Stress induced nuclear granules form in response to accumulation of misfolded proteins in Caenorhabditis elegans
© The Author(s). 2017
Received: 17 February 2017
Accepted: 7 April 2017
Published: 19 April 2017
Environmental stress can affect the viability or fecundity of an organism. Environmental stressors may affect the genome or the proteome and can cause cellular distress by contributing to protein damage or misfolding. This study examines the cellular response to environmental stress in the germline of the nematode, C. elegans.
Salt stress, oxidative stress, and starvation, but not heat shock, induce the relocalization of ubiquitin, proteasome, and the TIAR-2 protein into distinct subnuclear regions referred to as stress induced nuclear granules (SINGs). The SINGs form within 1 h of stress initiation and do not require intertissue signaling. K48-linked polyubiquitin chains but not K63 chains are enriched in SINGs. Worms with a mutation in the conjugating enzyme, ubc-18, do not form SINGs. Additionally, knockdown of ubc-20 and ubc-22 reduces the level of SING formation as does knockdown of the ubiquitin ligase chn-1, a CHIP homolog. The nuclear import machinery is required for SING formation. Stressed embryos containing SINGs fail to hatch and cell division in these embryos is halted. The formation of SINGs can be prevented by pre-exposure to a brief period of heat shock before stress exposure. Heat shock inhibition of SINGs is dependent upon the HSF-1 transcription factor.
The heat shock results suggest that chaperone expression can prevent SING formation and that the accumulation of damaged or misfolded proteins is a necessary precursor to SING formation. Thus, SINGs may be part of a novel protein quality control system. The data suggest an interesting model where SINGs represent sites of localized protein degradation for nuclear or cytosolic proteins. Thus, the physiological impacts of environmental stress may begin at the cellular level with the formation of stress induced nuclear granules.
KeywordsUbiquitin Proteasome Nuclear body Salt stress Oxidative stress Starvation
Organisms are faced with a variety of environmental conditions, some of which can adversely affect the status of proteins. Cells have multiple systems that monitor and maintain the proteome. Protein quality control systems help to refold proteins, to sequester proteins, or to degrade damaged or misfolded proteins. The ubiquitin proteasome system (UPS) is the major intracellular protein degradation pathway. This system is essential for removing damaged or misfolded proteins [1, 2]. In the current study, we investigate how the UPS is involved in the response to environmental stressors in the germline of the nematode, Caenorhabditis elegans. Ubiquitin is an 8.5 kDa polypeptide. Three distinct enzymatic activities link ubiquitin to the substrate protein via an isopeptide bond between the C-terminal glycine of ubiquitin and the amino group on a lysine residue of the substrate. This process is used to either add a single ubiquitin or a polyubiquitin chain. Different types of polyubiquitin chains form depending on the lysine linkage used. K48 polyubiquitin chains are recognized by the proteasome  and K63 chains are associated with protein trafficking, NFκB activation, and receptor endocytosis [4, 5].
Protein quality control systems exist for proteins in the cytosol, the mitochondria, and the endoplasmic reticulum . However, the control of protein quality in the nucleus is not well understood. Ubiquitin and proteasome are both found in the nucleus along with various chaperones . Proteasome activity has been detected in the nuclei of mammalian cells . Therefore, the machinery needed for protein quality control exists in the nucleus, but details on the pathway for triggering nuclear protein degradation is not known. The best described examples of nuclear protein degradation come from yeast where the San1p ubiquitin ligase is known to target unstable proteins for nuclear degradation . Also in yeast, misfolded cytoplasmic proteins can be imported into the nucleus for degradation . It is not presently clear if this same type of pathway exists in other organisms.
There are several documented nuclear changes in response to stress. The nuclei of cells in various model organisms are known to develop distinct nuclear bodies [11, 12]. These nuclear bodies often vary in size, lack a defining membrane, and are spherical in shape. Nuclear bodies that form in response to stress include promyelocytic leukemia bodies (PML), heat-shock bodies, paraspeckles, clastosomes, nucleoplasmic speckles, and insulator bodies [13–16]. Heat-shock bodies form as a result of elevated temperatures, which leads to the activation of HSF1 [14, 17]. PML bodies form in response to elevated levels of oxidative stress and increase in numbers and size as stress exposure is extended [18–20]. Osmotic stress also induces formation of clastosomes and insulator bodies [15, 16]. Some nuclear bodies are known to contain ubiquitin and proteasome components . Clastosomes contain both ubiquitin conjugates and 19S and 20S proteasome complexes, and disappear when proteasome inhibitors are added. These nuclear bodies are proposed to be active sites of proteolysis . Proteasome components have also been observed in nucleoplasmic speckles and PML nuclear bodies [22–24]. Currently, there is a poor understanding of nuclear bodies’ physiological role and how they are connected to the cellular stress response.
Previous studies in C. elegans have shown that exposure to hypertonic stress increases the level of ubiquitin conjugates . That study also showed that expression of the proteasome was required for worms to survive threshold levels of hypertonic stress indicating that protein quality control is a critical aspect of surviving environmental stress. The current study further elaborates on the role of the UPS in response to environmental stress in the germline of C. elegans. We show that ubiquitin and the proteasome respond to salt stress, oxidative stress, and starvation by forming Stress Induced Nuclear Granules (SINGs). These SINGs form quickly after stress is initiated and do not require intertissue signaling. The SINGs are enriched in K48 polyubiquitin chains suggesting that they may be sites of protein degradation. The expression of chaperones reduces the frequency of SINGs suggesting a model where the accumulation of misfolded proteins leads to SING formation.
Stress induces foci with K48 polyubiquitin chains and proteasomes in the nucleus
To test whether the SINGs are artifacts of the fluorescent protein fusions, we used antibodies specific for ubiquitin, the RPT-3 subunit of the 19S proteasome, and the PAS-6 (alpha 1) subunit of the 20S proteasome. The antibody stains show that both endogenous ubiquitin and proteasome localize to SINGs after exposure to 500 mM NaCl (Fig. 1c and e). This indicates that the fluorescent reporters act as credible indicators of the localization of endogenous ubiquitin and proteasome. As can be seen in the different panels in Fig. 1, the SINGs vary in size and fluorescent intensity. Generally, larger SINGs appear in the proximal oocytes but smaller SINGs exist in the distal region. However, even in proximal oocytes there can be both large SINGs and smaller SINGs as is seen in the example in Fig. 1b.
Exposure to high salt could cause cellular distress via a variety of mechanisms such as creating osmotic stress or by disrupting intermolecular and intramolecular charge-based interactions. In order to distinguish between these two scenarios, we used a non-ionic osmotic stress and looked for SING formation. Worms treated with a high concentration of sucrose also form SINGs (Additional file 1: Figure S1) suggesting that osmotic stress may be the instigator of SING formation. To explore whether SINGs might be sites of protein aggregation that form in response to salt stress, we performed FRAP analysis of SINGs and compared the behavior of GFP::Ub in SINGs to that of Q82::GFP, a polyglutamine containing protein that forms aggregates in the cytoplasm of muscle cells . The GFP::Ub in SINGs shows much greater mobility than that of Q82::GFP in aggregates indicating that SINGs are not likely to be sites of protein aggregation (Additional file 2: Figure S2).
Polyubiquitin chains form via an isopeptide linkage between the C-terminal glycine of one ubiquitin and a lysine side chain of another ubiquitin. These chains can be formed by attachment to one of seven different lysines in ubiquitin. All types of chains have been found in cells but the K48 and K63 chains are the most abundant . In order to examine the linkage specificity of ubiquitin in SINGs, we used anti-K48 and anti-K63 polyubiquitin antibodies. K48-linked chains localize to specific nuclear foci under salt stressed conditions in oocytes but K63 chains do not (Fig. 1d). The presence of K48-linked chains in nuclear foci of stressed individuals along with the presence of proteasome is consistent with a model suggesting that proteolysis may be occurring at SING sites and that these sites may be participating in a protein quality control system.
SINGs form in response to accumulation of misfolded proteins and require nuclear import
It is known that protein quality control systems change as an organism ages. In C. elegans, chaperone induction declines in aging worms  and the amount of protein aggregation increases . When 4 day old worms were subjected to salt stress, SING formation happened more rapidly than in younger adults (Fig. 2c). This result taken together with our finding that prior heat shock can prevent SING formation is consistent with a scenario where cellular stress that causes an increase in protein misfolding also leads to the formation of nuclear granules that contain ubiquitin and proteasome.
Thus, nuclear import is required for SING formation. However, at this time it is not clear whether ubiquitinated substrates and/or proteasomes themselves are imported into the nucleus or whether some other factor such as an E3 ligase that ubiquitinates substrates in the nucleus is being transported into the nucleus.
The UPS is required for SING formation
To further explore ubiquitin chain formation at SING sites, live imaging on worms expressing an unconjugatable ubiquitin (GFP::UbAA) was performed (Fig. 4c and d). GFP::UbAA has a dialanine at the C-terminus rather than the diglycine that is required for conjugation onto protein substrates. SINGs were not present during stress in the GFP::UbAA strain indicating the ubiquitin conjugation was required for SING localization. This result along with uba-1 result and the K48 antibody stain (Fig. 1d) indicate that SINGs contain ubiquitin that is conjugated onto substrates and that ubiquitination is required for ubiquitin and proteasome to localize to SINGs.
To evaluate whether proteasome activity is required for the formation of SINGs, proteasome inhibitors (MG132, Bortezomib, and Lactacystin) were used on worms expressing GFP::Ub and RPT-1::mCh. When worms were soaked in salt solution containing proteasome inhibitors, GFP::Ub and RPT-1::mCh fail to localize to SINGs (Fig. 4e and f). The result indicates that proteasome activity is required for ubiquitin and proteasome to localize to SINGs and was unexpected. Since ubiquitin localization to the SINGs precedes proteasomes (Additional file 4: Figure S3), it was anticipated that proteasomal activity would not be required for ubiquitin localization to SINGs. The proteasome inhibition results may indicate that there is a proteolytic event that is required in order to initiate the formation of the SINGs. Another likely explanation is that proteasome inhibition causes an overall decline in the amount of free ubiquitin and thus prevents any new ubiquitination events and SING formation.
Next, we explored the order of ubiquitin and proteasome appearance in the SINGs. To determine this, time-lapse studies of SING formation were conducted by collecting 30 consecutive frames with 1 min intervals on stressed C. elegans gonads from the strain expressing GFP::Ub and RPT-1::mCh. Each frame was assessed for the presence of ubiquitin, proteasome, or both in SINGs. SINGs with either ubiquitin only or both ubiquitin and proteasome were observed (Additional file 4: Figures S3). However, no SINGs were observed that contained only proteasome. Therefore, ubiquitin appears to precede the proteasome in SING formation.
Screens for E3s involved in the SING pathway revealed that knockdown of chn-1 inhibited the formation of SINGs (Fig. 5d and e). chn-1 is a homolog of CHIP which is a co-chaperone for Hsp70 and is known to be involved in the ubiquitination of misfolded proteins . Thus, UBC-18, UBC-20/-22, and CHN-1 are all implicated in the ubiquitination of proteins that are required for or targeted to SINGs.
SINGs are induced by a variety of stressors
In order to test whether ubc-18 was required for SING formation under these stress conditions, we treated worms with ubc-18 (RNAi) and exposed them to the same stress conditions. ubc-18 (RNAi) treated worms did not form SINGs under either stress condition (Fig. 6a and b). Therefore, ubc-18 appears to be a general requirement for the formation of SINGs and is not specific to salt stress.
Heat shock is also a type of cellular stress. In order to examine whether heat shock induces the formation of SINGs, GFP::Ub and RPT-1::mCh worms were moved to 37 °C for 60 min, and then observed under confocal microscopy for the presence of SINGs. As a control, worms expressing HSF-1::GFP were subjected to the same procedure (data not shown). Consistent with a previous report  heat shock bodies were observed in the muscle nuclei of HSF-1::GFP worms (12/20 muscle nuclei showed heat shock bodies). However, in worms expressing GFP::Ub, no SINGs were present in the nuclei of the oocytes after heat shock (Fig. 6c). Therefore, these heat shock conditions do not induce SING formation.
Other studies have identified the transcription factor SKN-1 as an important component of the stress response in C. elegans [39, 40]. We investigated whether skn-1 was required for the formation of SINGs. Antibody staining to detect SINGs was done on wild type and skn-1(zu129) mutant worms soaked in M9 buffer, 500 mM NaCl, or 10 mM H2O2. The nuclei of skn-1 mutants formed SINGs in response to both types of stress (Additional file 5: Figure S4). Our results show that the SINGs seen during osmotic and oxidative stress are working through a stress response pathway that does not require skn-1.
TIAR-2 is a component of SINGs
Other proteins were also tested for their presence in SINGs. Live imaging was conducted on fluorescently labeled nuclear pore proteins (NPPs), tubulin, SUMO, and the PGL-1 protein in the germline of C. elegans. Results showed that some NPPs did react to salt stress by forming perinuclear foci, but these areas did not colocalize with SINGs (Additional file 3: Figure S5A and S5B). Tubulin remained in the cytoplasm and exhibited minor rearrangement during salt stress (Additional file 3: Figure S5D). SUMO (SMO-1) and PGL-1, a P granule component also do not localize to SINGs after salt stress (Additional file 3: Figure S5C and S5D). Therefore, relocalization to SINGs is not a general phenomenon for all proteins in the oocyte. Also, SINGs do not contain high concentration of RNA based on staining with SYTO 14 after stress induction (Additional file 3: Figure S5E).
Embryos containing SINGs exhibit embryonic lethality and impaired cell division
Since ubc-18 loss of function reduces the formation of SINGs, we went on to test if ubc-18 embryos showed any resistance to stress conditions. Wild type and ubc-18 mutant embryos were placed under unstressed or salt stress conditions, and then scored for hatching after 48 h. 97% of the wild type unstressed embryos hatched and 61% of the wild type salt stressed embryos hatched (Fig. 10c). This result was expected because approximately half of stressed embryos exhibit SING formation. In unstressed ubc-18 mutants 86% of the embryos hatched; however, 89% of the stressed ubc-18 mutant embryos were able to hatch (Fig. 10c). These data suggest that ubc-18 embryos have a lower hatching rate than wild type, but that they are more resistant to salt stress. Prior studies with ubc-18 had shown a reduced brood size, but the actual hatching rate had not been reported . No association between ubc-18 and an elevated resistance to stress has been reported previously.
Similarly to ubc-18, RNAi knockdown of ubc-20 plus ubc-22 reduces SING formation (Fig. 5b and c). We found that RNAi of ubc-20/ubc-22 also increased embryonic resistance to salt stress (Fig. 10c). The correlation between the lack of SINGs and the increased embryonic survival in mutant or RNAi embryos supports the hypothesis that SINGs have a negative impact on cell survival or development. Interestingly, ubc-18 and ubc-20/22 embryos do not survive better than wild type under oxidative stress (Fig. 10c). This could be because oxidative stress has additional detrimental effects other than the induction of SINGs.
Movie S1. GFP::Ub and mCh::H2B embryos soaked in M9 buffer for 60 min. Unstressed embryos undergo complete cell division within 5 min. (AVI 2889 kb)
Movie S2. GFP::Ub and mCh::H2B embryos soaked in 500 mM NaCl for 60 min. Salt stressed embryos do not complete cell division. (AVI 4369 kb)
Exposure to stress is also known to affect the lifespan of an organism . The oocyte SINGs persist for up to 72 h post stress exposure (data not shown) and thus it is presumed that they may have long term effects on the organism. We tested whether salt stress had an effect on the lifespan of C. elegans. Life span studies were carried out on adult wild type and ubc-18 (RNAi) worms that had been exposed to 500 mM NaCl for 60 min as L4 larvae. Surprisingly, this treatment had no observable effect on the lifespan (Additional file 8: Figure S6).
Our results reveal a novel pathway that responds to collapse of protein quality in the germline and other tissues of C. elegans. Salt stress, oxidative stress, and starvation lead to the redistribution of ubiquitin, proteasome, and the TIAR-2 protein into structures we refer to as Stress Induced Nuclear Granules.
Environmental stress has previously been shown to induce various nuclear bodies. One example are the heat shock bodies containing HSF1. Insulator bodies form in Drosophila cells in response to osmotic stress however they localize to chromatin . The C. elegans SINGs are similar to clastosomes and PML bodies in that they contain ubiquitin and proteasome and are induced by stress [15, 19, 20, 48]. However, PML bodies have a high concentration of SUMO which is not present in SINGs. Clastosomes which were reported to form in mammalian cultured cells appear to be the most similar to the SINGs since they also require proteasome activity to form .
An interesting aspect of our findings is that proteasome and ubiquitin foci form inside the nucleus rather than in the cytoplasm. This result adds to a growing number of reports that suggest that protein degradation is taking place in the nucleus (reviewed in ). In yeast, it has been reported that degradation of some short-lived proteins occurs by first importing them into the nucleus where they are degraded by nuclear proteasomes . The presence of the proteasome and K48-linked ubiquitin chains is consistent with the idea that SINGs are sites of localized protein degradation. If protein degradation is occurring at the SINGs, it will be of great interest to determine whether there is a specific set of proteins that are degraded or whether SINGs are general sites of degradation for any K48 ubiquitinated protein. Follow up studies are underway in an attempt to discriminate between these two possibilities.
SING formation in embryos results in a cell cycle arrest. Thus, SINGs may interfere with cell division. Embryos that lack ubc-18 do not exhibit SINGs when stressed and they can proceed through the cell cycle normally. This may point to a direct role of the SINGs in compromising cell division. Further studies are needed in order to determine which aspects of cell division are affected when SINGs are present. This effect of stress on the cell cycle during embryogenesis may be a contributing factor to compromised reproductive capability in organisms exposed to environmental stress .
Our studies have identified a novel nuclear structure that we refer to as SINGs. Salt stress, osmotic stress, and starvation induce SING formation. SINGs share some features with previously identified nuclear bodies such as clastosomes and PML bodies. The appearance of SINGs correlates with conditions of protein misfolding. Thus, SINGs likely participate in a nuclear protein quality control pathway that is initiated when misfolded proteins accumulate. Ubiquitination and nuclear import are required for the formation of SINGs suggesting that misfolded proteins from the cytoplasm may be transported into the nucleus before localizing to SINGs. Future research will explore whether ubiquitination occurs prior to nuclear import or after.
C. elegans Strains and Maintenance
C. elegans strains used in the study
ruls57 [pie-1::GFP::tubulin + unc-119(+)]
sEx884 [rCesC12C8.1::GFP + pCeh361]
jyEx128 [vha-6p::GFP::Ub cb-unc-119(+)]; ttTi5605 II; unc-119(ed9)
skn-1(zu129) IV/nT1 [unc-?(n754) let-?] (IV; V).
fgpIs20 [(pFGP79) pie-1p::mCherry::smo-1(GG) + unc-119(+)]; ruIs32 [pie-1p::GFP::H2B + unc-119(+)] III
axEx73 [pie-1p::pie-1::GFP + rol-6(su1006) + N2 genomic DNA]
axIs1486 [pCG51; LAP::Y46G5A.13(tia-1.2) +unc-119(+)]
axls1595[pie-1p::GFP::npp-9(orf)::npp-9 3'UTR + unc-119(+)]
axIs1735[pie-1p::LAP tag::npp-10 (full length) + unc-119 (+)]
ltIs37 [(pAA64) pie-1p::mCherry::his-58 + unc-119(+)] IV; axIs1522 [pie-1p::GFP::pgl-1::pgl-1 3'UTR + unc-119(+)]
axIs1844[GFP:: npp-7 + unc-119(+)]
rcIs31 [pie-1p::GFP::Ub + unc-119(+)]; ltIs37 [pie-1p::mCherry::his-58]
rcIs31 [pie-1p::GFP::Ub + unc-119(+)]; rcSi1 [mex-5p::rpt-1::mCherry + unc-119] II; unc-119 (ed3)
avIs116 [pie-1p∷GFP∷UbAA + unc-119(+)]; ltIs37 [pie-1p::mCherry::his-58]
jjls1089 [npp-1::GFP + unc-119(+)]
drSi13 [hsf-1p::hsf-1::GFP::unc-54' 3'UTR + Cbr-unc-119(+)] II
qaIs3546 [unc-119(+) + pie-1::GFP::npp-8]; unc-119(ed3)
Fluorescence microscopy and time-lapse imaging
All fluorescent and time-lapse images were acquired using a ZEISS AxioObserver with a LSM 700 confocal module and a 63X/1.4 Plan-Apochromat oil DIC M27 objective. Images were analyzed with ZEN 2009 software. In live imaging and antibody staining experiments, the 488 nm laser was used for imaging both GFP and FITC, and the 555 nm laser was used for both mCherry and TRITC. DAPI was imaged using the 405 nm laser. Image settings on the microscope were kept constant for each set of experiments. Dimensions of images acquired were 512 × 512 pixels at a depth of 8 bits. Images were adjusted to be brighter for visual presentation in Photoshop using the brightness function.
A time-lapse series (30 frames with 1 min intervals) of the worm strain LN154 (GFP::Ub and RPT-1:: mCh) was used to investigate the timing of colocalization events. All time-lapse experiments were imaged on the confocal at room temperature. A time-lapse series (30 frames with 0.5 min intervals) of LN130 (GFP::Ub and mCh::H2B) was used for cell division experiments.
Primary antibodies used were rabbit anti-19S proteasome (sc-98797 from Santa Cruz), mouse monoclonal anti-20S proteasome (MCP20 from Enzo Lifesciences), mouse monoclonal anti-ubiquitin (P4D1 from Santa Cruz), rabbit anti-K48 ubiquitin (Apu2 from Millipore), and rabbit anti-K63 ubiquitin (Apu3 from Millipore). Secondary antibodies used were goat anti-mouse FITC (Abcam) and goat anti-rabbit TRITC (Jackson ImmunoResearch Labratories).
Immunostaining of C. elegans
Day 1 adult worms were placed in Egg Buffer and cut open on poly-L-lysine-coated slides to release the gonadal arms and embryos. Slides were placed in liquid nitrogen and fixed with 100% methanol at -20 °C for 20 min, followed by washing three times with PBST for 5 min. Slides were then blocked for 1 h with 30% normal goat serum in PBST at 23 °C, and incubated overnight with primary antibody (1:200) at 4 °C. Slides were washed with PBST, and incubated with secondary antibody (1:200) at 23 °C for 1.5 h. Vectashield plus DAPI (Vector Labs, Burlingame, CA) was used for mounting each slide prior to imaging with confocal microcopy.
Photobleaching experiments were performed using a Zeiss LSM700 laser scanning confocal system. Two pre-bleach images were acquired. Bleaching was done via 40 iterations of bleaching with 100% on the 488 nm laser. Fluorescence was bleached to ≤ 20% of initial intensity. Images were acquired every 1 s with the 488 nm laser set to 5% power. Each data point was normalized for background and photobleaching using the equation: [(ROIb – ROIbg)/(ROInb –ROIbg)]/[(pbROIb –pbROIbg)/(pbROInb –pbROIbg)].
RNAi by feeding
RNAi was achieved by feeding worms bacteria that express dsRNA for each gene. RNAi clones for the UBC genes were obtained from the Ahringer library or the Vidal ORF library. The ubc-18 RNAi is an ORF clone. Controls for the RNAi experiments used the L4440 plasmid (vector) without any gene insert in the HT115 bacterial strain. RNAi clones were streaked from glycerol stocks onto tryptic soy agar media with ampicillin (100 μg/mL) and tetracycline (10 μg/mL). Bacterial overnights were grown in tryptic soy broth with ampicillin and tetracycline. NGM plates containing ampicillin and 0.2% lactose were seeded with the bacterial overnights. L4s were transferred to above plates and grown for 22 h under ideal conditions for each respective strain.
Osmotic stress, oxidative stress, and starvation
C. elegans were grown on OP50 containing NGM at their appropriate temperatures until they reached 1 day adults. Worms from this population were then moved to control and stressed conditions. Conditions involving liquid (control, salt, osmotic, and oxidative stress) were performed by soaking day 1 adults in a watch glass containing 1 mL of solution at room temperature. Solution concentrations for each stress are as follows: 500 mM NaCl (salt stress), 815 mM Sucrose (osmotic stress), 10 mM H2O2 (oxidative stress). Control (unstressed) individuals were soaked M9 buffer for 60 min. The worm strain JH2099 (GFP:: TIAR-2) was salt stressed for 120 min. The GFP::Ub intestinal strain (ERT261) was stressed at 1 M NaCl for 60 min prior to imaging. Oxidative stress was induced by soaking worms in a 10 mM H2O2 solution for 60 min.
For starvation, synchronized L4 worms were moved to NGM plates without OP50 bacteria or peptone for a period of 48 h prior to being examined. To induce heat shock, day 1 adults grown on seeded NGM at 25 °C were shifted to a 37 °C for 60 min prior to imaging.
After treatment, worms were mounted on a 3% agar pad and examined with a laser scanning confocal microscope (Zeiss LSM700).
Treatment with proteasome inhibitors
Proteasome inhibitor solutions were prepared the same day as the experiment by adding proteasome inhibitor stock solution to either M9 buffer or 500 mM NaCl. Proteasome inhibitors and concentrations tested include 20 μM MG132, 10 μM Lactacystin, and 20 μM Bortezomib. In vivo proteasome inhibitor experiments were performed by soaking day 1 adult worms in 20 μM MG132 for 60 min at room temperature. Worms were then transferred over to a solution containing both MG132 and 500 mM NaCl. Control worms were soaked in a 500 mM NaCl solution with no proteasome inhibitor for 60 min before imaging.
Detection of DNA and RNA in C. elegans
The cell permeable SYTO14 green fluorescent nucleic acid stain (Life Technologies) was used to observe total RNA in oocytes. SYTO14-stained RNA excites at 521 nm and emits at 547 nm. Fresh SYTO14 solution was either made in M9 buffer (unstressed) or 500 mM NaCl (stressed). Worms were dissected in 5 μm SYTO14 solution for 15 min and then imaged by confocal microscopy.
DAPI (NucBlue Fixed Cell ReadyProbes, Life Technologies) was used in the SYTO14 experiment to visualize DNA. One drop of NucBlue was added directly to the top of the slide and incubated for 15 min prior to visualization.
Prior heat shock exposure
Day 1 adult worms grown at 25 °C were shifted to 34 °C for 60 min prior to 500 mM NaCl exposure and then imaged with confocal microscopy. Control individuals stayed at the initial growing temperature before being exposed to salt stress.
Embryonic lethality and cell division analysis
Wild type and ubc-18 (RNAi) embryos were cut out of day 1 adult worms and soaked in either M9 buffer (control), 500 mM NaCl, or 10 mM H2O2 for 60 min. After which, embryos were then pipetted onto unseeded NGM plates and scored for hatching at 48 h.
Cell division analysis was conducted by taking time-lapse movies on both control and salt stressed embryos expressing GFP::Ub and mCh::H2B. Time-lapse movies were described above in the Fluorescence Microscopy and Time-lapse Imaging section.
L1 worms were moved to either vector or RNAi seeded plates. Once worms reached the L4 stage they were subjected to 500 mM NaCl stress for 60 min and then moved to new RNAi seeded NGM plates. 80 worms were used for each condition.
Sample sizes and number of experiments performed are noted in each figure legend. In general, a minimum of 1020 oocytes were observed over three biological repeats unless otherwise noted in the figure legend. Two sample z-tests were performed using VasarStats on the 20S and TIAR-2 antibody stains as well as on live-cell images which include RNAi and stress experiments (Figs. 1e, 2b, 3b, d, 4b and 5c, e). The Fisher’s exact test was performed on data that was less than five (Figs. 2c, 4d, f, 6b and 10b, c, and Additional file 5: Figure S4B). Error bars presented in this paper represent a 95% confidence interval and were derived using the modified Wald method on GraphPad. Data was considered to be statistically significant if p < 0.05. A log-rank test was used to determine significant differences in survivorship curves in the lifespan experiment.
C-terminus of Hsc70 Interacting Protein
Fluorescence recovery after photobleaching
Green fluorescent protein
Heat shock factor 1
Nuclear factor kappa-light-chain-enhancer of activated B cells
Nuclear pore protein
Stress induced nuclear granule
Ubiquitin proteasome system
We would like to thank Bill Tansey, David Nelson, and Jennifer Schisa for the helpful discussions and comments on the manuscript. We thank Hilary Kemp, Emily Troemel, and Harold Smith for sharing strains. We are grateful to members of the Boyd laboratory as well as Matt Elrod-Erickson, David Nelson, and Jason Jessen for input on the project. We thank Jacob Sanders for initial observations regarding muscle SINGs and Sofia Lima for assistance with the longevity assays. Some nematode and bacterial strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
K.S. was funded via a Graduate Research award and M.S. was funded via a URECA award from the office of Vice President of Research at Middle Tennessee State University. Some of the funding for this study came from a grant to L. B. from the National Institute of Child Health and Human Development at NIH (R15 HD083882).
Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files]. All strains used in this study are available by request from Lynn Boyd.
KS carried out most of the experiments and wrote the manuscript. MR conducted the RNAi screen for ubiquitin conjugating enzymes. LB helped with experimental design and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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