Extended Data Fig. 4: OptoTDP-43 for the analysis of TDP-43 self-association in paraspeckle assembly.
From: Paraspeckle condensation is controlled via TDP-43 polymerization and linked to neuroprotection
a) Schematic representation of the optoTDP-43 construct. FL, full-length. b) OptoTDP-43 and Cry2olig-only distribution with and without blue light stimulation. Representative images are shown. Scale bars, 10 µm. c) OptoTDP-43 is expressed at near-endogenous levels. Representative images of TDP-43 GFP and optoTDP-43 expressing cells stained for TDP-43 and single-cell fluorescence intensity quantification relative to the endogenous level (measured in NT cells in the same FoV) are shown. C-terminal TDP-43 antibody was used. Asterisks indicate NT cells. 13 transfected and 25 non-transfected cells (n = 5 or 6 FoV) were analysed, from a representative experiment. Graph shows mean ± s.e.m. Scale bar, 10 µm. d) Endogenous TDP-43 downregulation in optoTDP-43 expressing cells. Cells expressing optoTDP-43 or Cry2olig were subjected to repetitive (x1/x6 pulses) or continuous (1-h long) blue light stimulation and analysed by western blot. Arrow indicates optoTDP-43. Note no endogenous TDP-43 or optoTDP-43 aggregation or fragmentation after blue light stimulation (that is no difference between Cry2olig vector and optoTDP-43 in the 35-kDa fragment abundance or protein amount retained in the well – complexes >250 kDa). Endogenous TDP-43 (43-kDa band) was quantified by densitometry (mean ± s.e.m.). N = 4, *p < 0.05 (p = 0.0143), one-tailed Mann-Whitney U test. e) Cry2olig-only oligomerisation does not affect paraspeckle clusters. Note that Cry2olig readily forms cytoplasmic aggregates upon blue light stimulation. Paraspeckle clusters (arrows) detected with a NEAT1_2 middle (core) probe in cells with or without continuous blue-light stimulation. Representative images and quantification (from a representative experiment) are shown. Number of cells analysed (n) is shown within bars. Scale bar, 10 µm. HeLa cells were used in these experiments. Analyses were performed 24 h post-transfection. Source numerical data and unprocessed blots are available in the Source data.
