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. 2022 Sep 2;12(1):15001.
doi: 10.1038/s41598-022-19055-1.

Adult fibroblasts use aggresomes only in distinct cell-states

Christopher S Morrow  1 , Zachary P Arndt  1 , Payton C Klosa  1 , Bo Peng  1 , Eden Y Zewdie  1 , Bérénice A Benayoun  2 , Darcie L Moore  3
Affiliations

Adult fibroblasts use aggresomes only in distinct cell-states

Christopher S Morrow et al. Sci Rep. .

Abstract

The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes, whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to provide a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for studying aggresome formation and function.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Aggresomes are formed by fibroblasts in response to proteasome inhibition in distinct cell-states. (A) A schematic depicting how the aggresome forms. When polyubiquitinated proteins accumulate, they are trafficked along microtubules to the centrosome. (B,C) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 h prior to fixation and immunostaining for K48 polyUb (green), vimentin (red) and nuclei (Hoechst; blue). Samples were imaged, and subsequently analyzed for the proportion of cells forming aggresomes discernable by an enrichment of K48 polyUb only (red bar), a vimentin cage only (green), both (blue) or neither (white) (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (D) Examples of vimentin cages surrounding the aggresome in quiescent and immortalized fibroblasts treated with 10 µM MG132 for 8 h prior to being fixed and immunostained as in (B,C). (E) Proliferating, senescent, quiescent and immortalized fibroblasts were treated with 10 µM MG132 for 8 h prior to being fixed and immunostained for K48 polyUb (green), γ-tubulin (red) and stained for nuclei (Hoechst; blue). (F,G) Quiescent and immortalized fibroblasts were treated with 10 µM MG132 for 8 h with either 0.1% DMSO or 2 mM nocodazole prior to being fixed and immunostained for K48 polyUb (green) and stained for nuclei (Hoechst; blue). Samples were quantified as in (C). (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). Arrows denote aggresomes (B,D,F) or centrosomes (E). Scale bars 10 µm.
Figure 2
Figure 2
Aggresomes are used within diverse proteostasis networks. (A,B) Proliferating, senescent, quiescent, and immortalized fibroblasts were pulsed with 10 µg/mL puromycin for 10 min prior to soluble protein extraction and analysis by western blot. Relative levels of puromycin incorporation and actin expression were visualized by western blot. Samples were analyzed for puromycin levels relative to actin. (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (C,D) Proliferating, senescent, quiescent, and immortalized fibroblasts were analyzed for soluble protein expression of HSP90β and actin by western blot (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (E,F) Proliferating, senescent, quiescent, and immortalized fibroblast protein lysates were prepared and analyzed for relative levels of trypsin-like proteasome activity by measuring AMC fluorescence as a function of time (N = 3; Two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (F) Normalized AMC accumulation at 60 min for each sample. (G,H) Proliferating, senescent, quiescent, and immortalized fibroblasts were stained and analyzed for lysosome content (Lysotracker; red) and nuclei (Hoechst; blue) (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). The dotted line in each panel separates cell-states that either do or do not form the aggresome. (I) Schematic summarizing the proteostasis network experiments in each fibroblast cell-state. Red indicates higher levels of each node of the proteostasis network respectively. Scale bars 10 µm. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Transcriptional profiling of the fibroblast cell-state specific response to proteasome inhibition reveals stress-activated MAPK signaling associated with aggresome formation. (A) MDS analysis of the global transcriptomes of proliferating (purple), senescent (blue), quiescent (red), and immortalized (orange) fibroblasts treated with either 0.1% DMSO (circles) or 10 µM MG132 (squares) for 8 h. Ovals denote clustering of DMSO or MG132 conditions. (B) MDS analysis of the proteostasis network’s (chaperone, proteasome and lysosome genes) transcriptional response to proteasome inhibition in proliferating, senescent, quiescent, and immortalized fibroblasts. Ovals denote clustering of aggresome-forming or -lacking cell-states. (C) Heat maps depicting differential expression of chaperone, proteasome, or autophagy genes in response to MG132 between proliferating, senescent, quiescent, or immortalized fibroblasts. (D) Average DEseq2 log2-fold change values for all chaperone, proteasome, or autophagy genes as proliferating, senescent, quiescent, and immortalized fibroblasts respond to MG132 treatment. (E) Transcript per million counts of representative genes implicated in aggresome formation in proliferating, senescent, quiescent, and immortalized fibroblasts treated with DMSO (circles) or MG132 (squares). (F,G) GO and KEGG analysis of the signaling pathways significantly differentially regulated in response to MG132 in proliferating, senescent, quiescent, and immortalized cells. FDR refers to false discovery rate and NES refers to normalized enrichment score. The dotted line in each panel separates cell-states that either do or do not form the aggresome.
Figure 4
Figure 4
p38α MAPK and p38β MAPK inhibition with TAK-715, and TAK1 inhibition with 5Z-7 suppress aggresome formation in fibroblasts. (A,B) Average DESeq2 log2(fold change) values for all MAPK cascade or Regulation of stress-activated MAPK cascade genes as proliferating (purple), senescent (blue), quiescent (red), and immortalized (orange) fibroblasts respond to MG132 treatment. (C) A schematic summarizing the interaction of TAK1, MKK3/6 and p38 MAPKs in stress-activated MAPK signaling. Inhibitors are shown in red. (D–G) Quiescent and immortalized fibroblasts were treated with 5 µg/mL 5Z-7 or 0.05% DMSO for 24 h, and then additionally treated with 10 µM MG132 for 7 h still in the presence of 5Z-7 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (N = 3; Student’s t-test; mean ± SD). (H–K) Quiescent and immortalized fibroblasts were treated with 10 µM MG132 and/or 5 µg/mL 5Z-7-Oxozeaenol for 8 h as above and then analyzed for expression of p38 MAPKs, actin, and P-p38 MAPKs (T180/Y182) by western blot. (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (L–O) Quiescent and immortalized fibroblasts were treated with 50 µM TAK-715 or 1% DMSO for 24 h, then additionally treated with 10 µM MG132 for 7 h still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (N = 3; Student’s t-test; mean ± SD). (P,Q) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 h prior to analysis of p38 MAPKs, and P-p38 MAPKs (T180/Y182) by western blot. A ratio of P-p38/total p38 was used to measure activation of p38 (N = 3; Two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (R) A schematic depicting a working model for p38 MAPK’s role in aggresome formation in primary mouse dermal fibroblasts, suggesting p38 activity is necessary but not sufficient. Scale bars 10 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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