Characterisation of the aggregation capacity of CREST. (A) In cells with diffuse distribution of CREST, it re-localizes to nucleolar caps (arrowheads) in response to transcriptional inhibition. (B) The pool of CREST in nuclear dot-like aggregates fails to redistribute to nucleolar caps upon inhibition of transcription, and the aggregates persist under these conditions, although weakly CREST-positive nuclear caps can be observed (arrowheads). In A and B SH-SY5Y cells were exposed to actinomycin D for 1 hour. (C) CREST is recovered in detergent-insoluble fractions. HEK293 cells expressing untagged CREST were subjected to sequential protein extraction as described in Materials and methods. For total lysate (L) and high-salt (HS) fraction 10% of the amount relative to other fractions was loaded. Bar chart shows relative protein amounts (±s.e.m.) in each fraction quantified by densitometry. (D) Untagged CREST, Flag-CREST and GFP-CREST do not form SDS-resistant oligomeric forms. Cleared lysates of CREST-expressing SH-SY5Y cells were run in SDS-containing agarose gel; all variants were visualized using anti-CREST antibody. Mutant tau protein from spinal cord lysate of a transgenic P301S mouse (detected by phospho-tau-specific antibody) was used to demonstrate typical behavior of amyloid species in this assay. (E) Schematic representation of CREST deletion constructs used in the study. All variants were expressed as GFP-fusion proteins. (F, G) Distribution of CREST deletion mutants in SH-SY5Y cells. CR_dNT and CR_dNT-Met were shifted to the cytoplasm and formed nuclear dot-like aggregates less frequently than full-length protein (G). Bar chart in G shows the fraction of cells (mean ± s.e.m.) with nuclear aggregates for each variant (*** - p < 0.001; at least 150 cells counted per variant in each of the three independent experiments). CREST was expressed for 24 hours prior to actinomycin D exposure, cell lysis or fixation. Scale bars, 10 μm.