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Stress Induced Nuclear Granules Form in Response to Accumulation of Misfolded Proteins in Caenorhabditis Elegans

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Stress Induced Nuclear Granules Form in Response to Accumulation of Misfolded Proteins in Caenorhabditis Elegans

Katherine M Sampuda et al. BMC Cell Biol.

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Nuclear body; Oxidative stress; Proteasome; Salt stress; Starvation; Ubiquitin.

Figures

Fig. 1
Fig. 1
Salt stress induces redistribution of ubiquitin and proteasome into nuclear stress bodies. a Reproductive tissue of C. elegans. The C. elegans gonad is a U-shaped organ with mitotically dividing cells at the distal tip. The distal region of the gonad is syncytial and oocytes become increasingly cellularized and larger as they progress through the proximal region towards the spermatheca. b Live imaging of GFP::Ub (green) and RPT-1::mCh (red) in proximal oocytes. During unstressed (M9 buffer) conditions ubiquitin and 19S proteasome were present in both nucleus and cytoplasm, but appeared to be more concentrated in the nucleus. The merged image shows ubiquitin, proteasome and DAPI channels. In stressed (500 mM NaCl) conditions ubiquitin and RPT-1 concentrate in stress bodies in the nucleus. A single oocyte is shown in each row. SINGs were found in salt stress (66/80 oocytes), but were absent in unstressed conditions (0/80) oocytes). Data were collected from 2 independent experiments (n = 20 worms). Scale bar indicates 10 μm. c Antibody staining of ubiquitin and 19S proteasome in the distal gonad. Gonads of unstressed and stressed young adult C. elegans were dissected out and stained with an ubiquitin antibody (green) and a 19S proteasome antibody (red). The merged image shows the ubiquitin, proteasome, and DAPI channels. Both ubiquitin and proteasome where found to be diffuse within the nucleus in unstressed conditions (0/600 oocytes with SINGs), but localized into SINGs during salt stress (468/600 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm. d K63 and K48 polyubiquitin antibody staining in the distal gonad. During exposure to 500 mM NaCl K48 chains localized into SINGs (552/600 oocytes), whereas, K63 chains show no localization to SINGs in response to stress (0/600 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm. e 20S proteasome subunit antibody staining. An antibody to the alpha 1 subunit of the 20S proteasome (green) was used to stain stressed (500 mM NaCl) and unstressed (M9) gonads. A region of the distal gonad is shown for each. The merged image shows the ubiquitin, proteasome, and DAPI channels. In unstressed conditions, SINGs containing 20S proteasome do not form (10/200 oocytes). Under stressed conditions the 20S proteasome subunit colocalizes with K48 ubiquitin chains (red) in SINGs (153/200 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm
Fig. 2
Fig. 2
SING formation correlates with reduced protein quality control. a Worms expressing GFP::Ub and RPT-1::mCh were incubated for 60 min at either 25 °C or 34 °C prior to soaking in 500 mM NaCl for 60 min. The merged image is shown. Areas that appear yellow have both GFP and mCherry. The brief heat shock reduced the occurrence of SING. Cytoplasmic granules also appear as a result of the heat shock. When hsf-1 was knocked down via RNAi, SING formation was normal even after heat shock. Scale bar indicates 10 μm. b The percentage of oocytes forming SINGs as described in A. A total of 1020 oocytes were observed for each condition from 3 independent experiments (n = 30 worms). Statistically relevant differences ***p < 0.0002, two-tailed z test. c Percentage of oocytes forming SINGs in day 1 and day 4 adults after exposure to 500 mM NaCl for the times indicated. A total of 510 oocytes were observed for each time point from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a Fisher’s Exact test: **p < 0.01 and ****p < 0.0001
Fig. 3
Fig. 3
SING formation required the nuclear import pathway. a RNAi of ima-1, ran-1, and arx-5 reduces SING formation. Worms expressing GFP::Ub in the germline were soaked in 500 mM NaCl for 60 min. Proximal oocytes are shown. Control worms treated with vector RNAi formed SINGS normally. Scale bar indicates 10 μm. b The percentage of oocytes forming SINGs after treatment as described in A. Oocytes were scored for both GFP::Ub and RPT-1::mCh at SINGs. A total of 1020 oocytes were analyzed for each condition from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: **p < 0.01 and ***p < 0.001. c RNAi of smo-1 reduces SING formation in a worm strain expressing GFP::Ub in the germline. Proximal oocytes are shown. Scale bar indicates 10 μm. d The percentage of oocytes forming SINGs after treatment as described in C. A total of 1020 oocytes were analyzed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001
Fig. 4
Fig. 4
SING formation requires ubiquitination and proteasome activity. a Worms expressing RPT-1::mCh were subjected to uba-1 RNAi for 24 h prior to soaking in 500 mM NaCl for 60 min. RPT-1::mCh does not localize to SINGs when uba-1 is knocked down. Scale bar indicates 10 μm. b The percentage of oocytes forming SINGs after uba-1 RNAi treatment as described in A. A total of 1020 oocytes were analyzed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001. c Unconjugated ubiquitin does not localize to SINGs. A worm strain expressing GFP::Ub (green) and mCh::H2B (red) shows localization of GFP::Ub to SINGs after soaking in 500 mM NaCl for 60 min. Whereas, GFP::UbAA does not localize to SINGs. GFP::UbAA lacks a C-terminal diglycine that is required for ubiquitin conjugation. Scale bar indicates 10 μm. d The percentage of oocytes forming SINGs in control and salt stress treated worms as described in C. GFP::Ub control and GFP::UbAA control or salt stressed worms showed no signs of SING formation, whereas, GFP::Ub salt stressed did exhibit SING formation after stress exposure. A total of 510 oocytes were analyzed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. e Young adult C. elegans expressing GFP::Ub and RPT-1::mCh were either soaked in M9 buffer or 500 mM NaCl for 60 min. Worms soaked in M9 did not form SINGs. Under salt stress conditions, worms without the proteasome inhibitor MG132 formed SINGs, whereas, in the presence of the proteasome inhibitor MG132, SING formation was reduced. Scale bar indicates 10 μm. f Proteasome inhibitors MG132, bortezomib, and lactacystin inhibit the formation of SINGs in response to salt stress. The percentage of oocytes forming SINGs after proteasome inhibitor treatment. A minimum of 240 oocytes were collected from 3 independent experiments (n = 24 worms). Statistical significance was calculated between salt stress and salt stress with proteasome inhibitor by a Fisher’s Exact test: ****p < 0.0001
Fig. 5
Fig. 5
Ubiquitination pathway components participating in SING formation. a SINGs in uba-1 and ubc-18 mutants. Antibodies to ubiquitin and the 19S proteasome were used to stain wild type worms, a temperature sensitive mutant of uba-1, and a loss of function mutation in ubc-18. The merged images show the ubiquitin, proteasome, and DAPI channels. uba-1 worms grown at 16 °C showed the presence of SINGs (551/600 oocytes) in response to salt stress (500 mM NaCl for 60 min). However, uba-1 worms grown at 25 °C showed a reduction in SING formation (12/600 oocytes) when exposed to salt stress. SINGs were not induced in ubc-18 mutants during salt stress (0/600 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm. b Worms expressing RPT-1::mCh were subjected to either control RNAi (vector) or RNAi of the ubc-20 plus ubc-22 E2 enzymes. This combined RNAi reduced the appearance of SINGs. Scale bar indicates 10 μm. c Quantification of the percentage of oocytes forming SINGs after control RNAi and chn-1 RNAi treatment as described in B. A total of 1020 oocytes were observed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001. d Worms expressing RPT-1::mCh were subjected to either control RNAi (vector) or RNAi of the chn-1 E3 enzyme. Knockdown of chn-1 reduced the appearance of SINGs. Scale bar indicates 10 μm. e Quantification of the percentage of oocytes forming SINGs after treatment as described in D. A total of 1020 oocytes were observed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a two-tailed z test: ***p < 0.001
Fig. 6
Fig. 6
Oxidative stress and Starvation induce SING formation. a Oxidative stress and starvation in gonads. Worms expressing GFP::Ub and mCh::H2B were grown on bacteria with control or ubc-18 RNAi and subjected to 10 mM H2O2 for 30 min or starvation for 48 h. Distal gonads are shown. Oxidative stress and starvation induced SING formation in vector treated worms. SINGs did not form in ubc-18 worms soaked in H2O2 or starved. Scale bar indicates 10 μm. b Quantification of the percentage of oocytes forming SINGs after treatment as described in A. A total of 250 oocytes were collected from 3 independent experiments (n = 15 worms). Statistical significance was calculated by a two-tailed z test: ****p < 0.0001. c Heat shock does not induce SINGs. Worms expressing GFP::Ub and mCh::H2B were subjected to heat shock by placing them at 37 °C for one hour. In control worms incubated at 25 °C for 1 h, 0/600 oocytes showed SINGs. In the heat shock group, 0/600 oocytes showed SINGs. Data were collected from 2 independent experiments (n = 20 worms). Scale bar indicates 10 μm
Fig. 7
Fig. 7
SINGs form in intestinal cells. a A strain expressing GFP::Ub in intestinal cells was soaked in 1 M NaCl for 1 h prior to imaging with a laser scanning confocal microscope. The salt stress induced SING formation in intestinal nuclei. Worms that had been grown under ubc-18 (RNAi) did not form SINGs upon salt stress. b Quantification of the intestinal nuclear response to salt stress. Three trials were done for each condition including 10 worms per trial and 10 intestinal nuclei per worm (n = 30 worms). Statistical significance was calculated by a Fisher’s Exact test: ***p < 0.001
Fig. 8
Fig. 8
SING formation does not require intertissue signaling. a Young adult worms expressing RPT-1::mCh were cut open in either M9 buffer or 500 mM NaCl to extrude the gonads from the body. Carcasses containing the other tissues were quickly removed. Gonads were imaged after 60 min. All gonads soaked in 500 mM NaCl formed SINGs (134/300 nuclei; 10 gonads). No SINGs were formed in the gonads soaked in M9 (0/300 nuclei; 10 gonads). Data were collected from 3 independent experiments (n = 10 worms). b Intestinal cells also form SINGs when separated from the body. Intestinal nuclei are large and polyploid and have an elongated shape. Worms expressing GFP::Ub in the intestine were cut open in either M9 buffer or 1 M NaCl to extrude the intestine from the body. Carcasses containing the other tissues were quickly removed. Intestines were imaged after 60 min. No intestines soaked in M9 formed SINGs (0/40 nuclei; 8 intestines). For intestines soaked in 1 M NaCl, all intestines showed SINGs (14/30 nuclei; 8 intestines). Data were collected from 2 independent experiments (n = 8 worms). Scale bar indicates 10 μm
Fig. 9
Fig. 9
SINGs contain the TIAR-2 protein. a Worms expressing GFP::TIAR-2 were soaked in M9 or 500 mM NaCl for 60 min and then stained with an antibody to the 19S proteasome. The merged image shows the TIAR-2, proteasome, and DAPI channels. In M9 treated worms, few oocytes showed nuclei with TIAR-2 and proteasome colocalization (38/600). Whereas, in salt stressed worms, the number of TIAR-2 and proteasome colocalization events increased (212/600 oocytes). Data were collected from 3 independent experiments (n = 30 worms). Scale bar indicates 10 μm. b Day 4 adults expressing TIAR-2::GFP were soaked in a watch glass containing 500 mM NaCl with or without MG132 for 60 min. Worms that were not treated with MG132 showed TIAR-2 in SINGs, whereas MG132 treated worms showed fewer TIAR-2 containing SINGs. c Quantification of the percentage of TIAR-2 in SINGs as described in B. A total of 90 proximal oocytes were observed from 3 independent experiments (n = 30 worms). Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001
Fig. 10
Fig. 10
SINGs correlate with embryonic lethality and impaired cell division. a Stress induces SING formation in embryos. Embryos were obtained from worms expressing GFP::Ub and mCh::H2B and placed in 500 mM NaCl for 1 h or 10 mM H202 for 30 min. Embryos formed SINGs under both types of stress conditions. Scale bar indicates 10 μm. b Hatching rate of embryos that contain SINGs. Embryos were dissected from gravid adults that were soaked in M9 (unstressed) or 500 mM NaCl (stressed) for 1 h. Stressed embryos were screened under the microscope for the presence of SINGs. Embryos with SINGs showed a 0% hatch rate versus a 100% hatch rate for unstressed embryos or stressed embryos without SINGs. A total of 40 embryos were analyzed for each condition from 3 independent experiments. Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. c Hatching rates of wild type and ubc-18 mutant embryos in unstressed and stressed conditions. Embryos were subjected to two different stresses: salt stress (500 mM NaCl for 60 min) and oxidative stress (10 mM H2O2 for 30 min). Both types of stress reduce the hatching rate of wild type embryos. However, in ubc-18 or ubc-20/ubc-22 (RNAi) embryos which have reduced SING formation, salt stress does not reduce the hatching rate. Oxidative stress does reduce hatching rate in ubc-18 and ubc-20/ubc-22 (RNAi) embryos. In ubc-18 and ubc-20/ubc-22 (RNAi) embryos, the numbers for the M9 and the 500 mM NaCl hatching rates showed no statistical difference. Experiments were repeated three times with 25 embryos per trial for a total of 75 embryos per condition. Statistical significance was calculated by a Fisher’s Exact test: ****p < 0.0001. d Time-lapse imaging on unstressed and stressed embryos expressing GFP::Ub and mCh::H2B. Ten embryos from the unstressed and ten embryos from the salt stressed groups were observed on the confocal microscope with time-lapse imaging for 15 min each. Many cells in each unstressed embryo were able to complete cell division within 5 min. Cells in salt stressed embryos showed no progression through the cell division cycle. The asterisk in the M9 embryo shows a cell that proceeds through prophase, metaphase, and anaphase of the cell cycle. The asterisk in the 500 mM NaCl embryo shows a nucleus that has SINGs and remains in prophase. The arrow shows a nucleus with SINGs that remains in metaphase throughout the 15 min observation period. A total of 10 embryos were collected from 3 independent experiments. Scale bar indicates 10 μm
Fig. 11
Fig. 11
Model for SING formation. Unstressed cells experience a healthy protein homeostasis. When cells are exposed to stressors that induce protein misfolding, the ubiquitin system adds K48-linked ubiquitin chains to the misfolded proteins. Ubiquitinated proteins and proteasomes localize to SINGs in the nucleus

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