Figure 2. Age-dependent protein aggregation can occur in different tissues. (A-B) Ppie-1::gfp::
rho-1-expressing animal, Day 1 (A) and Day 9 (B-C). In our proteomic analysis, we identified RHO-1 as a protein prone to aggregate with age both in the reproductive and somatic tissues. GFP::RHO-1, expressed in the germline, was localized to germline stem cell (open arrowhead) and oocyte (full arrowhead) membranes in young animals. With age, GFP::RHO-1 also accumulated in sclerotic oocytes in the uterus (full arrowhead). Scale bar: 20 mm. (D) FRAP-immobile GFP::RHO-1 in aged oocytes. GFP::RHO-1 is pseudocolored in magenta. Laser setting: 25% in 0.85
mm2 (white open box). Scale bar: 2 mm. (E) Quantification of relative fluorescence intensity (RFI) during recovery. We found no recovery of GFP::RHO-1 in sclerotic oocytes in aged animals (Day 12, N animals = 5, N puncta evaluated = 5) but rapid fluorescence recovery for GFP::RHO-1 localized to the germ-line cell membrane in young animals (
t1/2 = 35 s, Day 1, N animals = 5, N puncta evaluated = 5) and in aged animals (
t1/2 = 17 s, Day 12, N animals = 3, N puncta evaluated = 4). (F-K) Formation of KIN- 19::tagRFP puncta in the anterior pharyngeal bulb (metacorpus) with age. Pkin-19::
kin-19::tagrfp animals (F and G);
daf-2(
e1370); Pkin-19::
kin-19::tagrfp animals (H); control Pkin-19::tagrfp animals (I and J). (F-J) 10 ms exposure, 1006. (K) Schematic of C. elegans' pharynx, boxed area is shown in F-J. (L) FRAP-immobile KIN-19::tagRFP puncta in the anterior pharyngeal bulb of 15-day-old Pkin-19::
kin-19::tagrfp animal. Laser settings: 40% in 0.46
mm2 (white open box). Scale bar: 1 mm. (M) We found no fluorescence recovery of pharyngeal or BDU neuronal KIN-19::tagRFP in aged animals (Day 12, N animals = 5, N puncta evaluated = 5 and Day 12 and 14, N animals = 5, N puncta evaluated = 5, respectively). KIN-19::tagRFP was able to diffuse back at a slow rate in young animals both in the pharynx and neurons (
t1/2 = 107 s, Day 1, N animals = 4, N puncta evaluated = 5 and
t1/2 = 151 s, Day 3, N animals = 5, N puncta evaluated = 5, respectively). The few puncta formed by tagRFP alone contained highly mobile protein in aged animals (Day 12; N animals = 5, N puncta evaluated = 5).