Biphasic SMN condensates safeguard stress-induced cytoplasmic mislocalization of splicing RNPs

Figures

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  Fig. 1. SMN forms reversible cytoplasmic condensates upon hyperosmotic stress. Still images of live IMR90 cells stably expressing YFP-SMN, which were put under osmotic stress for 180 min and recovered under non-stressed conditions for another 180 min. Images were analysed using bleach correction to enable direct intensity comparison over time. Scalebar: 10 µm. The graphic sketches SMN condensation states during the time course

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  Fig. 2. S-bodies and SG assemble separately upon osmotic stress. IMR90 cells were put under osmotic stress for 180 min (A, C) and recovered under non-stressed conditions for another 180 min (B), fixed and stained for endogenous SMN, the SG marker G3BP1 and the P-body marker DDX6 (C). B: Automated quantification (see methods) of S-body (SMN, yellow) and SG (G3BP1, magenta) condensates per cell as well as their colocalization (grey) included at least 100 cells in each of three independent experiments. The graph shows mean values and -/+ S.D

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  Fig. 3. S-bodies are biomolecular condensates that bind to microtubules. IMR90 cells were fixed and analysed using antibodies against SMN (yellow in merges) and (A) fluorescently labelled Phalloidin (magenta) to detect F-actin or (B) antibodies against Tubulin (magenta) to detect MT. Scalebar: 10 µm; in insets 1 µm. (C) STORM image of S-bodies along MT in IMR90 cells during 180’ stress. (D) IMR90 cells were pretreated with nocodazole 60 min prior to and during osmotic stress (180’) and SMN and MT visualized as in (B). Scalebar: 10 µm; in insets 1 µm

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  Fig. 4. S-bodies comprise the SMN complex and mislocalized cytoplasmic UsnRNPs. IMR90 cells were put under osmotic stress for 180 min and SMN visualized via indirect immunofluorescence (yellow in merges) together (magenta in merges) with (A) Gemin 2 and DDX20, (B) CLNS1A/pICln and Sm B, (C) methylated Sm proteins (Y12 antibody) and the hypermethylated UsnRNA CAP structures (TMG antibody), (D) mRNAs (in situ hybridization using fluorescently labelled oligo dT) and U1 snRNA (in situ hybridization using fluorescently labelled U1 specific probe). Scalebar: 10 µm; in insets 1 µm

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  Fig. 5. Recovery from osmotic stress reshapes S-bodies into biphasic Janus bodies. IMR90 cells were put under osmotic stress for 180 min and relieved from stress for further 20 min. SMN was visualized via indirect immunofluorescence. (A) Examples of shapes of the SMN signal at 180 min (S-bodies) and after additional 20 min of recovery (Janus bodies). Scalebar: 1 µm. Schematic representation of cytoplasmic SMN (yellow) and CLNS1A (magenta) condensates. The graph shows the shape analysis of SMN in S-bodies and Janus bodies. Condensate roundness measures how closely particles resemble a perfect circle (1.0) or elongated shapes (approaching 0.0). Plotted data points are mean values from 10 condensates of the same experiment while the mean from the three independent experiments is shown. -/+ S.D. and p-value is indicated. (B-D) SMN (yellow in merges) was analysed in parallel to CLNS1A/pICln. (B) Image detail to show two phases of Janus bodies with SMN and CLNS1A/pICln. Scalebar: 1 µm. (C) Single cell overview. Scalebar: 10 µm; in inset 1 µm. (D) Osmotic stress and recovery were compared with assembled MT (ctrl) or after MT disassembly upon nocodazole treatment 60’ prior to stress onset.…

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  Fig. 6. SMN full length (SMNwt) and SMN∆exon7 (SMN∆7) expressing cells differ in coping with osmotic stress. (A) Immunoblot to document the expression of exogenous (eYFP-SMNwt or eYFP-SMN∆7) and endogenous SMN in the IMR90 cell lines used here. (B) IMR90 eYFP-SMNwt or eYFP-SMN∆7 expressing cells were put under osmotic stress for 180 min and subsequently allowed to recover from stress for further 180 min. nt: non-treated, ie time 0’. YFP-SMN (yellow in merges) was detected by direct fluorescence in parallel to G3BP1 (magenta in merges) with indirect immunofluorescence. Scalebar: 10 µm; in inset 1 µm

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  Fig. 7. Osmotic stress and recovery in SMA model cells. Mouse embryonic fibroblasts (MEFs) expressing endogenous murine Smn (wt) or only human SMN2 from a single allele (SMA) were put under osmotic stress for 180’ without (-) or with (+) a 24 h Risdiplam treatment. SMN condensation in Cajal bodies (CB, A) or S-bodies (SB, B) was monitored by indirect immunofluorescence using an antibody that recognized identical epitopes in the murine or the human SMN protein. The graphs show quantifications of the cell fraction displaying CB (A) or SB (B). Plotted data points are mean values from 100 cells of the same experiment while the means from the three independent experiments are plotted. -/+ S.D. and p-values are indicated. (C) Cells as in (A) and (B) were allowed to recover for additional 180’ after osmotic stress and SMN (yellow in merges) analysed in parallel to U1 snRNA (magenta in merges) to monitor cytoplasmic UsnRNPs. The graph shows a quantification of SMA cells with remaining cytoplasmic U1 snRNA without (-) or with (+) Risdiplam treatment. Plotted data points are mean values from 50 cells of the same experiment while means from three independent experiments are shown. -/+ S.D. and…