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