Figure 3. The age-associated protein deposit can be distinguished from Q-bodies, JUNQ/INQ, and IPOD.
(A) Cells were heat shocked (42°C for 30 min) to induce the formation of Q-bodies (stress induced Hsp104-labeled aggregates) and the kinetics of Q-bodies dissolution was followed with time-lapse microscopy of Hsp104-GFP. (B) Cells were imaged after heat shock (42°C for 30 min). Arrowhead in 3D-projected images depicts co-localization of Hsp104 with age-associated deposit-resident (Ssa1, Hsp42) and non-resident (Hsp82, Btn2) markers in Q-bodies (compare with Figure 1C). (C) The expression of JUNQ/INQ marker VHL-GFP was induced at the start of the imaging to follow its recognition by Hsp104-mCherry. (D) Quantification of VHL-GFP foci recognized by Hsp104-GFP within 30 min of their appearance (N = 29 cells). (E) 3D-projected images of RPN4 deleted cells expressing Hsp82-GFP and Hsp104-mCherry. Hsp82-GFP, which localizes to Q-bodies (Figure 3A) but not to age-associated protein deposits (Figure 1C), co-localizes with one of the two Hsp104-mCherry foci in aged cell (see fluorescence intensity line-scan over the two foci). (F) 3D-projected image of Btn2-GFP and Hsp104-mCherry expressing cell. Btn2 is typically very low abundant and does not accumulate (98.6% of cases, N = 72) to the age-associated deposit (white arrowhead). (G) Cell with a Btn2 focus, which typically (96.4% of cases, N = 30) did not display accumulation of Hsp104-mCherry). (H) Rnq1-overexpression was induced at the onset of imaging. The panel shows the z-sections displaying the age-associated deposit (white arrowhead in ‘merge’). (I) Quantification of newly formed Rnq1-GFP foci that accumulate Hsp104-mCherry within 30 min of their appearance (N = 53 cells). (J) Representative image of [PIN+, PSI+] cell harboring GFP-tagged endogenous Rnq1 and mCherry-tagged Hsp104. Arrowhead indicates the age-associated protein deposit. (K) Example of a [PIN+, PSI+] cell that displays an Rnq1-aggregate. Arrowhead indicates Hsp104-labeled foci, of which only one has accumulated Rnq1. Scale bars: A-B, E, J-K 5 μm, C, G 2 μm. Graphs display mean ± SEM.