    4. Pratzka J
    5. Luca FC

    (2011) The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

    Journal of Cell Biology 192:583–598.

    https://doi.org/10.1083/jcb.201011061

    • Google Scholar
53. 1. Lalli G

    (2014) Regulation of neuronal polarity

    Experimental Cell Research 328:267–275.

    https://doi.org/10.1016/j.yexcr.2014.07.033

    • Google Scholar
54. 1. Le Goff X
    2. Motegi F
    3. Salimova E
    4. Mabuchi I
    5. Simanis V

    (2000)

    The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p

    Journal of Cell Science 113 Pt 23:4157–4220.

    • Google Scholar
55. 1. Lee CF
    2. Brangwynne CP
    3. Gharakhani J
    4. Hyman AA
    5. Jülicher F

    (2013) Spatial organization of the cell cytoplasm by position-dependent phase separation

    Physical Review Letters 111:088101.

    https://doi.org/10.1103/PhysRevLett.111.088101

    • Google Scholar
56. 1. Lee HJ
    2. Kim JM
    3. Kang WK
    4. Yang H
    5. Kim JY

    (2015) The NDR Kinase Cbk1 Downregulates the Transcriptional Repressor Nrg1 through the mRNA-Binding Protein Ssd1 in Candida albicans

    Eukaryotic Cell 14:671–754.

    https://doi.org/10.1128/EC.00016-15

    • Google Scholar
57. 1. Li Y
    2. Yang KJ
    3. Park J
    4. Park J

    (2010) Multiple implications of 3-phosphoinositide-dependent protein kinase 1 in human cancer

    World Journal of Biological Chemistry 1:239–247.

    https://doi.org/10.4331/wjbc.v1.i8.239

    • Google Scholar
58. 1. Lin Y
    2. Protter DS
    3. Rosen MK
    4. Parker R

    (2015) Formation and maturation of phase-separated Liquid droplets by RNA-binding proteins

    Molecular Cell 60:208–227.

    https://doi.org/10.1016/j.molcel.2015.08.018

    • Google Scholar
59. 1. Lv H
    2. Zhu Y
    3. Qiu Y
    4. Niu L
    5. Teng M
    6. Li X

    (2015) Structural analysis of Dis3l2, an exosome-independent exonuclease from Schizosaccharomyces pombe

    Acta Crystallographica Section D Biological Crystallography 71:1284–1294.

    https://doi.org/10.1107/S1399004715005805

    • Google Scholar
60. 1. Makino DL
    2. Baumgärtner M
    3. Conti E

    (2013) Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex

    Nature 495:70–75.

    https://doi.org/10.1038/nature11870

    • Google Scholar
61. 1. Malecki M
    2. Viegas SC
    3. Carneiro T
    4. Golik P
    5. Dressaire C
    6. Ferreira MG
    7. Arraiano CM

    (2013) The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway

    The EMBO Journal 32:1842–1854.

    https://doi.org/10.1038/emboj.2013.63

    • Google Scholar
62. 1. Malinovska L
    2. Kroschwald S
    3. Alberti S

    (2013) Protein disorder, prion propensities, and self-organizing macromolecular collectives

    Biochimica Et Biophysica Acta 1834:918–931.

    https://doi.org/10.1016/j.bbapap.2013.01.003

    • Google Scholar
63. 1. Martin SG
    2. Arkowitz RA

    (2014) Cell polarization in budding and fission yeasts

    FEMS Microbiology Reviews 38:228–253.

    https://doi.org/10.1111/1574-6976.12055

    • Google Scholar
64. 1. Martin SG
    2. McDonald WH
    3. Yates JR
    4. Chang F

    (2005) Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity

    Developmental Cell 8:479–491.

    https://doi.org/10.1016/j.devcel.2005.02.008

    • Google Scholar
65. 1. Martín-Cuadrado AB
    2. Dueñas E
    3. Sipiczki M
    4. Vázquez de Aldana CR
    5. del Rey F

    (2003) The endo-beta-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe

    Journal of Cell Science 116:1689–1698.

    https://doi.org/10.1242/jcs.00377

    • Google Scholar
66. 1. Matsuo Y
    2. Asakawa K
    3. Toda T
    4. Katayama S

    (2006) A rapid method for protein extraction from fission yeast

    Bioscience, Biotechnology, and Biochemistry 70:1992–1994.

    https://doi.org/10.1271/bbb.60087

    • Google Scholar
67. 1. Matsusaka T
    2. Hirata D
    3. Yanagida M
    4. Toda T

    (1995)

    A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast

    The EMBO Journal 14:3325–3338.

    • Google Scholar
68. 1. Mazanka E
    2. Alexander J
    3. Yeh BJ
    4. Charoenpong P
    5. Lowery DM
    6. Yaffe M
    7. Weiss EL

    (2008) The NDR/LATS family kinase Cbk1 directly controls transcriptional asymmetry

    PLoS Biology 6:e203.

    https://doi.org/10.1371/journal.pbio.0060203

    • Google Scholar
69. 1. Meng Z
    2. Moroishi T
    3. Guan KL

    (2016) Mechanisms of Hippo pathway regulation

    Genes & Development 30:1–17.

    https://doi.org/10.1101/gad.274027.115

    • Google Scholar
70. 1. Millward TA
    2. Heizmann CW
    3. Schäfer BW
    4. Hemmings BA

    (1998) Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins

    The EMBO Journal 17:5913–5922.

    https://doi.org/10.1093/emboj/17.20.5913

    • Google Scholar
71. 1. Mitchison JM
    2. Nurse P

    (1985)

    Growth in cell length in the fission yeast Schizosaccharomyces pombe

    Journal of Cell Science 75:357–376.

    • Google Scholar
72. 1. Mora A
    2. Komander D
    3. van Aalten DM
    4. Alessi DR

    (2004) PDK1, the master regulator of AGC kinase signal transduction

    Seminars in Cell & Developmental Biology 15:161–170.

    https://doi.org/10.1016/j.semcdb.2003.12.022

    • Google Scholar
73. 1. Moreno S
    2. Klar A
    3. Nurse P

    (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe

    Methods in Enzymology 194:795–823.

    https://doi.org/10.1016/0076-6879(91)94059-L

    • Google Scholar
74. 1. Nilsson D
    2. Sunnerhagen P

    (2011) Cellular stress induces cytoplasmic RNA granules in fission yeast

    RNA 17:120–153.

    https://doi.org/10.1261/rna.2268111

    • Google Scholar
75. 1. Papadaki P
    2. Pizon V
    3. Onken B
    4. Chang EC

    (2002) Two ras pathways in fission yeast are differentially regulated by two ras guanine nucleotide exchange factors

    Molecular and Cellular Biology 22:4598–4606.

    https://doi.org/10.1128/MCB.22.13.4598-4606.2002

    • Google Scholar
76. 1. Passier R
    2. Zeng H
    3. Frey N
    4. Naya FJ
    5. Nicol RL
    6. McKinsey TA
    7. Overbeek P
    8. Richardson JA
    9. Grant SR
    10. Olson EN

    (2000) CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo

    Journal of Clinical Investigation 105:1395–1406.

    https://doi.org/10.1172/JCI8551

    • Google Scholar
77. 1. Patel A
    2. Lee HO
    3. Jawerth L
    4. Maharana S
    5. Jahnel M
    6. Hein MY
    7. Stoynov S
    8. Mahamid J
    9. Saha S
    10. Franzmann TM
    11. Pozniakovski A
    12. Poser I
    13. Maghelli N
    14. Royer LA
    15. Weigert M
    16. Myers EW
    17. Grill S
    18. Drechsel D
    19. Hyman AA
    20. Alberti S

    (2015) A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation

    Cell 162:1066–1143.

    https://doi.org/10.1016/j.cell.2015.07.047

    • Google Scholar
78. 1. Penzes P
    2. Cahill ME
    3. Jones KA
    4. Srivastava DP

    (2008) Convergent CaMK and RacGEF signals control dendritic structure and function

    Trends in Cell Biology 18:405–413.

    https://doi.org/10.1016/j.tcb.2008.07.002

    • Google Scholar
79. 1. Raj A
    2. van den Bogaard P
    3. Rifkin SA
    4. van Oudenaarden A
    5. Tyagi S

    (2008) Imaging individual mRNA molecules using multiple singly labeled probes

    Nature Methods 5:877–879.

    https://doi.org/10.1038/nmeth.1253

    • Google Scholar
80. 1. Ramaswami M
    2. Taylor JP
    3. Parker R

    (2013) Altered ribostasis: RNA-protein granules in degenerative disorders

    Cell 154:727–736.

    https://doi.org/10.1016/j.cell.2013.07.038

    • Google Scholar
81. 1. Robertson AM
    2. Hagan IM

    (2008) Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress in S. pombe

    Journal of Cell Science 121:4055–4068.

    https://doi.org/10.1242/jcs.034488

    • Google Scholar
82. 1. Robinson SR
    2. Oliver AW
    3. Chevassut TJ
    4. Newbury SF

    (2015) The 3' to 5' Exoribonuclease DIS3: From Structure and Mechanisms to Biological Functions and Role in Human Disease

    Biomolecules 5:1515–1539.

    https://doi.org/10.3390/biom5031515

    • Google Scholar
83. 1. Ross DT
    2. Scherf U
    3. Eisen MB
    4. Perou CM
    5. Rees C
    6. Spellman P
    7. Iyer V
    8. Jeffrey SS
    9. Van de Rijn M
    10. Waltham M
    11. Pergamenschikov A
    12. Lee JC
    13. Lashkari D
    14. Shalon D
    15. Myers TG
    16. Weinstein JN
    17. Botstein D
    18. Brown PO

    (2000) Systematic variation in gene expression patterns in human cancer cell lines

    Nature Genetics 24:227–235.

    https://doi.org/10.1038/73432

    • Google Scholar
84. 1. Rupes I
    2. Jia Z
    3. Young PG

    (1999) Ssp1 promotes actin depolymerization and is involved in stress response and new end take-off control in fission yeast

    Molecular Biology of the Cell 10:1495–1510.

    https://doi.org/10.1091/mbc.10.5.1495

    • Google Scholar
85. 1. Sawin KE
    2. Nurse P

    (1998) Regulation of cell polarity by microtubules in fission yeast

    Journal of Cell Biology 142:457–471.

    https://doi.org/10.1083/jcb.142.2.457

    • Google Scholar
86. 1. Schaeffer D
    2. Reis FP
    3. Johnson SJ
    4. Arraiano CM
    5. van Hoof A

    (2012) The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

    Nucleic Acids Research 40:9298–9307.

    https://doi.org/10.1093/nar/gks693

    • Google Scholar
87. 1. Shields JM
    2. Pruitt K
    3. McFall A
    4. Shaub A
    5. Der CJ

    (2000) Understanding Ras: 'it ain't over 'til it's over'

    Trends in Cell Biology 10:147–154.

    https://doi.org/10.1016/s0962-8924(00)01740-2

    • Google Scholar
88. 1. Snell V
    2. Nurse P

    (1994)

    Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth

    The EMBO Journal 13:2066–2074.

    • Google Scholar
89. 1. Stork O
    2. Zhdanov A
    3. Kudersky A
    4. Yoshikawa T
    5. Obata K
    6. Pape HC

    (2004) Neuronal functions of the novel serine/threonine kinase Ndr2

    Journal of Biological Chemistry 279:45773–45781.

    https://doi.org/10.1074/jbc.M403552200

    • Google Scholar
90. 1. Su SS
    2. Tanaka Y
    3. Samejima I
    4. Tanaka K
    5. Yanagida M

    (1996)

    A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation

    Journal of Cell Science 109:1347–1357.

    • Google Scholar
91. 1. Suárez MB
    2. Alonso-Nuñez ML
    3. del Rey F
    4. McInerny CJ
    5. Vázquez de Aldana CR

    (2015) Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe

    Cell Cycle 14:3124–3137.

    https://doi.org/10.1080/15384101.2015.1078035

    • Google Scholar
92. 1. Tahirovic S
    2. Bradke F

    (2009) Neuronal polarity

    Cold Spring Harbor Perspectives in Biology 1:a001644.

    https://doi.org/10.1101/cshperspect.a001644

    • Google Scholar
93. 1. Tanos B
    2. Rodriguez-Boulan E

    (2008) The epithelial polarity program: machineries involved and their hijacking by cancer

    Oncogene 27:6939–6957.

    https://doi.org/10.1038/onc.2008.345

    • Google Scholar
94. 1. Tatebe H
    2. Nakano K
    3. Maximo R
    4. Shiozaki K

    (2008) Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast

    Current Biology 18:322–330.

    https://doi.org/10.1016/j.cub.2008.02.005

    • Google Scholar
95. 1. Tatebe H
    2. Shimada K
    3. Uzawa S
    4. Morigasaki S
    5. Shiozaki K

    (2005) Wsh3/Tea4 is a novel cell-end factor essential for bipolar distribution of Tea1 and protects cell polarity under environmental stress in S. pombe

    Current Biology 15:1006–1015.

    https://doi.org/10.1016/j.cub.2005.04.061

    • Google Scholar
96. 1. Toda T
    2. Niwa H
    3. Nemoto T
    4. Dhut S
    5. Eddison M
    6. Matsusaka T
    7. Yanagida M
    8. Hirata D

    (1996)

    The fission yeast sts5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway

    Journal of Cell Science 109:2331–2342.

    • Google Scholar
97. 1. Toretsky JA
    2. Wright PE

    (2014) Assemblages: functional units formed by cellular phase separation

    Journal of Cell Biology 206:579–588.

    https://doi.org/10.1083/jcb.201404124

    • Google Scholar
98. 1. Uesono Y
    2. Toh-e A
    3. Kikuchi Y

    (1997) Ssd1p of Saccharomyces cerevisiae associates with RNA

    Journal of Biological Chemistry 272:16103–16109.

    https://doi.org/10.1074/jbc.272.26.16103

    • Google Scholar
99. 1. Vaggi F
    2. Dodgson J
    3. Bajpai A
    4. Chessel A
    5. Jordán F
    6. Sato M
    7. Carazo-Salas RE
    8. Csikász-Nagy A

    (2012) Linkers of cell polarity and cell cycle regulation in the fission yeast protein interaction network

    PLoS Computational Biology 8:e1002732.

    https://doi.org/10.1371/journal.pcbi.1002732

    • Google Scholar
100. 1. Verde F
     2. Mata J
     3. Nurse P

     (1995) Fission yeast cell morphogenesis: identification of new genes and analysis of their role during the cell cycle

     Journal of Cell Biology 131:1529–1538.

     https://doi.org/10.1083/jcb.131.6.1529

     • Google Scholar
101. 1. Verde F
     2. Wiley DJ
     3. Nurse P

     (1998) Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

     PNAS 95:7526–7531.

     https://doi.org/10.1073/pnas.95.13.7526

     • Google Scholar
102. 1. Vjestica A
     2. Zhang D
     3. Liu J
     4. Oliferenko S

     (2013) Hsp70-Hsp40 chaperone complex functions in controlling polarized growth by repressing Hsf1-driven heat stress-associated transcription

     PLoS Genetics 9:e1003886.

     https://doi.org/10.1371/journal.pgen.1003886

     • Google Scholar
103. 1. Wang CY
     2. Chen WL
     3. Wang SW

     (2013) Pdc1 functions in the assembly of P bodies in Schizosaccharomyces pombe

     Molecular and Cellular Biology 33:1244–1297.

     https://doi.org/10.1128/MCB.01583-12

     • Google Scholar
104. 1. Wang JT
     2. Smith J
     3. Chen BC
     4. Schmidt H
     5. Rasoloson D
     6. Paix A
     7. Lambrus BG
     8. Calidas D
     9. Betzig E
     10. Seydoux G

     (2014) Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans

     eLife 3:e04591.

     https://doi.org/10.7554/eLife.04591

     • Google Scholar
105. 1. Wanless AG
     2. Lin Y
     3. Weiss EL

     (2014) Cell morphogenesis proteins are translationally controlled through UTRs by the Ndr/LATS target Ssd1

     PLoS One 9:e85212.

     https://doi.org/10.1371/journal.pone.0085212

     • Google Scholar
106. 1. Wei B
     2. Hercyk BS
     3. Mattson N
     4. Mohammadi A
     5. Rich J
     6. DeBruyne E
     7. Clark MM
     8. Das M

     (2016) Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis

     Molecular Biology of the Cell 27:1235–1280.

     https://doi.org/10.1091/mbc.E15-10-0700

     • Google Scholar
107. 1. Wiley DJ
     2. Marcus S
     3. D'urso G
     4. Verde F

     (2003) Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

     Journal of Biological Chemistry 278:25256–25263.

     https://doi.org/10.1074/jbc.M209703200

     • Google Scholar
108. 1. Wilson-Grady JT
     2. Villén J
     3. Gygi SP

     (2008) Phosphoproteome analysis of fission yeast

     Journal of Proteome Research 7:1088–1185.

     https://doi.org/10.1021/pr7006335

     • Google Scholar
109. 1. Woodham EF
     2. Machesky LM

     (2014) Polarised cell migration: intrinsic and extrinsic drivers

     Current Opinion in Cell Biology 30:25–32.

     https://doi.org/10.1016/j.ceb.2014.05.006

     • Google Scholar
110. 1. Wu JQ
     2. Sirotkin V
     3. Kovar DR
     4. Lord M
     5. Beltzner CC
     6. Kuhn JR
     7. Pollard TD

     (2006) Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

     The Journal of Cell Biology 174:391–402.

     https://doi.org/10.1083/jcb.200602032

     • Google Scholar
111. 1. Yaffe MB
     2. Rittinger K
     3. Volinia S
     4. Caron PR
     5. Aitken A
     6. Leffers H
     7. Gamblin SJ
     8. Smerdon SJ
     9. Cantley LC

     (1997) The structural basis for 14-3-3:phosphopeptide binding specificity

     Cell 91:961–971.

     https://doi.org/10.1016/S0092-8674(00)80487-0

     • Google Scholar
112. 1. Yamashita YM
     2. Yuan H
     3. Cheng J
     4. Hunt AJ

     (2010) Polarity in stem cell division: asymmetric stem cell division in tissue homeostasis

     Cold Spring Harbor Perspectives in Biology 2:a001313.

     https://doi.org/10.1101/cshperspect.a001313

     • Google Scholar
113. 1. Yanagida M
     2. Ikai N
     3. Shimanuki M
     4. Sajiki K
     5. Nobuyasu I

     (2011) Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases

     Philosophical Transactions of the Royal Society B 366:3508–3520.

     https://doi.org/10.1098/rstb.2011.0124

     • Google Scholar
114. 1. Yanagida M

     (2009) Cellular quiescence: are controlling genes conserved?

     Trends in Cell Biology 19:705–715.

     https://doi.org/10.1016/j.tcb.2009.09.006

     • Google Scholar
115. 1. Yang R
     2. Kong E
     3. Jin J
     4. Hergovich A
     5. Püschel AW
     6. Pu AW

     (2014) Rassf5 and Ndr kinases regulate neuronal polarity through Par3 phosphorylation in a novel pathway

     Journal of Cell Science 127:3463–3476.

     https://doi.org/10.1242/jcs.146696

     • Google Scholar
116. 1. Yoshimura T
     2. Arimura N
     3. Kaibuchi K

     (2006) Molecular mechanisms of axon specification and neuronal disorders

     Annals of the New York Academy of Sciences 1086:116–125.

     https://doi.org/10.1196/annals.1377.013

     • Google Scholar
117. 1. Zallen JA
     2. Peckol EL
     3. Tobin DM
     4. Bargmann CI

     (2000) Neuronal cell shape and neurite initiation are regulated by the Ndr kinase SAX-1, a member of the Orb6/COT-1/warts serine/threonine kinase family

     Molecular Biology of the Cell 11:3177–3190.

     https://doi.org/10.1091/mbc.11.9.3177

     • Google Scholar
118. 1. Zhao S
     2. Chen D
     3. Geng Q
     4. Wang Z

     (2013) The highly conserved LAMMER/CLK2 protein kinases prevent germ cell overproliferation in Drosophila

     Developmental Biology 376:163–170.

     https://doi.org/10.1016/j.ydbio.2013.01.023

     • Google Scholar
119. 1. Zinn K

     (2004) Dendritic tiling; new insights from genetics

     Neuron 44:211–213.

     https://doi.org/10.1016/j.neuron.2004.09.024

     • Google Scholar

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Aaron Gitler, and the evaluation has been overseen by J. Paul Taylor as Reviewing Editor and James Manley as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that the current submission of your work cannot be considered further for publication in eLife, at least in its current form.

Please be aware that the Senior Editor, the Reviewing Editor and the 3 additional referees deliberated extensively over this submission. The mechanism described – phosphorylation-dependent control of RNA-binding protein assembly with P-bodies, with consequential spatial and temporal regulation of the expression of a subset of mRNAs – is of great interest to us. The major weakness is insufficient characterization of mRNA interactions with Sts5, demonstration of spatial redistribution of these mRNAs to P-bodies, and correlation with changes in mRNA stability. These concerns, in addition to those elaborated by the reviewers below, would require more extensive additional work than we typically allow for in a revision. We request that revised manuscripts be returned within 2 months. That said, if you are able to address these concerns fully, we would encourage submitting a new manuscript to eLife.

Reviewer #1:

Non membrane-delimited subcellular compartments play critical cellular functions essential for life. These include P-bodies and stress granules (SGs), which are composed of RNAs and RNA-binding proteins. Many of the protein constituents of SGs and P-bodies contain domains resembling yeast prions (called prion-like domains or low-complexity domains). These domains afford the protein a remarkable ability to undergo phase transitions. There has been an emergence of interest in this phenomenon since it has broad implications on gene regulation and many other facets of biology.

This new manuscript by Verde and colleagues presents a very exciting discovery showing how a similar phenomenon can be used to regulate polarized cell growth. They show that the fission yeast protein Sts5 forms RNA granule like accumulations during mitosis and this is negatively regulated by a kinase, Orb6. Remarkably, the aggregation of Sts5 serves to sequester certain key polarized growth protein encoding mRNAs. This is a novel example of how regulated aggregation can be harnessed by cell biology to regulate a fundamental biological process.

The authors have combined high quality imaging, biochemistry, and mRNA analyses to provide compelling evidence to support this novel cellular function for Orb6 and Sts5. In a broader way, they have opened up the possibility that similar phase separations can be harnessed in biology to regulate many other fundamental processes.

In my opinion, this manuscript will be of interest to the readers of eLife and will make an important conceptual advance. There is only one area that I suggest the authors consider:

Several recent studies have demonstrated that some RNA-binding proteins, such as hnRNPA1 and FUS can undergo liquid-liquid demixing in vitro (e.g., Patel et al., Cell 2015; Molliex et al., Cell 2015; Lin et al., Mol Cell 2015). I suggest the authors perform experiments in vitro to see if Sts5 can form similar liquid compartments and, if so, if the phosphorylation by Orb6 alters these properties. One part of this story that seems less well developed is the precise role of Orb6 in negatively regulating Sts5 aggregation. The in vitro experiments with defined components might help to better define this mechanism.

Reviewer #2:

This manuscript presents a novel (to my knowledge) study into links between polarized growth and mRNA regulation in S. pombe. The authors provide both genetic and biochemical evidence for negative regulation of the RNA-binding protein, Sts5, by the NDR kinase, Orb6. Orb6 inhibits Sts5 assembly into puncta, the association with P-bodies, and degradation of Sts5 target mRNAs. They show that Orb6 acts through regulating Sts5's interaction with Rad24.

This work describes a new role for Orb6 kinase in the control of polarized growth independent of Cdc42 pathways. The broad interest and novelty of the work are related to the molecular regulation of RNP body assembly and function by a conserved kinase and the ability to see this regulation highly spatially controlled in the cytoplasm. Below are suggested experiments that could solidify the links in the model but overall I am in favor of publishing this manuscript.

1) The evidence of translational control is limited to a single protein (Figure 3). The authors present evidence that Orb6 inhibits Sts5-association with P-bodies. This in turn promotes stability and perhaps translation of Sts5 bound mRNAs encoding polarity factors. Confidence in the generality of this mechanism would be increased levels of other putative proteins were measured and if Ssp1 was more deeply analyzed. Are Ssp1 protein levels changing in a different mutant that cannot form P-bodies? Do Ssp1 levels change during cell stress and cell division when Sts5 is clearly forming cytoplasmic puncta?

2) Are the mRNAs identified by microarray as controlled by Sts5 changing localization consistent with the model of spatial control of repression/degradation/translation? It would be good to have some other validation of mRNA regulation beyond just the microarrays. Would it be possible to try single molecular RNA FISH and see how distribution changes from clustered as would be if in large RNPs or distributed throughout cytosol? This tool would allow the visualization of local changes in localization of Sts5 mRNA targets in response to Orb6 inhibition or cellular stress and help distinguish, potentially, between the functions of the mitotic Sts5 puncta and P-bodies.

3) Is Sts5 an in vivo target of Orb6 in cells? The results suggest that Orb6-dependent phosphorylation of Sts5 regulates its association with P-bodies or Rad24. Although the authors present an in vitro kinase assay result to demonstrate that Orb6 can phosphorylate Sts5, further evidence of Sts5 as a direct substrate in vivo would be more convincing. This in turn would allow the generation of mutants that are not phosphorylatable to actually demonstrate that phosphorylation is the trigger of disassembly of cytoplasmic puncta. Alternatively, does Sst5 form "aggregates" in vitro and are these disassembled by Orb6 kinase?

4) Does Orb6 overexpression cause an inability of the cell to react properly to stress?

Reviewer #3:

In this manuscript, the authors show that the lethality of the NDR kinase Orb6 can be suppressed by loss of function in the RNA-binding protein Sts5. They show that Sts5 is phosphorylated by Orb6 kinase, which promotes its association with 14-3-3; forms aggregates upon starvation or upon loss of Orb6 activity; and associates with several mRNAs, including ssp1 mRNA. Sts5 loss of function leads to increased levels of these and other mRNAs, indicating Ssp5 promotes mRNA degradation. They propose that Orb6 phosphorylation of Sts5 promotes its association of 14-3-3 to antagonize its association with P-bodies, thus preventing the degradation of key mRNA for polarized growth. They further propose that this regulation is spatially regulated, with Sts5 aggregates forming preferentially at the non-growing cell end of monopolar cells; and temporally regulated, with Orb6-mediated inhibition taking place during mitosis and cytokinesis but being relieved upon contractile ring closure.

The ideas put forward by this study are interesting and largely novel. However, I find that several parts of the model are not well supported by the data shown, with some inconsistencies. Several key points are overstated in the text relative to the data shown, which in aggregate fail to convince me of the whole narrative. These are detailed below.

Genetic interaction of sts5 and orb6 (Figure 1 and Figure 1—figure supplement 1):

The authors identify sts5 loss-of-function as suppressor of orb6 mutation lethality and show some evidence that this suppression is separable from the role of Orb6 in regulating Cdc42 activity. The suppression data is convincing, but the separation of function is less so. In the growth assay shown in Figure 1—figure supplement 2A, single colonies cannot be seen in the higher dilutions and the gef1∆ single mutant strain appears very sick. From this assay, one could argue that the gef1∆ orb6-as double mutant actually grows better than the single mutant. This should be clarified. In addition, if the orb6-sts5 control of growth is really genetically separable from the spatial control of Cdc42, this would predict that Sts5 loss of function does not correct the round shape of orb6 mutants (at permissive temperature) because of Cdc42 activity presence on cell sides, i.e. sts5-276 orb6-as double mutant treated with PP1 inhibitor at 25°C should be round. Is this the case?

I am not sure I understand exactly what is quantified in Figure 1—figure supplement 1C. In Figure 1B, it is shown that 60% of orb6-as2 cells are septated. In Figure 1—figure supplement 1, only non-septated cells are shown with CRIB signal on cell sides, with quantification that amounts to 80%. Clearly 60+80>100, so I am not sure how to reconcile these numbers. If only a subset of cells is quantified, this should be specified. Also, in Figure 1B, the authors indicate a standard deviation from only n=2 experiments, which cannot be derived.

Formation of Sts5 punctae, association with P-bodies, mRNA degradation and regulation by Orb6:

The authors show that starvation or Orb6 kinase inhibition leads to formation of Sts5 punctae that colocalize with P-body marker Dcp1. Sts5 loss of function also leads to a reduction in the number of P-bodies after Orb6 inhibition, implicating Sts5 in the formation of P-bodies. However, they do not examine the role of Sts5 in P-body formation upon starvation, when P-body structures are much more prominent. Does Sts5 also play a role in the formation of P-bodies upon starvation? During N-starvation, it looks like there is only a partial colocalization of Sts5 with Dcp1. This should be stated in the text.

Regarding mRNA levels, the data on ssp1 mRNA level regulation is convincing, with epistasis of Sts5 over Orb6, indicating that Orb6 prevents Sts5-mediated degradation of Ssp1 mRNA. However, epistasis is not shown for any of the other transcripts examined. This would be required for the authors to make their claim of a general finding.

The link between aggregate formation, P-bodies and mRNA degradation is also not clear. The authors state, in the Results section, subsection “Orb6 kinase inhibits Sts5 aggregation and Sts5 localization to P-bodies”, that "Orb6 kinase prevents [...] Sts5 localization to P-bodies, with the effect of preventing the degradation of specific mRNAs" and, in the sixth paragraph of the Discussion section, that "Sts5 promotes mRNA degradation likely by recruiting transcripts to processing (P) bodies". However, the observed increased level of several mRNAs in the sts5∆ strain does not correlate with the colocalization of Sts5 with P-bodies: the latter is shown upon starvation, whereas the mRNA levels are examined in normal growth conditions. Similarly, in rad24∆ where there is no obvious colocalization of Sts5 with P-body markers, the authors claim Ssp1 levels are reduced (whether this is actually the case is also an issue – see below). From this, it appears that formation of aggregates and mRNA level regulation are not causally linked. It is equally possible that aggregation is a consequence of the sts5 interaction with/degradation of mRNAs or that the two effects or not directly causally related.

The kinase assay in Figure 6A lacks a negative control to state that Orb6 kinase phosphorylates Sts5. What is shown is that a Mob2-associated kinase phosphorylates Sts5. This may indeed be Orb6, but should be accurately described in the text.

Kinase assay and 14-3-3 role:

This part is not very convincing with text and figures showing a number of inconsistencies and over-interpretations.

The authors state (Results section, subsection “14-3-3 protein Rad24 negatively regulates Sts5 aggregation”) that "ssp1 mRNA levels are consistently reduced in rad24∆ mutants", but the data shown in Figure 6E does not state a statistically significant difference between wt and rad24∆ strains. The fact that the levels are significantly reduced in rad24∆ compared to sts5∆ or sts5∆ rad24∆ double mutants does not indicate anything about the possible role of Rad24, simply that Sts5 promotes Ssp1 degradation.

Similarly, the authors propose on later in the Results section that "Orb6 inhibits [...] Sts5 localization to P-bodies by promoting its interaction with 14-3-3 protein Rad24." However, the data shows that Sts5 forms aggregates in rad24∆ cells, but I do not see evidence that they contain Dcp1 P-body marker (see Figure 6C). The authors indeed actually write that P-body formation is not strongly induced in rad24∆.

Temporal regulation:

The proposed anti-correlation of Ssp5 aggregates with Orb6 activity during cell division is interesting, but is poorly supported. Previous work had shown that Orb6 activity is inhibited by SIN signaling until closure of the contractile ring. Here, the authors rely solely on this published evidence to propose a causal link between Ssp5 aggregate dissipation during septation and Orb6 activity. They should at least precisely quantify the number of aggregates over time during cytokinesis. For this a timelapse acquisition of Sts5-3xGFP would be best, relative to a cytokinesis marker to time the progression of ring constriction. They should also show that any disappearance of Sts5 aggregates is indeed due to Orb6 activity, using either of their orb6-as or orb6 ts alleles.

The localization of Orb6-GFP during cell division, claimed to be shown in Figure 7—figure supplement 2C, is not shown. That figure legend is also present at the bottom of Figure 7—figure supplement 1.

The cell lysis phenotype of sts5∆ cells should be quantified.

Spatial regulation:

In general, the spatial regulation of this mRNA stabilization control is not very convincing. The asymmetry in the aggregates of Sts5 is rather difficult to see. It is well documented in tea1∆ cells, but less so in wt cells. Could the authors provide more examples to show the Sts5 asymmetric localization in monopolar wt cells? The authors propose the existence of an Sts5 phosphorylation gradient. This is an interesting idea, with conceptual similarity to gradients of MEX-5 in C. elegans that contribute to the dissolution of P-granules, but is it actually possible at the small scale of a yeast cell? Diffusion rates in the cytosol, if Sts5 diffuses unimpeded, makes this unlikely, but it could be constrained through aggregate formation. Examination of the motility of Sts5 aggregates would help to resolve this point.

I am also not convinced that Sts5 has a direct role in regulating bipolar growth. As the sts5∆ mutant cells are longer at all densities examined, the increased proportion of bipolar cells could simply be a result of the longer cell length. It is well established that fission yeast cells have a cell size-dependent regulation of bipolarity. The authors instead seem to argue that the increased length is a consequence of increased bipolarity, but I do not see evidence for this. For cells of equal initial length, are the sts5∆ mutant cells growing faster? Are cells of same length more bipolar if sts5∆?

Allele nomenclature and temperature sensitivity:

The nomenclature of the alleles used is confusing. It should be clearly stated in the text that orb4 and sts5 are two names for the same gene (and as much as possible use a single gene name throughout the manuscript), and that the alleles used are both early stop codons. The authors also use a deletion of the gene, but it is not very clear which strain is used in which experiment, and why different strains were used. For instance, in second paragraph of Results section, the authors say they "examined whether sts5 deletion also suppresses the loss of polarity observed upon Orb6 kinase inhibition", but the figure referred to (Figure 1—figure supplement 1B-C) states use of orb4-A9 allele.

More generally, I do not understand the use of different "permissive", "semi-permissive" and "restrictive" temperatures to examine the phenotype of sts5 loss of function. In what way are the alleles used conditional if these are either early stop codons or deletions? This should be clearly stated and discussed. For instance, the increased levels of Ssp1-HA in sts5 mutants is only shown to happen at 36°C, but the text states that the increased levels are "exaggerated" at 36°C (Results section, subsection “Loss of Sts5 leads to increased levels of mRNAs involved in growth control and bipolar growth activation”), which implies it is also the case at lower temperature.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Aaron D Gitler (Reviewer #1), and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We hope you will be able to submit the revised version within two months, so please let us know if you have any questions first.

Summary:

This manuscript presents novel insight into a link between mRNA regulation and polarized growth in S. pombe. The authors present genetic and biochemical evidence indicating that NDR kinase Orb6 negatively regulates the RNA-binding protein Sts5. Orb6 activity alters an interaction of Sts5 with 14-3-3, regulating the ability of Sts5 to assemble into cytoplasmic P-bodies, which sequester and influence the stability of Sts5 target mRNAs. There was consensus among the reviewers that the additional data and analyses provided in the revised manuscript addressed most of the concerns from the original review, but several issues remain that must be addressed.

Essential revisions:

1. The FISH experiment showing colocalization of ssp1 mRNA with Sts5 (and Dcp1) granules is a nice addition. However, in addition to showing representative images the extent of colocalization should quantified.

2. Similarly, the analysis of Efc25 protein levels is a nice addition, though it needs to be quantified. An epistasis experiment to test whether deletion of sts5 abrogates the reduction in Efc25 levels observed in orb6 mutant (as shown for Ssp1) would further support the conclusion.

3. In Figure 6E, the statistical comparisons should be done between rad24∆ and wt, not between rad24∆ and sts5∆.

[Editors’ note: the author responses to the first round of peer review follow.]

Thank you for submitting your work entitled "Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Aaron Gitler, and the evaluation has been overseen by J. Paul Taylor as Reviewing Editor and James Manley as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that the current submission of your work cannot be considered further for publication in eLife, at least in its current form.

Please be aware that the Senior Editor, the Reviewing Editor and the 3 additional referees deliberated extensively over this submission. The mechanism described – phosphorylation-dependent control of RNA-binding protein assembly with P-bodies, with consequential spatial and temporal regulation of the expression of a subset of mRNAs – is of great interest to us. The major weakness is insufficient characterization of mRNA interactions with Sts5, demonstration of spatial redistribution of these mRNAs to P-bodies, and correlation with changes in mRNA stability. These concerns, in addition to those elaborated by the reviewers below, would require more extensive additional work than we typically allow for in a revision. We request that revised manuscripts be returned within 2 months. That said, if you are able to address these concerns fully, we would encourage submitting a new manuscript to eLife.

Reviewer #1:

[…] In my opinion, this manuscript will be of interest to the readers of eLife and will make an important conceptual advance. There is only one area that I suggest the authors consider:

Several recent studies have demonstrated that some RNA-binding proteins, such as hnRNPA1 and FUS can undergo liquid-liquid demixing in vitro (e.g., Patel et al., Cell 2015; Molliex et al., Cell 2015; Lin et al., Mol Cell 2015). I suggest the authors perform experiments in vitro to see if Sts5 can form similar liquid compartments and, if so, if the phosphorylation by Orb6 alters these properties. One part of this story that seems less well developed is the precise role of Orb6 in negatively regulating Sts5 aggregation. The in vitro experiments with defined components might help to better define this mechanism.

Sts5 protein contains a predicted unstructured N terminal domain in the first 301 amino acids. We have included this finding in the current version of the paper (Figure 2—figure supplement 1). The role of unstructured domains in the formation of liquid compartments and cytoplasmic phase transition is a biological problem of fundamental importance, and we plan to devote substantial effort in the future to reconstitute the behavior of Sts5 in vitro. At the moment however, we feel these experiments are beyond the scope of this paper, and it would require an entire additional manuscript to present them completely and well controlled. Also, the phosphorylation studies are complicated by the existence of other phosphorylated sites in the Sts5 molecule by which other kinases may contribute to its regulation (results of proteomic analysis: Wilson-Grady et al., 2008; Koch et al., 2011; Carpy et al., 2014; Kettenbach et al., 2015), and that may, at this time, confound the in vitro experiments.

Reviewer #2:

[…] This work describes a new role for Orb6 kinase in the control of polarized growth independent of Cdc42 pathways. The broad interest and novelty of the work are related to the molecular regulation of RNP body assembly and function by a conserved kinase and the ability to see this regulation highly spatially controlled in the cytoplasm. Below are suggested experiments that could solidify the links in the model but overall I am in favor of publishing this manuscript.

1) The evidence of translational control is limited to a single protein (Figure 3). The authors present evidence that Orb6 inhibits Sts5-association with P-bodies. This in turn promotes stability and perhaps translation of Sts5 bound mRNAs encoding polarity factors. Confidence in the generality of this mechanism would be increased levels of other putative proteins were measured and if Ssp1 was more deeply analyzed. Are Ssp1 protein levels changing in a different mutant that cannot form P-bodies? Do Ssp1 levels change during cell stress and cell division when Sts5 is clearly forming cytoplasmic puncta?

We further characterized Efc25, the protein encoded by Sts5-bound efc25 mRNA, whose levels increase in sts5Δ cells and decrease in orb6 mutant cells. We found that Efc25 protein levels increase in sts5Δ cells and decrease in orb6 mutants, similarly to Ssp1 protein (see Figure 3—figure supplement 1; Figure 5—figure supplement 1E).

As far as Ssp1 protein levels in mutants that cannot form P-bodies areconcerned, we have tested Ssp1 levels in pdc1Δ and edc3Δ mutant cells that encode Pbody components that regulate mRNA decapping. We found that Ssp1 protein levels are increased in the pdc1Δ and edc3Δ backgrounds (see Figure 3 E and F).

2) Are the mRNAs identified by microarray as controlled by Sts5 changing localization consistent with the model of spatial control of repression/degradation/translation? It would be good to have some other validation of mRNA regulation beyond just the microarrays. Would it be possible to try single molecular RNA FISH and see how distribution changes from clustered as would be if in large RNPs or distributed throughout cytosol? This tool would allow the visualization of local changes in localization of Sts5 mRNA targets in response to Orb6 inhibition or cellular stress and help distinguish, potentially, between the functions of the mitotic Sts5 puncta and P-bodies.

Consistent with Sts5 controlling the localization of ssp1 mRNA, we found that ssp1 fluorescent RNA FISH probes (Stellaris) colocalize with a subset of Sts5-GFP and Dcp1- mCherry containing punta in glucose-starved cells (see Figure 3G and Figure 3—figure supplement 1 D). Our results are consistent with experiments published by other investigators, for example by the laboratory of Roy Parker, whose protocol we adapted (Brengues and Parker, 2007).

3) Is Sts5 an in vivo target of Orb6 in cells? The results suggest that Orb6-dependent phosphorylation of Sts5 regulates its association with P-bodies or Rad24. Although the authors present an in vitro kinase assay result to demonstrate that Orb6 can phosphorylate Sts5, further evidence of Sts5 as a direct substrate in vivo would be more convincing. This in turn would allow the generation of mutants that are not phosphorylatable to actually demonstrate that phosphorylation is the trigger of disassembly of cytoplasmic puncta. Alternatively, does Sst5 form "aggregates" in vitro and are these disassembled by Orb6 kinase?

Sts5 protein contains an unstructured N-terminal domain comprising the first 301 amino acids (Figure 2—figure supplement 1). However it is not recruited into RNPs in the absence of the mRNA-binding domain. The Sts5 sequence presents several canonical Orb6 consensus sites and other lower affinity Orb6 consensus sites that may also need to be modified to ensure maximal response. Also, the phosphorylation studies are complicated by the existence of other phosphorylated sites by other kinases in different domains of the Sts5 molecule that may contribute to its regulation (results of proteomic analysis (Wilson-Grady et al., 2007; Koch et al., 2011; Carpy et al., 2014; Kettenbach et al., 2015), and that may confound in vitro experiments. These are interesting experiments however, and are going to be the subject of extensive efforts in the lab in the future.

4) Does Orb6 overexpression cause an inability of the cell to react properly to stress?

Nutritional stress induces Sts5 recruitment to RNPs and P-body formation. Thus, we used Sts5 recruitment to RNPs and P-body formation as a read-out of stress. We have over-expressed Orb6 kinase and monitored P-body formation and Sts5 recruitment to RNPs upon glucose deprivation. We found that Orb6 overexpression significantly decreases the intensity of Sts5 puncta and P-bodies (see Figure 5— figure supplement 2A).

Reviewer #3:

In this manuscript, the authors show that the lethality of the NDR kinase Orb6 can be suppressed by loss of function in the RNA-binding protein Sts5. They show that Sts5 is phosphorylated by Orb6 kinase, which promotes its association with 14-3-3; forms aggregates upon starvation or upon loss of Orb6 activity; and associates with several mRNAs, including ssp1 mRNA. Sts5 loss of function leads to increased levels of these and other mRNAs, indicating Ssp5 promotes mRNA degradation. They propose that Orb6 phosphorylation of Sts5 promotes its association of 14-3-3 to antagonize its association with P-bodies, thus preventing the degradation of key mRNA for polarized growth. They further propose that this regulation is spatially regulated, with Sts5 aggregates forming preferentially at the non-growing cell end of monopolar cells; and temporally regulated, with Orb6-mediated inhibition taking place during mitosis and cytokinesis but being relieved upon contractile ring closure.

The ideas put forward by this study are interesting and largely novel. However, I find that several parts of the model are not well supported by the data shown, with some inconsistencies. Several key points are overstated in the text relative to the data shown, which in aggregate fail to convince me of the whole narrative. These are detailed below.

Genetic interaction of sts5 and orb6 (Figure 1 and Figure 1—figure supplement 1):

The authors identify sts5 loss-of-function as suppressor of orb6 mutation lethality and show some evidence that this suppression is separable from the role of Orb6 in regulating Cdc42 activity. The suppression data is convincing, but the separation of function is less so. In the growth assay shown in Figure 1—figure supplement 2A, single colonies cannot be seen in the higher dilutions and the gef1∆ single mutant strain appears very sick. From this assay, one could argue that the gef1∆ orb6-as double mutant actually grows better than the single mutant. This should be clarified.

The growth delay of the gef1Δ strain observed in Figure 1—figure supplement 2A, as compared to gef1Δ orb6-as2, in the control DMSO condition, was due to a difference in the background of the strains. The gef1Δ strain was burdened by ura4Δ and leu1-32 auxotrophic markers, which produced a growth difference even in the presence of exogenously added supplements. We have repeated these experiments with a different gef1Δ strain that does not contain these mutations, and found no difference in growth between gef1Δ and gef1Δ orb6-as2 in DMSO. (See Figure 1—figure supplement 2A)

In addition, if the orb6-sts5 control of growth is really genetically separable from the spatial control of Cdc42, this would predict that Sts5 loss of function does not correct the round shape of orb6 mutants (at permissive temperature) because of Cdc42 activity presence on cell sides, i.e. sts5-276 orb6-as double mutant treated with PP1 inhibitor at 25°C should be round. Is this the case?

Indeed, we have analyzed sts5Δ orb6-as2 double mutant cells, treated with 1- Na-PP1 inhibitor at 25°C for 5 hours, and found them to be round. We show this experiment in the current version of the paper (see Figure 1—figure supplement 1B)

I am not sure I understand exactly what is quantified in Figure 1—figure supplement 1C. In Figure 1B, it is shown that 60% of orb6-as2 cells are septated. In Figure 1—figure supplement 1, only non-septated cells are shown with CRIB signal on cell sides, with quantification that amounts to 80%. Clearly 60+80>100, so I am not sure how to reconcile these numbers. If only a subset of cells is quantified, this should be specified.

In Figure 1—figure supplement 1C we quantified the extent of CRIB-GFP mis-localization only in non-septating cells (cells that do not contain a septum, which indeed is a subset). We have clarified this in the corresponding legend of the Figure 1—figure supplement 1C and D. Also, ectopic localization of CRIB-GFP is a rapid response that can be observed as early as 15 minutes after Orb6-as2 inhibition with 1-NA-PP1 (Das et al., 2009). Thus, the experimental conditions in the experiment depicted in Figure 1—figure supplement 1C and 1D are different than in Figure 1B, in which we quantified septation index after prolonged Orb6- as2 inhibition (2 hours) and the proportion of septated cells was different in the two experiments.

Also, in Figure 1B, the authors indicate a standard deviation from only n=2 experiments, which cannot be derived.

As also mentioned below, we counted almost 400 cells for each condition, giving us confidence in our conclusion. The reviewer, however, is correct to point out the issue with the standard deviation. In the revised version, we show in Figure 1A an entirely different set of three experiments using the sts5Δ strain, instead of the orb4-A9 allele. This was done in response to point 6 of this reviewer, to simplify the use of different alleles. The results are entirely consistent with the previous experiment, and the 6 experiments were performed three times (see Figure 1A).

Formation of Sts5 punctae, association with P-bodies, mRNA degradation and regulation by Orb6:

The authors show that starvation or Orb6 kinase inhibition leads to formation of Sts5 punctae that colocalize with P-body marker Dcp1. Sts5 loss of function also leads to a reduction in the number of P-bodies after Orb6 inhibition, implicating Sts5 in the formation of P-bodies. However, they do not examine the role of Sts5 in P-body formation upon starvation, when P-body structures are much more prominent. Does Sts5 also play a role in the formation of P-bodies upon starvation?

As mentioned above, to address the role of Sts5 during nutritional stress we have performed additional experiments that were not included in the original version of the paper. In the current version, we show that loss of sts5 (sts5Δ) significantly decreases the intensity of P-bodies formed following glucose depletion. We added these experiments to the supplement section of the paper (Figure 5—figure supplement 2 B-D).

During N-starvation, it looks like there is only a partial colocalization of Sts5 with Dcp1. This should be stated in the text.

We have mentioned this fact in the text (Results section, subsection “Sts5 proteins are recruited into cytoplasmic puncta during mitosis and during nutritional starvation”).

Regarding mRNA levels, the data on ssp1 mRNA level regulation is convincing, with epistasis of Sts5 over Orb6, indicating that Orb6 prevents Sts5-mediated degradation of Ssp1 mRNA. However, epistasis is not shown for any of the other transcripts examined. This would be required for the authors to make their claim of a general finding.

As mentioned above, we now characterize also efc25, another mRNA that copurifies with Sts5 protein, whose levels increase in sts5Δ cells and decrease in orb6 mutant cells. Similarly to Ssp1 protein, Efc25 protein levels increase in sts5Δ cells and decrease in orb6 mutants (see Figure 3—figure supplement 1; Figure 5—figure supplement 1, E).

The link between aggregate formation, P-bodies and mRNA degradation is also not clear. The authors state, in the Results section, subsection “Orb6 kinase inhibits Sts5 aggregation and Sts5 localization to P-bodies”, that "Orb6 kinase prevents [...] Sts5 localization to P-bodies, with the effect of preventing the degradation of specific mRNAs" and, in the sixth paragraph of the Discussion section, that "Sts5 promotes mRNA degradation likely by recruiting transcripts to processing (P) bodies". However, the observed increased level of several mRNAs in the sts5∆ strain does not correlate with the colocalization of Sts5 with P-bodies: the latter is shown upon starvation, whereas the mRNA levels are examined in normal growth conditions. Similarly, in rad24∆ where there is no obvious colocalization of Sts5 with P-body markers, the authors claim Ssp1 levels are reduced (whether this is actually the case is also an issue – see below). From this, it appears that formation of aggregates and mRNA level regulation are not causally linked. It is equally possible that aggregation is a consequence of the sts5 interaction with/degradation of mRNAs or that the two effects or not directly causally related.

Indeed, our data indicates that in the absence of Sts5, levels of certain Sts5- associated mRNAs are increased even in the absence of extensive or visible P-body formation. Previous findings in S. cerevisiae indicate that mRNA degradation proceeds to a certain extent even in the absence of substantial P-body formation. Processes such as mRNA decay and RNA-mediated gene silencing (Eulalio et al., 2007) and mRNA 7 decapping and translational repression (Decker et al., 2007) are functional in cells lacking detectable microscopic P-bodies indicating that even though P-body components play crucial roles in mRNA silencing and decay, localization of mRNAs into large P-bodies is not necessary for function. Consistent with this idea, we find that loss of P-body components Pdc1 and Edc3 leads to a significant increase in Ssp1 protein levels, even in the absence of stress, when P-bodies are not visible (see Figure 3E and F).

As the reviewer mentions, our data also show that Sts5 recruitment into large RNPs in the rad24Δ strain does not trigger a substantial decrease in ssp1 mRNA levels. Ssp1 mRNA levels, however, are decreased upon Orb6 kinase inhibition, which induces both P-body formation and Sts5 protein recruitment, in addition to other effects, such as Cdc42 activation. Thus, our data suggest that additional yet unknown signals, dependent on Orb6 kinase, are required for inducing mRNA degradation. We have clarified our conclusions in the current version of the paper (e.g. Results section, subsection “14-3-3 protein Rad24 negatively regulates Sts5 recruitment into cytoplasmic puncta”; fourth paragraph of Discussion section).

The kinase assay in Figure 6A lacks a negative control to state that Orb6 kinase phosphorylates Sts5. What is shown is that a Mob2-associated kinase phosphorylates Sts5. This may indeed be Orb6, but should be accurately described in the text.

We have changed the text to reflect this caveat (Results section, subsection “14-3-3 protein Rad24 negatively regulates Sts5 recruitment into cytoplasmic puncta”).

Kinase assay and 14-3-3 role:

This part is not very convincing with text and figures showing a number of inconsistencies and over-interpretations.

The authors state (Results section, subsection “14-3-3 protein Rad24 negatively regulates Sts5 aggregation”) that "ssp1 mRNA levels are consistently reduced in rad24∆ mutants", but the data shown in Figure 6E does not state a statistically significant difference between wt and rad24∆ strains. The fact that the levels are significantly reduced in rad24∆ compared to sts5∆ or sts5∆ rad24∆ double mutants does not indicate anything about the possible role of Rad24, simply that Sts5 promotes Ssp1 degradation.

Similarly, the authors propose on later in the Results section that "Orb6 inhibits.… Sts5 localization to P-bodies by promoting its interaction with 14-3-3 protein Rad24." However, the data shows that Sts5 forms aggregates in rad24∆ cells, but I do not see evidence that they contain Dcp1 P-body marker (see Figure 6C). The authors indeed actually write that P-body formation is not strongly induced in rad24∆.

Our conclusion from experiments detailed in Figure 6C is that one of the roles of Orb6 kinase is to regulate association of Sts5 with Rad24 and prevent Sts5 recruitment into RNPs. Our experiments with rad24Δ mutant cells indeed show that Sts5 recruitment into RNPs occurs independently of substantial P-body formation, since these cells are not starved. We tried to be clearer in the current version of the paper. We speculate from our findings and propose that Sts5 protein regulation by Orb6 kinase is part of a mechanism ultimately regulating mRNA association with the P-bodies, since we see P-body formation upon Orb6 kinase inhibition. We also rephrased this part.

Temporal regulation:

The proposed anti-correlation of Ssp5 aggregates with Orb6 activity during cell division is interesting, but is poorly supported. Previous work had shown that Orb6 activity is inhibited by SIN signaling until closure of the contractile ring. Here, the authors rely solely on this published evidence to propose a causal link between Ssp5 aggregate dissipation during septation and Orb6 activity. They should at least precisely quantify the number of aggregates over time during cytokinesis. For this a timelapse acquisition of Sts5-3xGFP would be best, relative to a cytokinesis marker to time the progression of ring constriction. They should also show that any disappearance of Sts5 aggregates is indeed due to Orb6 activity, using either of their orb6-as or orb6 ts alleles.

We have quantified the number of Sts5 puncta during cytokinesis in the presence or absence of Orb6 kinase. As a marker for cytokinesis we used Rlc1, in addition to calcofluor. Rlc1, myosin II regulatory light chain (Le Goff et al., 2000) (Wu et al., 2006), disappears from the site of division when the actomyosin ring has fully contracted and the septum is closed (Wei, 2016). We found that Sts5 puncta are present as long as Rlc1 is present at the site of division (a sign that septum closure in not complete) but disappear in cells where Rlc1 has left the site of division, after septum closure. Indicating that disappearance of Sts5 puncta is indeed due to Orb6 activity, we find that Sts5 puncta are still visible in cells that have completed septum closure (and where Rlc1 has left the site of division), when Orb6 activity is inhibited. We show these experiments in the current version of the paper (see Figure 7G and H; Figure 7—figure supplement 2).

The localization of Orb6-GFP during cell division, claimed to be shown in Figure 7—figure supplement 2, is not shown. That figure legend is also present at the bottom of Figure —figure supplement 1.

We indeed showed the localization of Orb6-GFP at the septum in Figure 7—figure supplement 1A (c), as the reviewer mentions. Figure 7—figure supplement 2C was a single typo, for which we apologize, as it should have read Figure 7—figure supplement 1A (c). We corrected this typo in the text.

The cell lysis phenotype of sts5∆ cells should be quantified.

We quantified the lysis phenotype of sts5Δ in triplicate experiments, and found that, under our experimental conditions, 43% of septating sts5Δ cells lyse at 36° C, as compared to 8% septating control cells. We added this information in the corresponding legend (see Figure 7—figure supplement 2).

Spatial regulation:

In general, the spatial regulation of this mRNA stabilization control is not very convincing. The asymmetry in the aggregates of Sts5 is rather difficult to see. It is well documented in tea1∆ cells, but less so in wt cells. Could the authors provide more examples to show the Sts5 asymmetric localization in monopolar wt cells? The authors propose the existence of an Sts5 phosphorylation gradient. This is an interesting idea, with conceptual similarity to gradients of MEX-5 in C.elegans that contribute to the dissolution of P-granules, but is it actually possible at the small scale of a yeast cell? Diffusion rates in the cytosol, if Sts5 diffuses unimpeded, makes this unlikely, but it could be constrained through aggregate formation. Examination of the motility of Sts5 aggregates would help to resolve this point.

Indeed, to verify the observation of asymmetric Sts5 puncta in small monopolar cells, first observed in wild-type cells, we used tea1Δ cells since they are 9 longer, stay monopolar, and localize Orb6-GFP at only one tip. We have added more examples of small wild-type cells that reflect the average representative localization under conditions of active cell growth (see Figure 7—figure supplement 1A,D,E and F). To avoid confusion or overstatements, we removed the word “gradient” that was originally proposed in the discussion.

I am also not convinced that Sts5 has a direct role in regulating bipolar growth. As the sts5∆ mutant cells are longer at all densities examined, the increased proportion of bipolar cells could simply be a result of the longer cell length. It is well established that fission yeast cells have a cell size-dependent regulation of bipolarity. The authors instead seem to argue that the increased length is a consequence of increased bipolarity, but I do not see evidence for this. For cells of equal initial length, are the sts5∆ mutant cells growing faster? Are cells of same length more bipolar if sts5∆?

In response to this reviewer’s comments, we have performed two additional sets of experiments. We show that sts5Δ cells display increased bipolarity even during exponential cell growth, when sts5Δ cells display the same size (average cell length at division; see Figure 8C). As this reviewer suggested, we measured the percentage of bipolar cells in cells of similar length (less than 10 micrometers; from 10 to 12; more than 12), and found that sts5Δ cells display an increased percentage of bipolar cells as compared to controls (see Figure 8—figure supplement 1, A) even if sts5Δ cells display the same growth rate as control sts5+ cells (Figure 8—figure supplement 1B).

Allele nomenclature and temperature sensitivity:

The nomenclature of the alleles used is confusing. It should be clearly stated in the text that orb4 and sts5 are two names for the same gene (and as much as possible use a single gene name throughout the manuscript), and that the alleles used are both early stop codons. The authors also use a deletion of the gene, but it is not very clear which strain is used in which experiment, and why different strains were used. For instance, in second paragraph of Results section, the authors say they "examined whether sts5 deletion also suppresses the loss of polarity observed upon Orb6 kinase inhibition", but the figure referred to (Figure 1—figure supplement 1B-C) states use of orb4-A9 allele.

We have redone the experiments in Figure 1A, using the sts5Δ allele, and obtained identical results to the orb4-A9 allele. Aside from growth suppression experiments detailed in Figure 1E (done some time ago with the sts5-276 allele by our collaborators at Hiroshima University) and one control experiment with CRIB-GFP (which was part of the initial screening with orb4-A9 allele; Figure 1—figure supplement 1C,D), all other experiments were performed using the sts5Δ allele. As the reviewer mentions, these are all early stop codons, and we never found the corresponding mutants to behave differently from the sts5Δ deletion mutant cells. We mentioned this aspect in the legend of Figure 1—figure supplement 1.

More generally, I do not understand the use of different "permissive", "semi-permissive" and "restrictive" temperatures to examine the phenotype of sts5 loss of function. In what way are the alleles used conditional if these are either early stop codons or deletions? This should be clearly stated and discussed. For instance, the increased levels of Ssp1-HA in sts5 mutants is only shown to happen at 36°C, but the text states that the increased levels are "exaggerated" at 36°C (Results section, subsection “Loss of Sts5 leads to increased levels of mRNAs involved in growth control and bipolar growth activation”), which implies it is also the case at lower temperature.

In the current version of the paper, for clarity, we decided not use “restrictive” or “permissive” definitions and just state the temperature at which experiments are performed. The use of “restrictive” or “permissive” temperature refers to conditional, temperature-sensitive phenotypes, and thus it is a functional definition. Indeed sts5Δ displays a morphological phenotype that is temperature-sensitive: it is cylindrical in shape (albeit with an increase in the extent of cell growth at the new end) at 25°C, while it is enlarged and round at 36°C. These phenotypes likely reflect a decreased ability for sts5Δ cells to adapt to higher temperatures. We changed the wording in seventh paragraph, Results section.

[Editors’ note: the author responses to the re-review follow.]

Thank you for submitting your article "Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Aaron D Gitler (Reviewer #1), and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We hope you will be able to submit the revised version within two months, so please let us know if you have any questions first.

Summary:

This manuscript presents novel insight into a link between mRNA regulation and polarized growth in S. pombe. The authors present genetic and biochemical evidence indicating that NDR kinase Orb6 negatively regulates the RNA-binding protein Sts5. Orb6 activity alters an interaction of Sts5 with 14-3-3, regulating the ability of Sts5 to assemble into cytoplasmic P-bodies, which sequester and influence the stability of Sts5 target mRNAs. There was consensus among the reviewers that the additional data and analyses provided in the revised manuscript addressed most of the concerns from the original review, but several issues remain that must be addressed.

Essential revisions:

1. The FISH experiment showing colocalization of ssp1 mRNA with Sts5 (and Dcp1) granules is a nice addition. However, in addition to showing representative images the extent of colocalization should quantified.

We have quantified the co-localization of ssp1 mRNA and Sts5 (and Dcp1) granules, and added these data in Figure 4—figure supplement 3B and C. We used the ImageJ plugin JACoP (Just Another Colocalization Plugin) to perform object-based quantification based on the distance between centers of mass of puncta (Bolte and Cordelieres, 2006). We measured, respectively, the proportion of ssp1 puncta colocalizing with Sts5-3xGFP or Dcp1-mCh (Figure 4—figure supplement 3B), and the proportion of Sts5-3xGFP or Dcp1-mCh puncta colocalizing with ssp1 puncta (Figure 4—figure supplement 3C), in three independent experiments, in the presence or absence of glucose.

2. Similarly, the analysis of Efc25 protein levels is a nice addition, though it needs to be quantified. An epistasis experiment to test whether deletion of sts5 abrogates the reduction in Efc25 levels observed in orb6 mutant (as shown for Ssp1) would further support the conclusion.

We have performed additional experiments, and showed that Efc25 levels decrease in inhibitor sensitive orb6-as2 mutants (Figure 6—figure supplement 1E). We also found that sts5 deletion abolishes the reduction of Efc25 levels in orb6-as2 mutants (Figure 6—figure supplement 1E). We have quantified the results of three independent experiments in Figure 6—figure supplement 1E, right panel.

3. In Figure 6E, the statistical comparisons should be done between rad24∆ and wt, not between rad24∆ and sts5∆.

We have changed the Figure 7E (Figure 8E in the current manuscript)appropriately to reflect the message of the experiment, that there is not a significant difference in ssp1 mRNA levels between rad24∆ and wt. P values were determined using analysis of variance (ANOVA) with SPSS statistics package 22.0, followed by Games-Howell post-hoc test. We also removed the double mutant sts5∆ rad24∆, because as stated by the reviewers, it wasn’t adding to the point of the figure.

Author details

1. Illyce Nuñez

   Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, United States

   Contribution

   IN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Marbelys Rodriguez Pino

   Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, United States

   Contribution

   MRP, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. David J Wiley

   Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, United States

   Contribution

   DJW, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Maitreyi E Das

   Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, United States

   Contribution

   MED, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Chuan Chen

   Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, United States

   Contribution

   CC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Tetsuya Goshima

   National Research Institute of Brewing, Higashi-Hiroshima, Japan

   Contribution

   TG, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Kazunori Kume

   Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan

   Contribution

   KK, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Dai Hirata

   Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan

   Contribution

   DH, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Takashi Toda

   1. Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Japan
   2. The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, London, United Kingdom

   Contribution

   TT, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Fulvia Verde

    1. Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, United States
    2. Marine Biological Laboratory, Woods Hole, United States

    Contribution

    FV, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    fverde@miami.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-2575-0823

• 2,010

  Page views

• 432

  Downloads

• 13

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.