Fig. 1: TDP-43 regulates paraspeckle condensation.
From: Paraspeckle condensation is controlled via TDP-43 polymerization and linked to neuroprotection
a, Non-transfected (NT) cells with intact paraspeckle clusters (inset 1) and TDP-43-OE cells with dispersed paraspeckles (inset 2); the arrow indicates a TDP-43-OE cell with residual NEAT1-positive clusters. b, An example of low-expressing cells; cells were classified as high-, medium- or low-expressing. c, The quantification is for HeLa, from a representative experiment; the number of cells analysed (n) is indicated within bars. FL, full-length. d, The profiles were drawn from images in a (HeLa). In a–d, the paraspeckle dispersal in TDP-43 overexpressing cells. TDP-43 with a GFP tag on its N-terminus was used. Scale bars, 15 µm. e, The NEAT1_2 arrangement in the paraspeckle spheroid and positions of probes used for dual NEAT1 RNA-FISH. f, Paraspeckle spheroids and their higher-order assemblies in HeLa cells as detected by dual NEAT1 RNA-FISH and SIM. g,h, The spheroid disruption and precursor accumulation in HeLa cells with TDP-43 OE: diffuse (g) and clustered (h) TDP-43 distribution. s, singlet, d, doublet, t, triplet, m, multiplet. A doublet with disrupted NEAT1_2 end joining is circled. Representative images and quantification from a representative experiment are shown. The number of cells quantified (n) is indicated within bars. i, The TDP-43 GFP enrichment in NEAT1 RNPs. j, The TDP-43 OE confirmation in human motor neurons (hMNs) using lentiviral (LV) delivery. Representative images are shown. Untagged TDP-43 and day 36 hMNs were used. A representative western blot is shown (mock and TDP-43 LV samples were run on the same gel). Scale bar, 10 μm. k, The increased TDP-43 dosage in hMNs leads to paraspeckle dispersal. RNAscope-ISH with a NEAT1_2 probe was used for amplified paraspeckle detection. Untagged TDP-43 and day 36 hMNs were used. Representative images and quantification from a representative experiment are shown. The number of cells quantified (n) is indicated within bars. Scale bar, 50 μm. l,m, The paraspeckle precursor: the spheroid ratio is cell line-specific: smaller NEAT1-positive particles corresponding to NEAT1 RNPs and bundles are more abundant than paraspeckle spheroids (l), and NEAT1_2 is more extractable (m) in SH-SY5Y cells as compared with HeLa. Arrows indicate spheroids. In l, quantification was performed on n = 10 cells from a representative experiment. ****P < 0.0001, one-tailed Mann–Whitney U test. In m, the semi-extractability was measured by RT–qPCR. Graphs show mean ± s.d. N = 4; *P < 0.05 (P = 0.0286), one-tailed Mann–Whitney U test. n, TDP-43 depletion shifts the equilibrium towards paraspeckle spheroids. Representative images and quantification of particle size in TDP-43-sufficient and -deficient SH-SY5Y cells. Shell (5′-end NEAT1) probe was used; s, spheroid; 288 and 216 particles were quantified for scrambled and TDP-43 siRNAs, respectively, from n ≥ 10 fields of view, from a representative experiment. ****P < 0.0001, one-tailed Student’s t-test. Representative images are shown in a, b, f and h.
