298 | Nature | Vol 578 | 13 February 2020
Article
in a proteasome activity-dependent manner after 4 h (Extended Data
Fig. 4c, d). Northern blot analysis revealed that hypertonic stress caused
a reduction in the Pol I-transcribed pre-rRNA and its processed forms^15
(Extended Data Fig. 4e). In addition, stably expressed RPs in cells were
constitutively ubiquitylated, and notably, ubiquitylation levels were
further increased by sucrose treatment (Fig. 2b). Thus, hyperosmotic
stress induces acute nucleolar stress, thereby causing failure of pre-
ribosome assembly.
To determine whether RPs localize to proteasome foci, we performed
immunofluorescence staining, and found that several RPs (RPL7A,
RPL15, RPL29 and RPS2) modestly colocalized with proteasome foci
(Fig. 2c, Extended Data Fig. 5a). In time-lapse imaging of the PSMB2–
FusionRed cells stably expressing RPL29–eGFP, we observed a rapid
emergence of RPL29 condensates and their degradation at proteasome
foci (Fig. 2d, Extended Data Fig. 5b, Supplementary Video 2). RPL29
structures were also observed in the presence of MLN-7243, but these
were smaller, and their clearance was markedly delayed (Fig. 2d, Sup-
plementary Video 3). Treatment with the Pol I inhibitor CX-5461 or
transient overexpression of RPs further increased the size of protea-
some foci, suggesting that the condensates formed from unassembled
orphan RPs produced in the nucleoplasm (Extended Data Fig. 5c, d).Proteasome foci are liquid droplets
Time-lapse imaging of the PSMB2–FusionRed cells stably expressing
eGFP–ubiquitin revealed that proteasome foci have liquid droplet-like
properties: small foci that contained proteasomes and ubiquitin sus-
pended in the nucleoplasm fused into larger foci, and their circularity
was 0.998 (Fig. 3a, Supplementary Video 4). The number of preformed
foci decreased upon addition of 1,6-hexanediol, an aliphatic alcohol
that destabilizes liquid droplets, but not by ammonium acetate, which
disrupts RNA gelation^20 ,^21 (Fig. 3b). Fluorescence recovery after pho-
tobleaching (FRAP) experiments revealed that approximately 90% ofRPL290.42RPL15PSMB20.37 Distance(μm) Distance(μm)RPL15
DAPI
Normalizedintensity^0048120.51.0PSMB2–eGFPPSMB2RPL29
DAPI
Normalizedintensity^0048120.51.0a0 min 30 min0.2 M sucrosePSMB2–eGFPCytosoNul c CytosoNulcb98-188-62-
49-
38-–+None
0.2 M sucrose: –+L29
–+L15
–+S262-
49-IP: Flag (SDS denatured)PSMB2–FusionRedRP–eGFP3FBlot:
ubiquitinBlot:
c Flagd
PSMB2–FusionRed RPL29–eGFP
0 min51 min min 10 min 20 min 30 min 40 min 50 min 60 minControl0.2 M sucroseMLN-7243PSMB2RPL29RPL29PSMB2PSMB2RPL29RPL29PSMB2(kDa)Fig. 2 | Ribosomal proteins are degraded in proteasome foci. a, Changes in
the ultrastructural appearance of type 2 granule matrix in the nucleolus and
nucleoplasm, as determined by transmission electron microscopy. Dense
fibrillar compartment structures (arrow) in the nucleolus disappeared and very
dense granules (arrowhead) in the nucleoplasm were observed after
stimulation of PSMB2–eGFP cells with 0.2 M sucrose. Nuc, nucleus. Scale bars,
5 μm. Representative images from two independent experiments. b, PSMB2–
FusionRed cells stably expressing eGFP–3×Flag-fused RPL29 (L29), RPL15 (L15)
or RPS2 (S2) were stimulated with or without 0.2 M sucrose for 30 min,
immunoprecipitated (IP) with Flag antibody and immunoblotted as indicated.
Representative result from three independent experiments. For gel source
data, see Supplementary Fig. 1. c, Colocalization of proteasome foci and
endogenous ribosomal proteins. PSMB2–eGFP cells were stimulated with 0.2 M
sucrose for 30 min, and endogenous ribosomal proteins were detected with
specific antibodies. The mean value of the Pearson correlation coefficient in
the nucleoplasm is shown in the image (n = 10 cells in two fields of view). Scale
bars, 10 μm. Each graph represents the normalized f luorescence distribution
over the white dashed lines. Representative results from two independent
experiments. See Extended Data Fig. 5a for additional ribosomal components.
d, Time-lapse images of single foci in live HCT116 cells stably expressing
PSMB2–FusionRed and eGFP–RPL29 (PSMB2–FusionRed/RPL29–eGFP). Cells
were stimulated with 0.2 M sucrose, with or without pretreatment with the E1
inhibitor MLN-7243 (1 μM, 1 h prior). Scale bars, 0.5 μm. Representative results
from five (control) or three (MLN-7243) independent experiments. See also
Supplementary Videos 2 and 3.
****b
0.2 M sucrose
0 min 5 min1,6-HDNHOAc 410 minPSMB2–eGFP10 min 30 min30 minPSMB2DAPIFoci number per cell040801201,6-HDNH^4OAc
Control 1,6-HDNH^4OAc
Control****NS NSc
Pre Bleach 5 s 15 s 30 s 100 sPSMB2–eGFP 0.2 M sucroseeGFP–UbiquitinaPSMB2–FusionRed00.51.0CircularityPSMB2Ubiquitin 0.998 ± 0.0240.2 M sucrose
0 s 20 s 30 s 40 s 60 sFociFluorescence recovery^00.51.0Bleach0204060
Time(s)Plateau 0.88
FastSlow t1/2t 0.43
1/213.71Fig. 3 | Proteasome foci are liquid droplets. a, Foci fusion in living HCT116 cells
stably expressing PSMB2–FusionRed and eGFP–ubiquitin after stimulation
with 0.2 M sucrose. Scale bar, 10 μm. Bottom, enlarged time-course views of the
square in the above image. Scale bars, 0.5 μm. The graph indicates the
circularity of individual foci 30 min after sucrose stimulation. Data are
mean ± s.d., n = 1,312 foci from 98 cells in two fields of view. b, PSMB2–eGFP
cells were treated with 1% 1,6-hexanediol (1,6-HD) or 0.1 M NH 4 OAc 2 min after
induction of foci formation by 0.2 M sucrose. Scale bars, 10 μm. The graph
shows the number of foci per cell at the indicated times. n represents cell
numbers (control: 166 cells (10 min), 147 cells (30 min); 1,6-HD, 147 cells (10 min),
174 cells (30 min); NH 4 OAc, 223 cells (10 min), 283 cells (30 min)). Data are
mean ± s.d. and were analysed by Kruskal–Wallis with Dunn’s multiple
comparison test. P = 0.7949 (NH 4 OAc, 10 min), P = 0.2274 (NH 4 OAc,
30 min), ****P < 0.0001. NS, not significant. c, Left, FR AP (red outline, 1 μm
circle) in a 0.2 M sucrose-induced proteasome focus in a PSMB2–eGFP cell.
Scale bars, 5 μm (top) and 1 μm (bottom). Right, quantification of f luorescence
recovery of the proteasome focus and double-exponential fitting curve (dark
green). Representative results from six independent experiments.