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Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe
Journal Pre-proof Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe Jing Zhang, Ji-Long Liu PII:
S1673-8527(19)30151-1
DOI:
https://doi.org/10.1016/j.jgg.2019.09.002
Reference:
JGG 724
To appear in:
Journal of Genetics and Genomics
Received Date: 19 April 2019 Revised Date:
29 August 2019
Accepted Date: 9 September 2019
Please cite this article as: Zhang, J., Liu, J.-L., Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe, Journal of Genetics and Genomics, https://doi.org/10.1016/ j.jgg.2019.09.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
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Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe
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Jing Zhang a, Ji-Long Liu a,b,*
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a
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Oxford, Oxford, OX1 3PT, United Kingdom
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b
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
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*Corresponding author. Email address: [email protected] or [email protected]
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Abstract
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The metabolic enzyme CTP synthase (CTPS) is able to compartmentalize into filaments, termed
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cytoophidia, in a variety of organisms including bacteria, budding yeast, fission yeast, fruit flies and
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mammals. A previous study in budding yeast shows that the filament-forming process of CTPS is
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not sensitive to temperature shift. Here we study CTPS filamentation in the fission yeast
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Schizosaccharomyces pombe. To our surprise, we find that both the length and the occurrence of
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cytoophidia in S. pombe decrease upon cold shock or heat shock. The temperature-dependent
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changes of cytoophidia are fast and reversible. Taking advantage of yeast genetics, we demonstrate
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that heat-shock proteins are required for cytoophidium assembly in S. pombe. Temperature
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sensitivity of cytoophidia makes S. pombe an attractive model system for future investigations of
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this novel membraneless organelle.
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Keywords: CTP synthase, cytoophidium, Schizosaccharomyces pombe, heat shock protein (HSP),
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nucleoside/nucleotide metabolism, cell biology, yeast genetics, cell compartmentalization
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1. Introduction
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CTP synthase (CTPS) is an enzyme involved in nucleotide biosynthesis in almost all
28
organisms. Recently multiple studies have demonstrated that CTPS forms filamentous structures
29
termed cytoophidia in Drosophila (Liu, 2010), bacteria (Ingerson-Mahar et al., 2010), yeast (Noree
30
et al., 2010; Zhang et al., 2014) and human cells (Carcamo et al., 2011; Chen et al., 2011) (reviews
31
see Aughey and Liu, 2015; Liu, 2010, 2016). In humans and the budding yeast Saccharomyces
32
cerevisiae, there are two CTPS isoforms (encoded by CTPS1 and CTPS2 in humans; encoded by
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URA7 and URA8 in S. cerevisiae), while in the fission yeast Schizosaccharomyces pombe, only one
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CTPS isoform exists and is encoded at a single locus cts1 on chromosome I (Kim et al., 2010;
35
Hayles et al., 2013). We have shown that among the three CTPS isoforms (CTPS isoform-A, B and
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C) in Drosophila only CTPS isoform-A is able to compartmentalize into filaments (Azzam and Liu,
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2013). We also have identified that the two human CTPS isoforms, CTPS1 and CTPS2, can both
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compartmentalize into cytoophidia (Gou et al., 2014). The lack of CTPS redundancy in S. pombe
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gives this fission yeast an advantage as a model for the study of cytoophidia.
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Noree et al. (2010) reported that CTPS and several other proteins can form filamentous
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structures in budding yeast. They also found that filamentation of these proteins is regulated by
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various environmental factors such as nutrition availability, carbon source depletion, energy status
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and the presence of protein synthesis inhibitors (Noree et al., 2010). In comparison with cells
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cultured at 30°C, budding yeast cells grown at low temperature (0°C) for 15 min showed no
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difference in the morphology and frequency of CTPS filaments (Noree et al., 2010). They
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concluded that filamentation of CTPS is not sensitive to temperature shift.
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In Drosophila S2R+ cells, nutrient depletion leads to an increase in number of cytoophidia
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(Aughey et al., 2014), which is parallel with the results observed in budding yeast. Similarly,
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cytoophidium assembly can also be induced in Drosophila larvae by nutrient restriction (Aughey et
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al., 2014). In Drosophila larval post-embryonic neuroblasts (pNbs), cytoophidia can be induced by
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nutritional restriction during the late L2 stage (Aughey et al., 2014; Tastan and Liu, 2015).
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Cytoophidia can also be observed in imaginal discs (eye, wing, leg and haltere) of third instar larvae
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fed with low-nutrient food (Aughey et al., 2014). A recent study in adult fly ovaries showed that
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cytoophidia increase their length upon starvation or during apoptosis (Wu and Liu, 2019).
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In this study, we use the fission yeast S. pombe as a model system and study the response of
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cytoophidia to environmental stresses. Surprisingly, we find that cytoophidia are very sensitive to
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cold shock and heat shock. Our results suggest that cytoophidium assembly is a
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temperature-sensitive process in S. pombe, in contrast to previous observations in S. cerevisiae.
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Furthermore, we generate six heat-shock mutant strains and find that both the length and the
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occurrence of cytoophidia decrease significantly when one of these heat-shock proteins is disrupted.
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Our study highlights the importance of exploring cytoophidium assembly in multiple systems.
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2. Results
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2.1. Cytoophidium assembly is regulated throughout growing stages
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S. pombe cells undergo four phases such as lag phase, exponential phase, stationary phase and
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death phase, with differing metabolic conditions. To examine whether the different phases have an
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effect on cytoophidia, the CTPS-YFP expressing cells were fixed at various points in the growing
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stages and observed under confocal microscope. Cell growth was monitored by measuring the
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OD600 value using the spectrometer. Each counting group contains more than 500 cells. Software
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ImageJ was used to measure the cytoophidium number per cell and the cytoophidium length (see
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details in Materials and methods). During the early-to-middle exponential phase (OD600 = 0.1–1.0),
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cytoophidia were highly abundant, being present in more than 90% of cells (Fig. 1A, B and F). As
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the cells grew and OD600 increased above 1.0, fewer cytoophidia were observed compared with the
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mid-exponential phase (Fig. 1C, D, and G). When cells entered the stationary phase, most of the
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cytoophidia disappeared, dispersing into the cytoplasm (Fig. 1E and G). This result indicates that
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cytoophidium regulation is closely linked to the metabolic conditions within S. pombe cells.
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Next, we wanted to know if the dispersal of cytoophidia at the stationary phase is reversible.
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During the early-to-middle exponential phase (OD600 = 0.1–1.0), a high percentage of cells
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contained cytoophidia with a length of over 1.0 µm (Fig. 1G). When cells entered the stationary
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phases, the cytoophidia dispersed into the cytoplasm (Fig. 2A). When shifted to rich media and
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grown for another 2 h, the cells entered the exponential phase again and the number of cytoophidia 4/19
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increased (Fig. 2B), indicating that stationary-phase-induced cytoophidium dispersal is reversible in
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S. pombe.
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2.2. Cytoophidia respond to a glutamine analog
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After treatment with the CTPS inhibitor 6-diazo-5-oxo-L-norleucine (DON), the average
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length of the cytoophidia was increased significantly (Fig. 3A–C), which is similar to the outcome
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observed in Drosophila and human HeLa cells (Chen et al., 2011; Aughey et al., 2014), while the
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cytoophidium number per cell in S. pombe did not changed significantly (Fig. 3D). Interestingly, the
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CTPS protein level was not changed after DON treatment (Fig. 3E).
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2.3. Cold shock disassembles cytoophidia
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When fission yeast cells were incubated at 32°C, we found that >90% of cells at the
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early-to-middle exponential phase contained obvious cytoophidia (Fig. 4A and E). When cells were
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incubated on ice (0°C) for 30 min, only 20% contained cytoophidia (Fig. 4B and E). Moreover, the
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length of the cytoophidia had decreased dramatically (Fig. 4B and F). After 1 h at 0°C, almost all
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cells did not have cytoophidia, and we could only detect residual cytoophidia of very short length
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(Fig. 4C, E and F). After 2 h at 0°C, CTPS showed a diffused pattern without any sign of
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cytoophidia (Fig. 4D–F).
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We previously showed that cytoophidia are inherited asymmetrically between two daughter
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cells during cell cycle division (Zhang et al., 2014). One daughter cell inherits the cytoophidium
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from the mother cell, while the other daughter cell does not. Upon cold shock, even without
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cytoophidia, the asymmetric distribution of CTPS is obvious in many paired cells, with one cell
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having a much higher signal than the other, indicating that the protein level of CTPS is very
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different (Fig. 4B–D). The differential distribution of CTPS between two daughter cells is
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presumably achieved via asymmetric inheritance of cytoophidia.
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2.4. Disassembly of cytoophidia induced by cold shock is fast
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To further investigate how quickly cold shock affects cytoophidium disassembly, we incubated
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fission yeast cells on ice for shorter time lengths. We found that treating cells at 0°C for only 5 min
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had a profound effect on cytoophidium formation (Fig. 5A and B). Although the percentage of cells
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containing cytoophidia did not change significantly (Fig. 5C), we did observe that the length of 5/19
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cytoophidia decreased dramatically, from 1.4 µm on average to below 1 µm (Fig. 5D). The
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cytoophidium number per cell also significantly decreased (Fig. 5E). These results suggest that
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assembly of cytoophidia in S. pombe is extremely temperature sensitive.
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2.5. Cold shock does not remove CTPS protein
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In Drosophila, the level of CTPS protein is critical for cytoophidium assembly.
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Overexpression of CTPS induces long and thick cytoophidia, while knockdown of CTPS
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disassembles cytoophidia (Azzam and Liu, 2013). To determine whether cold-shock-induced
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cytoophidium disassembly is due to the change of CTPS protein level, we performed Western blot
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of cells collected at different time points after cold-shock treatment. Anti-CTPS antibody was used
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for Western blot and histone H3 was used as the endogenous control. Samples were taken at 5, 15,
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30, 60 min and 2 h after cells were incubated on ice. We found that cold shock for up to 2 h did not
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remove CTPS protein (Fig. 6).
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2.6. Cold-shock-induced cytoophidium disassembly is reversible
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Next we asked whether cold-shock-induced cytoophidium disassembly is reversible. We
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incubated yeast cells at 0°C overnight. As expected, cytoophidia disassembled completely, with
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CTPS showing a diffuse distribution. We then returned the yeast cells to 32°C and observed the
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cells via live imaging. Within 5 min, we saw that CTPS started to form punctate structures, which
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grew longer over time (Fig. 7). After 30 min, we observed that many cells had cytoophidia of a size
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comparable to those in the control group (without cold shock) (data not shown). These results
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indicate that cold-shock-induced cytoophidium disassembly is reversible.
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2.7. DON treatment does not prevent cold-shock-induced disassembly of cytoophidia
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In eukaryotic cells, CTPS uses glutamine as a nitrogen donor. The glutamine analog DON
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binds to CTPS irreversibly. We have previously shown that DON treatment promotes cytoophidium
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assembly in Drosophila and human cells (Chen et al., 2011). DON treatment also promotes
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cytoophidium assembly in S. pombe. Treating fission yeast cells with DON for 2 h increased the
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cytoophidium length from 1.3 µm to 1.6 µm. On average, fewer cytoophidia were observed in
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DON-treated cells, suggesting that DON treatment induces fusion of small cytoophidia.
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To determine whether DON-induced cytoophidium assembly is reversible by cold-shock
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induction, we incubated fission yeast cells in DON for 2 h at 32°C followed by incubating them at
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0°C for 2 h. We found that cytoophidia were no longer detectable after the incubation at 0°C (Fig.
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8). These results indicate that 1) DON-induced cytoophidium assembly is reversible; and 2) DON
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treatment cannot prevent cold-shock-induced cytoophidium disassembly.
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2.9. Heat shock disassembles cytoophidia
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Having observed that cytoophidium assembly is sensitive to cold shock, we next asked
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whether heat shock affects cytoophidium assembly in fission yeast. At 32°C, the majority of S.
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pombe cells in the early-to-middle exponential phase contain obvious cytoophidia (Fig. 9A). When
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exposed to a higher temperature (i.e., 37°C or 42°C), cytoophidia showed a decrease in both the
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average length and the number of cells containing cytoophidia (Fig. 9B–F).
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Similar to cold shock, heat shock affects cytoophidium assembly very quickly (Fig. 10A and
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B). Cytoophidia significantly decreased in both the length and the number of cytoophidia per cell
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after only 5 min of heat shock treatment at 42°C, compared with cells cultured at 32°C (Fig. 10A‒
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D). However, the percentage of cells containing cytoophidia was unchanged after 5 min heat shock
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(Fig. 10E).
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2.10. Heat-shock proteins are required for cytoophidium assembly
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Heat-shock proteins are a family of proteins produced in response to environmental stresses,
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especially heat-shock stress. We already knew that cytoophidia respond to temperature changes. To
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determine whether small heat-shock proteins are required for cytoophidium assembly, we selected
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several small heat-shock proteins in S. pombe cells for further investigation. We constructed a series
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of deletion strains expressing CTPS-YFP, namely hsp9∆, hsp16∆, hsp20∆, hsp104∆, pdr13∆ and
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sks2∆. Since ppk15, a serine/threonine protein kinase, is not related to heat-shock proteins or
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heat-shock response, we used the ppk15∆ strain as an extra control alongside the wild-type control.
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We found that both the length and the occurrence of cytoophidia decreased significantly in all six
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heat-shock mutant strains that we investigated (Fig. 11). Our results indicate that heat-shock
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proteins are required for cytoophidium assembly.
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3. Discussion 7/19
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3.1. Comparison of cytoophidia between S. cerevisiae and S. pombe
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Several studies have shown that CTPS compartmentalizes into filamentous structures in
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various organisms, including bacteria, fruit fly, rat and human cells (Ingerson-Mahar et al., 2010;
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Liu, 2010; Noree et al., 2010; Carcamo et al., 2011; Chen et al., 2011). It has also been reported that
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CTPS filaments can be observed in budding yeast (Noree et al., 2010; Noree et al., 2014; Shen et al.,
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2016; Zhang et al., 2018). We have previously identified that CTPS forms cytoophidia in S. pombe
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(Zhang et al., 2014; Li et al., 2018). These observations suggest that cytoophidia are evolutionarily
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conserved from bacteria to humans.
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Both budding yeast and fission yeast are good model organisms to study cytoophidia. It has
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already been reported that the expression products of both CTPS genes (URA7 and URA8) can
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compartmentalize into filaments in S. cerevisiae (Noree et al., 2010). In budding yeast, cytoophidia
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respond to several drugs and environment stresses (Noree et al., 2010). Here we show that
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cytoophidia are also sensitive to DON and stresses in fission yeast. However, there are some
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notable differences in cytoophidium behavior between S. cerevisiae and S. pombe.
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In budding yeast, cytoophidia can be detected mostly in the stationary phase but not in the
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exponential phase (Noree et al., 2010; Shen et al., 2016). In S. pombe, we show that only
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exponential cells contain large numbers of cytoophidia while few or no cytoophidia can be observed
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during the stationary stage. Moreover, carbon deprivation induces cytoophidium assembly in
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budding yeast (Noree et al., 2010), while here we found that the lack of a carbon source causes the
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dispersal of cytoophidia in fission yeast. These opposing results suggest that the regulatory
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mechanism of cytoophidium assembly differs between S. pombe and S. cerevisiae.
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3.2. The effect of DON on cytoophidium assembly
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DON is an inhibitor for many glutamine-utilizing enzymes including CTPS. It has been
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reported that the majority of cytoophidia in human cells and Drosophila are an inactive form of
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CTPS (Aughey et al., 2014; Barry et al., 2014; Noree et al., 2014), leading to a hypothesis that
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cytoophidia act as a CTPS storage depot as well as a regulator of CTPS metabolic activities. When
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treated with DON, S. pombe showed an increase of the average length of cytoophidia. Similar
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results can be detected in human HeLa cells, Drosophila S2 cells and Drosophila tissues (Chen et 8/19
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al., 2011; Aughey et al., 2014;), indicating that the promotion of cytoophidium assembly by DON is
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highly conserved.
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Interestingly, although the average length of cytoophidia was increased after DON treatment,
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the average number of cytoophidia per cell did not change dramatically in S. pombe. The small
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cytoophidia may have fused into longer and thicker filaments, and therefore increased in filament
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length. This is similar to the maturation process of cytoophidia observed in mammalian culture cells
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(Gou et al., 2014; Chang et al., 2018; Keppeke et al., 2018).
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3.3. Cytoophidia respond to temperature shift
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Our study has revealed that cytoophidium maintenance is sensitive to temperature shift, both to
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cold shock and to heat shock. Given that the cytoophidium formation is growth phase-dependent in
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fission yeast, there is an argument that the temperature sensitivity is due to the cells switching
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between the exponential phase and the stationary phase upon temperature shift. The response of
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cytoophidia to heat or cold stress is fast (within a few minutes), while the time course of growth
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phase changes is slow (from a few hours to a few days). Therefore we think the dynamics of
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cytoophidia during temperature shift cannot be simply explained by growth phase changes.
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Using yeast genetics, we have shown the involvement of heat-shock proteins, hsp9, hsp16,
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hsp20, hsp104, pdr13 (ssz1) and sks2, in cytoophidium assembly. Among these six proteins, only
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hsp9 has been reported to be involved in vegetative growth metabolism under normal growing
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temperature conditions, while the other five proteins play roles during various stress responses.
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Heat-shock proteins are a family of proteins that are produced by cells in response to exposure
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to stressful conditions. They were first described in relation to heat shock, but are now known to be
213
expressed during other stresses including exposure to cold and UV light, and during wound healing
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or tissue remodeling (Li and Srivastava, 2004). Many members of this protein family perform a
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chaperone function by stabilizing new proteins to ensure correct folding or by helping to refold
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proteins that were damaged by the cell stress. This increase of expression is transcriptionally
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regulated (Wu, 1995). The dramatic upregulation of the heat-shock proteins is a key part of the
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heat-shock response and is induced primarily by heat-shock factor (HSF) (Wu, 1995). Heat-shock
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proteins are found in virtually all living organisms, from bacteria to humans. 9/19
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3.4. Potential functions of cytoophidia in S. pombe
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Recently, an increasing number of metabolic enzymes have been identified as having the
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ability to self-assemble and to form filamentous structures, including CTPS, suggesting that
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intracellular compartmentation of metabolic enzymes is more general than previously thought. A
224
large-scale screen of a yeast GFP library identifies that over 20% of the examined proteins were
225
able to from distinct intracellular structures (Narayanaswamy et al., 2009). Another screen of 40%
226
of a budding yeast GFP strain collection shows that nine proteins form four types of filamentous
227
structure (Noree et al., 2010). A further comprehensive screening of 4159 proteins in budding yeast
228
reveals that at least 23 proteins have filament-forming capacity (Shen et al., 2016). Several studies
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demonstrate that polymerisation of CTPS into cytoophidia downregulates enzymatic activity in
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bacteria (Barry et al., 2014), budding yeast (Noree et al., 2014), fruit flies and human cell lines
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(Aughey et al., 2014). Additional studies suggest forming filaments can upregulate enzymatic
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activities (Strochlic et al., 2014; Chang et al., 2015; Lynch et al., 2017). Thus, cytoophidium
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formation seems to be a general strategy to regulate enzymatic activity. The choice of upregulation
234
or downregulation may reflect the context of developmental stages or metabolic statuses.
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This study demonstrates that cytoophidium assembly is a temperature-sensitive process in S.
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pombe. The assembly and disassembly of the cytoophidium and its kind provide a potential
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mechanism to maintain cellular homeostasis during development and in mediating metabolic
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responses (Aughey et al., 2014; Barry et al., 2014; Noree et al., 2014; Petrovska et al., 2014;
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Strochlic et al., 2014). Yet it is still not clear whether the downregulation of cytoophidia is directly
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or indirectly caused by the action of heat-shock proteins. More recently, we found that forming
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cytoophidia prolongs the half-life of CTPS in human cell lines (Sun and Liu, 2019). Further studies
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are needed to determine how various heat-shock proteins cooperate in the processes of assembly,
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maintenance and disassembly of cytoophidia.
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4. Materials and methods
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4.1. S. pombe strains
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The generation of the CTPS-YFP-expressing S. pombe strain has been described in our previous study (Zhang et al., 2014). 10/19
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4.2. S. pombe cell culture
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If not mentioned, all cells were cultured at 32°C in YES media using a shaking incubator. Cell
250
growth was monitored by OD600, with a 0.1–1.5 OD600 value indicating exponentially growing cells
251
and over 1.5 OD600 indicating stationary cells. Two wild-type S. pombe strains, 001 (h+;
252
ade6-M216, leu1-32, ura4-D18) and 002 (h-; ade6 M216, leu1-32, ura4-D18), were obtained from
253
the Stephen Kearsey Laboratory (University of Oxford). The 003 (h+; ade6-M216, leu1 32, ura+,
254
cts1-yfp) strain was used as the control unless stated otherwise.
255
4.3. Site-directed mutagenesis
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In-fusion Cloning (Clontech, USA) and QuikChange Site-Directed Mutagenesis (Agilent, USA)
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technologies were utilized in this study to generate target point mutated plasmids. The standard
258
commercial protocol was used for the detailed manipulation of plasmid construction. The primers
259
used in In-fusion and QuickChange are designed on the website. For the overlap PCR, the overlaps
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of the primers were designed with 25 bp at least.
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4.4. Light microscopy
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Cells were cultured overnight before being fixed and scanned, and were guaranteed to be fresh
263
and in the exponential phase. Cells were fixed with 4% paraformaldehyde in phosphate-buffered
264
saline for 10 min and Hoechst 33342 (1 µg/mL) was used to label DNA. For live imaging, S. pombe
265
cells were cultured in glass-bottomed Petri dishes with YES and kept at 32°C. Imaging of fixed
266
cells or time lapse for live cells was acquired using a Leica TCS SP5 II confocal microscope or
267
Nikon A1R+ confocal microscope. Image processing and analysis were conducted using ImageJ or
268
GraphPad Prism 7.
269
4.5. S. pombe whole-genome DNA purification
270
S. pombe cells were grown overnight and washed once with sterile water. Cells were
271
re-suspended in 0.2 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM
272
Tris-HCl pH 8.0, 1 mM EDTA pH 8.0), 0.2 mL of phenol:chloroform together with 0.5 mL of glass
273
beads (425–600 µm; G8772, Sigma, USA), and vortexed for 10 min. Then, 0.2 mL of TE buffer (10
274
mM Tris-HCl pH 8.0, 1 mM Na-EDTA pH 8.0) was added and the cells were centrifuged at 15,871
275
×g for 5 min. The supernatant was transferred into a new 1.7-mL Eppendorf tube and 1 mL of 100% 11/19
276
ethanol was added. Cells were centrifuged at 15,871 ×g for 5 min and washed with 100% ethanol
277
once. Supernatant was discarded and the pellet was dried at room temperature. The pellet was
278
resuspended in 50–100 µL of EB buffer.
279
4.6. Preparation of S. pombe total proteins
280
The TCA protein extraction method was used. Cells were grown to an OD600 of 0.2 and
281
centrifuged at 3500 ×g for 5 min. Then, they were resuspended in 1 mL of 1.2 M sorbitol and
282
transferred to a 2-mL Eppendorf tube before centrifugation at full speed for 30 s. The supernatant
283
was discarded and 100 µL of 20% TCA (100 g TCA in 227 mL water) was added to resuspend the
284
cells. Then, 0.5 mL of glass beads (425–600 µm; G8772, Sigma) were added, and the cells were put
285
under vortex movement using Genie 2 for 10 min at 4°C, left to stand for 1 min and put under
286
vortex movement again for 1 min. They were vortexed again briefly after adding 900 µL of 5%
287
TCA (25 g TCA in 227 mL water). An 800-µL extract was transferred to a new 1.7-mL Eppendorf
288
tube and centrifuged for 10 min at 1000 ×g. The supernatant was discarded, and 250 µL of 1×
289
Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol,
290
0.05% BPB) was added followed by resuspension (more 1 M Tris-HCl pH 8.0 could be added if
291
solution turned yellow). After boiling for 3 min and cooling on ice, the extract was centrifuged at
292
1000 ×g for 10 min. Then, the supernatant was transferred to a new 1.7-mL Eppendorf tube, kept
293
frozen at −20°C, and thawed by boiling for 3 min every time a sample was used.
294
4.7. SDS-PAGE and Western blot
295
The NuPAGE MOPS System (Thermo Fisher Scientific, USA) was used to perform the
296
SDS-PAGE and Western blot. For antibody staining, membrane was incubated in primary antibody
297
solution (5% milk in TBST + 1:200 antibodies; the dilutions can vary among different antibodies)
298
overnight at 4°C. It was washed with TBST three times and each time for 10 min (shaking),
299
followed by incubation with secondary antibody solution (5% milk in TBST + 1:5000 antibodies) at
300
room temperature for 1 h. SuperSignal West Pico PLUS Kit (Thermo Fisher Scientific) was used
301
for visualization.
302
4.8. Statistical analysis
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The statistical analysis was performed with Prism (v7, GraphPad, CA, USA) and used to
304
produce graphs. All data were expressed as mean ± SEM. Unpaired two-tailed Student's t-test or
305
one-way ANOVA and Tukey's test were used for the data analysis. Significant differences were
306
attributed for * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.
307 308
Acknowledgments
309
We thank members of the Liu group, especially Drs. Andrew Bassett and Lydia Hulme, for
310
helpful discussion. This work was supported by the UK Medical Research Council (Grant No.
311
MC_UU_12021/3 and MC_U137788471), University of Oxford and ShanghaiTech University.
312 313
References
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cytoophidia in
Figure legends
Fig. 1. Conditions of the cell-cycle phase affect cytoophidia in S. pombe. A–E: CTPS distribution in S. pombe cells at different growth phases. F and G: Cytoophidium number (F) and length (G) vary with growth phases. In F and G, 278‒1750 cells were counted for each tested point. Error bars show SEM. Fig. 2. Stationary phase-induced CTPS disassembly is reversible in S. pombe. A: Cytoophidia disperse during the stationary phase. B: CTPS assembles back into cytoophidia when cells are shifted to rich media and cultured for 2 h. Fig. 3. DON treatment causes an increase in the length of cytoophidia in S. pombe. A and B: Cells without DON treatment (A) or after DON treatment for 4 h (B). C and D: DON treatment increases cytoophidium length (C) but does not change the number of cytoophidia dramatically (D). In C and D, untreated cells, n = 367; DON-treated cells, n = 691. Error bars show SEM. Significant deference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant. E: Western blot of CTPS protein shows no obvious change upon DON treatment. Fig. 4. Low temperature causes decrease of cytoophidia in S. pombe. A‒D: When exposed to an ice-cold environment, most cytoophidia disassemble after 30 min and totally disappear after 2 h. Arrows indicate pairs of cells with asymmetric CTPS distribution. E and F: During cold shock, cytoophidia show a dramatic decrease in occurrence (E) and average length (F). In E and F, 217‒ 940 cells were counted for each tested condition. Error bars show SEM. Fig. 5. Cytoophidia respond quickly to cold stress. A and B: The length of cytoophidia is decreased significantly when S. pombe cells are exposed to 0°C for 5 min. C: Percentage of cells containing cytoophidia is unchanged after 5 min cold shock. D and E: The average length of cytoophidia (D) and the number of cytoophidia per cell (E) have significantly decreased after 5 min cold shock, indicating that cytoophidia are extremely sensitive to temperature changes. In C–E, control (32°C), n = 258 cells; cold shock (0°C, 5min), n = 283 cells. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant.
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Fig. 6. Cold shock does not remove CTPS protein. A: Western blot of CTPS protein under cold stress. Lanes 1‒5, samples taken after cells put on ice for 0, 5, 15, 30, 60 min, respectively. Lanes 6‒8, samples taken after cells were put on ice for 2 h. B: Quantification of the CTPS protein levels shown in A indicates that CTPS protein does not disappear within 2 h of cold stress. Fig. 7. Cold-induced dispersal of cytoophidia is reversible and fast. It takes 3 min and 45 s to prepare the sample before confocal capture on the video. This time period should be added prior to the starting time point. The cells are transferred from 0ºC to 32ºC to perform the recovery experiment. Within 5 min cytoophidia (CTPS, green) start to emerge, indicating a quick response to temperature changes in S. pombe. Fig. 8. DON-induced cytoophidia can be disassembled by cold stress. A: Control group, with cells grown at 32ºC in YES media. B: Cytoophidia increase in length after cells are treated with 10 µM DON for 2 h. C: Cytoophidia are disassembled after cells are put on ice for 2 h. White, CTPS. D and E: Quantification of cytoophidium length (D) and cytoophidium number per cell (E) in each group. In D and E, 247–841 cells were counted for each tested group. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant. Fig. 9. High temperature causes decrease of cytoophidia in S. pombe. A–E: Cytoophidia are sensitive to temperature shift from 32°C to 37°C. F and G: When exposed to 37°C, both cytoophidium length (F) and percentage of cells containing cytoophidia (G) decrease. In F and G, 398–1132 cells were counted for each tested group. Fig. 10. Cytoophidia respond quickly to heat stress. A and B: Cytoophidium length decreases in response to heat shock after only 5 min, comparing cells cultured at 32°C (A) and 42°C (B). White, CTPS. C and D: Average length of cytoophidia (C) and average number of cytoophidia per cell (D) are decreased significantly when S. pombe cells are exposed to 42°C. E: Percentage of cells containing cytoophidia is unchanged after 5 min heat shock. In C–E, control (32°C) n = 258 cells; heat shock (42°C, 5min), n = 261 cells. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant.
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Fig. 11. Cytoophidium morphology is affected in heat-shock protein deletion strains. A and B: Average length of cytoophidia (A) and percentage of cells containing cytoophidia (B) significantly decrease in all six heat-shock protein deletion strains tested. For each tested group, 224–722 cells were counted. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001, ** P < 0.01). ns, not significant. C–J: Different deletion strains show different CTPS-YFP distribution patterns. ppk15∆ cells are used as a second control group.
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DNA+CTPS
OD600 =0. 36
D
B
DNA+CTPS
OD600 =0. 72
DNA+CTPS
OD600 =1. 45
E
C
DNA+CTPS
OD600 =1. 26
DNA+CTPS
St at i onar y
G
OD600
St at i on ar y
Cyt oophi di um numberperc el l
Cyt oophi di um l engt h( μm）r
F
OD600
St at i on ar y
A
Cyt oophi di um numberpercel l
S1673-8527(19)30151-1
DOI:
https://doi.org/10.1016/j.jgg.2019.09.002
Reference:
JGG 724
To appear in:
Journal of Genetics and Genomics
Received Date: 19 April 2019 Revised Date:
29 August 2019
Accepted Date: 9 September 2019
Please cite this article as: Zhang, J., Liu, J.-L., Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe, Journal of Genetics and Genomics, https://doi.org/10.1016/ j.jgg.2019.09.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Copyright © 2019, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
1
Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe
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Jing Zhang a, Ji-Long Liu a,b,*
4 5
a
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Oxford, Oxford, OX1 3PT, United Kingdom
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b
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
8 9 10
*Corresponding author. Email address: [email protected] or [email protected]
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1/19
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Abstract
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The metabolic enzyme CTP synthase (CTPS) is able to compartmentalize into filaments, termed
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cytoophidia, in a variety of organisms including bacteria, budding yeast, fission yeast, fruit flies and
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mammals. A previous study in budding yeast shows that the filament-forming process of CTPS is
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not sensitive to temperature shift. Here we study CTPS filamentation in the fission yeast
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Schizosaccharomyces pombe. To our surprise, we find that both the length and the occurrence of
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cytoophidia in S. pombe decrease upon cold shock or heat shock. The temperature-dependent
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changes of cytoophidia are fast and reversible. Taking advantage of yeast genetics, we demonstrate
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that heat-shock proteins are required for cytoophidium assembly in S. pombe. Temperature
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sensitivity of cytoophidia makes S. pombe an attractive model system for future investigations of
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this novel membraneless organelle.
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Keywords: CTP synthase, cytoophidium, Schizosaccharomyces pombe, heat shock protein (HSP),
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nucleoside/nucleotide metabolism, cell biology, yeast genetics, cell compartmentalization
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1. Introduction
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CTP synthase (CTPS) is an enzyme involved in nucleotide biosynthesis in almost all
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organisms. Recently multiple studies have demonstrated that CTPS forms filamentous structures
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termed cytoophidia in Drosophila (Liu, 2010), bacteria (Ingerson-Mahar et al., 2010), yeast (Noree
30
et al., 2010; Zhang et al., 2014) and human cells (Carcamo et al., 2011; Chen et al., 2011) (reviews
31
see Aughey and Liu, 2015; Liu, 2010, 2016). In humans and the budding yeast Saccharomyces
32
cerevisiae, there are two CTPS isoforms (encoded by CTPS1 and CTPS2 in humans; encoded by
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URA7 and URA8 in S. cerevisiae), while in the fission yeast Schizosaccharomyces pombe, only one
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CTPS isoform exists and is encoded at a single locus cts1 on chromosome I (Kim et al., 2010;
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Hayles et al., 2013). We have shown that among the three CTPS isoforms (CTPS isoform-A, B and
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C) in Drosophila only CTPS isoform-A is able to compartmentalize into filaments (Azzam and Liu,
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2013). We also have identified that the two human CTPS isoforms, CTPS1 and CTPS2, can both
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compartmentalize into cytoophidia (Gou et al., 2014). The lack of CTPS redundancy in S. pombe
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gives this fission yeast an advantage as a model for the study of cytoophidia.
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Noree et al. (2010) reported that CTPS and several other proteins can form filamentous
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structures in budding yeast. They also found that filamentation of these proteins is regulated by
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various environmental factors such as nutrition availability, carbon source depletion, energy status
43
and the presence of protein synthesis inhibitors (Noree et al., 2010). In comparison with cells
44
cultured at 30°C, budding yeast cells grown at low temperature (0°C) for 15 min showed no
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difference in the morphology and frequency of CTPS filaments (Noree et al., 2010). They
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concluded that filamentation of CTPS is not sensitive to temperature shift.
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In Drosophila S2R+ cells, nutrient depletion leads to an increase in number of cytoophidia
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(Aughey et al., 2014), which is parallel with the results observed in budding yeast. Similarly,
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cytoophidium assembly can also be induced in Drosophila larvae by nutrient restriction (Aughey et
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al., 2014). In Drosophila larval post-embryonic neuroblasts (pNbs), cytoophidia can be induced by
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nutritional restriction during the late L2 stage (Aughey et al., 2014; Tastan and Liu, 2015).
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Cytoophidia can also be observed in imaginal discs (eye, wing, leg and haltere) of third instar larvae
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fed with low-nutrient food (Aughey et al., 2014). A recent study in adult fly ovaries showed that
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cytoophidia increase their length upon starvation or during apoptosis (Wu and Liu, 2019).
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In this study, we use the fission yeast S. pombe as a model system and study the response of
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cytoophidia to environmental stresses. Surprisingly, we find that cytoophidia are very sensitive to
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cold shock and heat shock. Our results suggest that cytoophidium assembly is a
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temperature-sensitive process in S. pombe, in contrast to previous observations in S. cerevisiae.
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Furthermore, we generate six heat-shock mutant strains and find that both the length and the
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occurrence of cytoophidia decrease significantly when one of these heat-shock proteins is disrupted.
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Our study highlights the importance of exploring cytoophidium assembly in multiple systems.
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2. Results
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2.1. Cytoophidium assembly is regulated throughout growing stages
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S. pombe cells undergo four phases such as lag phase, exponential phase, stationary phase and
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death phase, with differing metabolic conditions. To examine whether the different phases have an
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effect on cytoophidia, the CTPS-YFP expressing cells were fixed at various points in the growing
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stages and observed under confocal microscope. Cell growth was monitored by measuring the
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OD600 value using the spectrometer. Each counting group contains more than 500 cells. Software
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ImageJ was used to measure the cytoophidium number per cell and the cytoophidium length (see
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details in Materials and methods). During the early-to-middle exponential phase (OD600 = 0.1–1.0),
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cytoophidia were highly abundant, being present in more than 90% of cells (Fig. 1A, B and F). As
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the cells grew and OD600 increased above 1.0, fewer cytoophidia were observed compared with the
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mid-exponential phase (Fig. 1C, D, and G). When cells entered the stationary phase, most of the
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cytoophidia disappeared, dispersing into the cytoplasm (Fig. 1E and G). This result indicates that
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cytoophidium regulation is closely linked to the metabolic conditions within S. pombe cells.
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Next, we wanted to know if the dispersal of cytoophidia at the stationary phase is reversible.
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During the early-to-middle exponential phase (OD600 = 0.1–1.0), a high percentage of cells
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contained cytoophidia with a length of over 1.0 µm (Fig. 1G). When cells entered the stationary
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phases, the cytoophidia dispersed into the cytoplasm (Fig. 2A). When shifted to rich media and
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grown for another 2 h, the cells entered the exponential phase again and the number of cytoophidia 4/19
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increased (Fig. 2B), indicating that stationary-phase-induced cytoophidium dispersal is reversible in
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S. pombe.
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2.2. Cytoophidia respond to a glutamine analog
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After treatment with the CTPS inhibitor 6-diazo-5-oxo-L-norleucine (DON), the average
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length of the cytoophidia was increased significantly (Fig. 3A–C), which is similar to the outcome
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observed in Drosophila and human HeLa cells (Chen et al., 2011; Aughey et al., 2014), while the
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cytoophidium number per cell in S. pombe did not changed significantly (Fig. 3D). Interestingly, the
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CTPS protein level was not changed after DON treatment (Fig. 3E).
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2.3. Cold shock disassembles cytoophidia
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When fission yeast cells were incubated at 32°C, we found that >90% of cells at the
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early-to-middle exponential phase contained obvious cytoophidia (Fig. 4A and E). When cells were
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incubated on ice (0°C) for 30 min, only 20% contained cytoophidia (Fig. 4B and E). Moreover, the
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length of the cytoophidia had decreased dramatically (Fig. 4B and F). After 1 h at 0°C, almost all
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cells did not have cytoophidia, and we could only detect residual cytoophidia of very short length
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(Fig. 4C, E and F). After 2 h at 0°C, CTPS showed a diffused pattern without any sign of
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cytoophidia (Fig. 4D–F).
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We previously showed that cytoophidia are inherited asymmetrically between two daughter
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cells during cell cycle division (Zhang et al., 2014). One daughter cell inherits the cytoophidium
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from the mother cell, while the other daughter cell does not. Upon cold shock, even without
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cytoophidia, the asymmetric distribution of CTPS is obvious in many paired cells, with one cell
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having a much higher signal than the other, indicating that the protein level of CTPS is very
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different (Fig. 4B–D). The differential distribution of CTPS between two daughter cells is
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presumably achieved via asymmetric inheritance of cytoophidia.
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2.4. Disassembly of cytoophidia induced by cold shock is fast
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To further investigate how quickly cold shock affects cytoophidium disassembly, we incubated
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fission yeast cells on ice for shorter time lengths. We found that treating cells at 0°C for only 5 min
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had a profound effect on cytoophidium formation (Fig. 5A and B). Although the percentage of cells
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containing cytoophidia did not change significantly (Fig. 5C), we did observe that the length of 5/19
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cytoophidia decreased dramatically, from 1.4 µm on average to below 1 µm (Fig. 5D). The
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cytoophidium number per cell also significantly decreased (Fig. 5E). These results suggest that
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assembly of cytoophidia in S. pombe is extremely temperature sensitive.
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2.5. Cold shock does not remove CTPS protein
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In Drosophila, the level of CTPS protein is critical for cytoophidium assembly.
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Overexpression of CTPS induces long and thick cytoophidia, while knockdown of CTPS
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disassembles cytoophidia (Azzam and Liu, 2013). To determine whether cold-shock-induced
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cytoophidium disassembly is due to the change of CTPS protein level, we performed Western blot
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of cells collected at different time points after cold-shock treatment. Anti-CTPS antibody was used
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for Western blot and histone H3 was used as the endogenous control. Samples were taken at 5, 15,
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30, 60 min and 2 h after cells were incubated on ice. We found that cold shock for up to 2 h did not
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remove CTPS protein (Fig. 6).
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2.6. Cold-shock-induced cytoophidium disassembly is reversible
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Next we asked whether cold-shock-induced cytoophidium disassembly is reversible. We
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incubated yeast cells at 0°C overnight. As expected, cytoophidia disassembled completely, with
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CTPS showing a diffuse distribution. We then returned the yeast cells to 32°C and observed the
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cells via live imaging. Within 5 min, we saw that CTPS started to form punctate structures, which
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grew longer over time (Fig. 7). After 30 min, we observed that many cells had cytoophidia of a size
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comparable to those in the control group (without cold shock) (data not shown). These results
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indicate that cold-shock-induced cytoophidium disassembly is reversible.
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2.7. DON treatment does not prevent cold-shock-induced disassembly of cytoophidia
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In eukaryotic cells, CTPS uses glutamine as a nitrogen donor. The glutamine analog DON
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binds to CTPS irreversibly. We have previously shown that DON treatment promotes cytoophidium
132
assembly in Drosophila and human cells (Chen et al., 2011). DON treatment also promotes
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cytoophidium assembly in S. pombe. Treating fission yeast cells with DON for 2 h increased the
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cytoophidium length from 1.3 µm to 1.6 µm. On average, fewer cytoophidia were observed in
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DON-treated cells, suggesting that DON treatment induces fusion of small cytoophidia.
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To determine whether DON-induced cytoophidium assembly is reversible by cold-shock
137
induction, we incubated fission yeast cells in DON for 2 h at 32°C followed by incubating them at
138
0°C for 2 h. We found that cytoophidia were no longer detectable after the incubation at 0°C (Fig.
139
8). These results indicate that 1) DON-induced cytoophidium assembly is reversible; and 2) DON
140
treatment cannot prevent cold-shock-induced cytoophidium disassembly.
141
2.9. Heat shock disassembles cytoophidia
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Having observed that cytoophidium assembly is sensitive to cold shock, we next asked
143
whether heat shock affects cytoophidium assembly in fission yeast. At 32°C, the majority of S.
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pombe cells in the early-to-middle exponential phase contain obvious cytoophidia (Fig. 9A). When
145
exposed to a higher temperature (i.e., 37°C or 42°C), cytoophidia showed a decrease in both the
146
average length and the number of cells containing cytoophidia (Fig. 9B–F).
147
Similar to cold shock, heat shock affects cytoophidium assembly very quickly (Fig. 10A and
148
B). Cytoophidia significantly decreased in both the length and the number of cytoophidia per cell
149
after only 5 min of heat shock treatment at 42°C, compared with cells cultured at 32°C (Fig. 10A‒
150
D). However, the percentage of cells containing cytoophidia was unchanged after 5 min heat shock
151
(Fig. 10E).
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2.10. Heat-shock proteins are required for cytoophidium assembly
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Heat-shock proteins are a family of proteins produced in response to environmental stresses,
154
especially heat-shock stress. We already knew that cytoophidia respond to temperature changes. To
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determine whether small heat-shock proteins are required for cytoophidium assembly, we selected
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several small heat-shock proteins in S. pombe cells for further investigation. We constructed a series
157
of deletion strains expressing CTPS-YFP, namely hsp9∆, hsp16∆, hsp20∆, hsp104∆, pdr13∆ and
158
sks2∆. Since ppk15, a serine/threonine protein kinase, is not related to heat-shock proteins or
159
heat-shock response, we used the ppk15∆ strain as an extra control alongside the wild-type control.
160
We found that both the length and the occurrence of cytoophidia decreased significantly in all six
161
heat-shock mutant strains that we investigated (Fig. 11). Our results indicate that heat-shock
162
proteins are required for cytoophidium assembly.
163
3. Discussion 7/19
164
3.1. Comparison of cytoophidia between S. cerevisiae and S. pombe
165
Several studies have shown that CTPS compartmentalizes into filamentous structures in
166
various organisms, including bacteria, fruit fly, rat and human cells (Ingerson-Mahar et al., 2010;
167
Liu, 2010; Noree et al., 2010; Carcamo et al., 2011; Chen et al., 2011). It has also been reported that
168
CTPS filaments can be observed in budding yeast (Noree et al., 2010; Noree et al., 2014; Shen et al.,
169
2016; Zhang et al., 2018). We have previously identified that CTPS forms cytoophidia in S. pombe
170
(Zhang et al., 2014; Li et al., 2018). These observations suggest that cytoophidia are evolutionarily
171
conserved from bacteria to humans.
172
Both budding yeast and fission yeast are good model organisms to study cytoophidia. It has
173
already been reported that the expression products of both CTPS genes (URA7 and URA8) can
174
compartmentalize into filaments in S. cerevisiae (Noree et al., 2010). In budding yeast, cytoophidia
175
respond to several drugs and environment stresses (Noree et al., 2010). Here we show that
176
cytoophidia are also sensitive to DON and stresses in fission yeast. However, there are some
177
notable differences in cytoophidium behavior between S. cerevisiae and S. pombe.
178
In budding yeast, cytoophidia can be detected mostly in the stationary phase but not in the
179
exponential phase (Noree et al., 2010; Shen et al., 2016). In S. pombe, we show that only
180
exponential cells contain large numbers of cytoophidia while few or no cytoophidia can be observed
181
during the stationary stage. Moreover, carbon deprivation induces cytoophidium assembly in
182
budding yeast (Noree et al., 2010), while here we found that the lack of a carbon source causes the
183
dispersal of cytoophidia in fission yeast. These opposing results suggest that the regulatory
184
mechanism of cytoophidium assembly differs between S. pombe and S. cerevisiae.
185
3.2. The effect of DON on cytoophidium assembly
186
DON is an inhibitor for many glutamine-utilizing enzymes including CTPS. It has been
187
reported that the majority of cytoophidia in human cells and Drosophila are an inactive form of
188
CTPS (Aughey et al., 2014; Barry et al., 2014; Noree et al., 2014), leading to a hypothesis that
189
cytoophidia act as a CTPS storage depot as well as a regulator of CTPS metabolic activities. When
190
treated with DON, S. pombe showed an increase of the average length of cytoophidia. Similar
191
results can be detected in human HeLa cells, Drosophila S2 cells and Drosophila tissues (Chen et 8/19
192
al., 2011; Aughey et al., 2014;), indicating that the promotion of cytoophidium assembly by DON is
193
highly conserved.
194
Interestingly, although the average length of cytoophidia was increased after DON treatment,
195
the average number of cytoophidia per cell did not change dramatically in S. pombe. The small
196
cytoophidia may have fused into longer and thicker filaments, and therefore increased in filament
197
length. This is similar to the maturation process of cytoophidia observed in mammalian culture cells
198
(Gou et al., 2014; Chang et al., 2018; Keppeke et al., 2018).
199
3.3. Cytoophidia respond to temperature shift
200
Our study has revealed that cytoophidium maintenance is sensitive to temperature shift, both to
201
cold shock and to heat shock. Given that the cytoophidium formation is growth phase-dependent in
202
fission yeast, there is an argument that the temperature sensitivity is due to the cells switching
203
between the exponential phase and the stationary phase upon temperature shift. The response of
204
cytoophidia to heat or cold stress is fast (within a few minutes), while the time course of growth
205
phase changes is slow (from a few hours to a few days). Therefore we think the dynamics of
206
cytoophidia during temperature shift cannot be simply explained by growth phase changes.
207
Using yeast genetics, we have shown the involvement of heat-shock proteins, hsp9, hsp16,
208
hsp20, hsp104, pdr13 (ssz1) and sks2, in cytoophidium assembly. Among these six proteins, only
209
hsp9 has been reported to be involved in vegetative growth metabolism under normal growing
210
temperature conditions, while the other five proteins play roles during various stress responses.
211
Heat-shock proteins are a family of proteins that are produced by cells in response to exposure
212
to stressful conditions. They were first described in relation to heat shock, but are now known to be
213
expressed during other stresses including exposure to cold and UV light, and during wound healing
214
or tissue remodeling (Li and Srivastava, 2004). Many members of this protein family perform a
215
chaperone function by stabilizing new proteins to ensure correct folding or by helping to refold
216
proteins that were damaged by the cell stress. This increase of expression is transcriptionally
217
regulated (Wu, 1995). The dramatic upregulation of the heat-shock proteins is a key part of the
218
heat-shock response and is induced primarily by heat-shock factor (HSF) (Wu, 1995). Heat-shock
219
proteins are found in virtually all living organisms, from bacteria to humans. 9/19
220
3.4. Potential functions of cytoophidia in S. pombe
221
Recently, an increasing number of metabolic enzymes have been identified as having the
222
ability to self-assemble and to form filamentous structures, including CTPS, suggesting that
223
intracellular compartmentation of metabolic enzymes is more general than previously thought. A
224
large-scale screen of a yeast GFP library identifies that over 20% of the examined proteins were
225
able to from distinct intracellular structures (Narayanaswamy et al., 2009). Another screen of 40%
226
of a budding yeast GFP strain collection shows that nine proteins form four types of filamentous
227
structure (Noree et al., 2010). A further comprehensive screening of 4159 proteins in budding yeast
228
reveals that at least 23 proteins have filament-forming capacity (Shen et al., 2016). Several studies
229
demonstrate that polymerisation of CTPS into cytoophidia downregulates enzymatic activity in
230
bacteria (Barry et al., 2014), budding yeast (Noree et al., 2014), fruit flies and human cell lines
231
(Aughey et al., 2014). Additional studies suggest forming filaments can upregulate enzymatic
232
activities (Strochlic et al., 2014; Chang et al., 2015; Lynch et al., 2017). Thus, cytoophidium
233
formation seems to be a general strategy to regulate enzymatic activity. The choice of upregulation
234
or downregulation may reflect the context of developmental stages or metabolic statuses.
235
This study demonstrates that cytoophidium assembly is a temperature-sensitive process in S.
236
pombe. The assembly and disassembly of the cytoophidium and its kind provide a potential
237
mechanism to maintain cellular homeostasis during development and in mediating metabolic
238
responses (Aughey et al., 2014; Barry et al., 2014; Noree et al., 2014; Petrovska et al., 2014;
239
Strochlic et al., 2014). Yet it is still not clear whether the downregulation of cytoophidia is directly
240
or indirectly caused by the action of heat-shock proteins. More recently, we found that forming
241
cytoophidia prolongs the half-life of CTPS in human cell lines (Sun and Liu, 2019). Further studies
242
are needed to determine how various heat-shock proteins cooperate in the processes of assembly,
243
maintenance and disassembly of cytoophidia.
244
4. Materials and methods
245
4.1. S. pombe strains
246 247
The generation of the CTPS-YFP-expressing S. pombe strain has been described in our previous study (Zhang et al., 2014). 10/19
248
4.2. S. pombe cell culture
249
If not mentioned, all cells were cultured at 32°C in YES media using a shaking incubator. Cell
250
growth was monitored by OD600, with a 0.1–1.5 OD600 value indicating exponentially growing cells
251
and over 1.5 OD600 indicating stationary cells. Two wild-type S. pombe strains, 001 (h+;
252
ade6-M216, leu1-32, ura4-D18) and 002 (h-; ade6 M216, leu1-32, ura4-D18), were obtained from
253
the Stephen Kearsey Laboratory (University of Oxford). The 003 (h+; ade6-M216, leu1 32, ura+,
254
cts1-yfp) strain was used as the control unless stated otherwise.
255
4.3. Site-directed mutagenesis
256
In-fusion Cloning (Clontech, USA) and QuikChange Site-Directed Mutagenesis (Agilent, USA)
257
technologies were utilized in this study to generate target point mutated plasmids. The standard
258
commercial protocol was used for the detailed manipulation of plasmid construction. The primers
259
used in In-fusion and QuickChange are designed on the website. For the overlap PCR, the overlaps
260
of the primers were designed with 25 bp at least.
261
4.4. Light microscopy
262
Cells were cultured overnight before being fixed and scanned, and were guaranteed to be fresh
263
and in the exponential phase. Cells were fixed with 4% paraformaldehyde in phosphate-buffered
264
saline for 10 min and Hoechst 33342 (1 µg/mL) was used to label DNA. For live imaging, S. pombe
265
cells were cultured in glass-bottomed Petri dishes with YES and kept at 32°C. Imaging of fixed
266
cells or time lapse for live cells was acquired using a Leica TCS SP5 II confocal microscope or
267
Nikon A1R+ confocal microscope. Image processing and analysis were conducted using ImageJ or
268
GraphPad Prism 7.
269
4.5. S. pombe whole-genome DNA purification
270
S. pombe cells were grown overnight and washed once with sterile water. Cells were
271
re-suspended in 0.2 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM
272
Tris-HCl pH 8.0, 1 mM EDTA pH 8.0), 0.2 mL of phenol:chloroform together with 0.5 mL of glass
273
beads (425–600 µm; G8772, Sigma, USA), and vortexed for 10 min. Then, 0.2 mL of TE buffer (10
274
mM Tris-HCl pH 8.0, 1 mM Na-EDTA pH 8.0) was added and the cells were centrifuged at 15,871
275
×g for 5 min. The supernatant was transferred into a new 1.7-mL Eppendorf tube and 1 mL of 100% 11/19
276
ethanol was added. Cells were centrifuged at 15,871 ×g for 5 min and washed with 100% ethanol
277
once. Supernatant was discarded and the pellet was dried at room temperature. The pellet was
278
resuspended in 50–100 µL of EB buffer.
279
4.6. Preparation of S. pombe total proteins
280
The TCA protein extraction method was used. Cells were grown to an OD600 of 0.2 and
281
centrifuged at 3500 ×g for 5 min. Then, they were resuspended in 1 mL of 1.2 M sorbitol and
282
transferred to a 2-mL Eppendorf tube before centrifugation at full speed for 30 s. The supernatant
283
was discarded and 100 µL of 20% TCA (100 g TCA in 227 mL water) was added to resuspend the
284
cells. Then, 0.5 mL of glass beads (425–600 µm; G8772, Sigma) were added, and the cells were put
285
under vortex movement using Genie 2 for 10 min at 4°C, left to stand for 1 min and put under
286
vortex movement again for 1 min. They were vortexed again briefly after adding 900 µL of 5%
287
TCA (25 g TCA in 227 mL water). An 800-µL extract was transferred to a new 1.7-mL Eppendorf
288
tube and centrifuged for 10 min at 1000 ×g. The supernatant was discarded, and 250 µL of 1×
289
Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol,
290
0.05% BPB) was added followed by resuspension (more 1 M Tris-HCl pH 8.0 could be added if
291
solution turned yellow). After boiling for 3 min and cooling on ice, the extract was centrifuged at
292
1000 ×g for 10 min. Then, the supernatant was transferred to a new 1.7-mL Eppendorf tube, kept
293
frozen at −20°C, and thawed by boiling for 3 min every time a sample was used.
294
4.7. SDS-PAGE and Western blot
295
The NuPAGE MOPS System (Thermo Fisher Scientific, USA) was used to perform the
296
SDS-PAGE and Western blot. For antibody staining, membrane was incubated in primary antibody
297
solution (5% milk in TBST + 1:200 antibodies; the dilutions can vary among different antibodies)
298
overnight at 4°C. It was washed with TBST three times and each time for 10 min (shaking),
299
followed by incubation with secondary antibody solution (5% milk in TBST + 1:5000 antibodies) at
300
room temperature for 1 h. SuperSignal West Pico PLUS Kit (Thermo Fisher Scientific) was used
301
for visualization.
302
4.8. Statistical analysis
12/19
303
The statistical analysis was performed with Prism (v7, GraphPad, CA, USA) and used to
304
produce graphs. All data were expressed as mean ± SEM. Unpaired two-tailed Student's t-test or
305
one-way ANOVA and Tukey's test were used for the data analysis. Significant differences were
306
attributed for * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.
307 308
Acknowledgments
309
We thank members of the Liu group, especially Drs. Andrew Bassett and Lydia Hulme, for
310
helpful discussion. This work was supported by the UK Medical Research Council (Grant No.
311
MC_UU_12021/3 and MC_U137788471), University of Oxford and ShanghaiTech University.
312 313
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cytoophidia in
Figure legends
Fig. 1. Conditions of the cell-cycle phase affect cytoophidia in S. pombe. A–E: CTPS distribution in S. pombe cells at different growth phases. F and G: Cytoophidium number (F) and length (G) vary with growth phases. In F and G, 278‒1750 cells were counted for each tested point. Error bars show SEM. Fig. 2. Stationary phase-induced CTPS disassembly is reversible in S. pombe. A: Cytoophidia disperse during the stationary phase. B: CTPS assembles back into cytoophidia when cells are shifted to rich media and cultured for 2 h. Fig. 3. DON treatment causes an increase in the length of cytoophidia in S. pombe. A and B: Cells without DON treatment (A) or after DON treatment for 4 h (B). C and D: DON treatment increases cytoophidium length (C) but does not change the number of cytoophidia dramatically (D). In C and D, untreated cells, n = 367; DON-treated cells, n = 691. Error bars show SEM. Significant deference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant. E: Western blot of CTPS protein shows no obvious change upon DON treatment. Fig. 4. Low temperature causes decrease of cytoophidia in S. pombe. A‒D: When exposed to an ice-cold environment, most cytoophidia disassemble after 30 min and totally disappear after 2 h. Arrows indicate pairs of cells with asymmetric CTPS distribution. E and F: During cold shock, cytoophidia show a dramatic decrease in occurrence (E) and average length (F). In E and F, 217‒ 940 cells were counted for each tested condition. Error bars show SEM. Fig. 5. Cytoophidia respond quickly to cold stress. A and B: The length of cytoophidia is decreased significantly when S. pombe cells are exposed to 0°C for 5 min. C: Percentage of cells containing cytoophidia is unchanged after 5 min cold shock. D and E: The average length of cytoophidia (D) and the number of cytoophidia per cell (E) have significantly decreased after 5 min cold shock, indicating that cytoophidia are extremely sensitive to temperature changes. In C–E, control (32°C), n = 258 cells; cold shock (0°C, 5min), n = 283 cells. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant.
17/19
Fig. 6. Cold shock does not remove CTPS protein. A: Western blot of CTPS protein under cold stress. Lanes 1‒5, samples taken after cells put on ice for 0, 5, 15, 30, 60 min, respectively. Lanes 6‒8, samples taken after cells were put on ice for 2 h. B: Quantification of the CTPS protein levels shown in A indicates that CTPS protein does not disappear within 2 h of cold stress. Fig. 7. Cold-induced dispersal of cytoophidia is reversible and fast. It takes 3 min and 45 s to prepare the sample before confocal capture on the video. This time period should be added prior to the starting time point. The cells are transferred from 0ºC to 32ºC to perform the recovery experiment. Within 5 min cytoophidia (CTPS, green) start to emerge, indicating a quick response to temperature changes in S. pombe. Fig. 8. DON-induced cytoophidia can be disassembled by cold stress. A: Control group, with cells grown at 32ºC in YES media. B: Cytoophidia increase in length after cells are treated with 10 µM DON for 2 h. C: Cytoophidia are disassembled after cells are put on ice for 2 h. White, CTPS. D and E: Quantification of cytoophidium length (D) and cytoophidium number per cell (E) in each group. In D and E, 247–841 cells were counted for each tested group. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant. Fig. 9. High temperature causes decrease of cytoophidia in S. pombe. A–E: Cytoophidia are sensitive to temperature shift from 32°C to 37°C. F and G: When exposed to 37°C, both cytoophidium length (F) and percentage of cells containing cytoophidia (G) decrease. In F and G, 398–1132 cells were counted for each tested group. Fig. 10. Cytoophidia respond quickly to heat stress. A and B: Cytoophidium length decreases in response to heat shock after only 5 min, comparing cells cultured at 32°C (A) and 42°C (B). White, CTPS. C and D: Average length of cytoophidia (C) and average number of cytoophidia per cell (D) are decreased significantly when S. pombe cells are exposed to 42°C. E: Percentage of cells containing cytoophidia is unchanged after 5 min heat shock. In C–E, control (32°C) n = 258 cells; heat shock (42°C, 5min), n = 261 cells. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001). ns, not significant.
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Fig. 11. Cytoophidium morphology is affected in heat-shock protein deletion strains. A and B: Average length of cytoophidia (A) and percentage of cells containing cytoophidia (B) significantly decrease in all six heat-shock protein deletion strains tested. For each tested group, 224–722 cells were counted. Error bars show SEM. Significant difference was determined by unpaired two-tailed Student’s t-test (**** P < 0.0001, ** P < 0.01). ns, not significant. C–J: Different deletion strains show different CTPS-YFP distribution patterns. ppk15∆ cells are used as a second control group.
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DNA+CTPS
OD600 =0. 36
D
B
DNA+CTPS
OD600 =0. 72
DNA+CTPS
OD600 =1. 45
E
C
DNA+CTPS
OD600 =1. 26
DNA+CTPS
St at i onar y
G
OD600
St at i on ar y
Cyt oophi di um numberperc el l
Cyt oophi di um l engt h( μm）r
F
OD600
St at i on ar y
A
Cyt oophi di um numberpercel l