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Figure 3 | Molecular Neurodegeneration

Figure 3

From: Calcium-responsive transactivator (CREST) protein shares a set of structural and functional traits with other proteins associated with amyotrophic lateral sclerosis

Figure 3

CREST is targeted to stress granules by various stresses. (A) CREST-Flag (top panel) and CREST-GFP (three bottom panels) are detected in stress granules induced by oxidative stress (sodium arsenite, SA), ER stress (thapsigargin, thaps) or inhibition of eIF4E (15d-PGJ2) and visualized with stress granule markers TIAR, FMRP and G3BP1. SA and 15d-PGJ2 were applied to SH-SY5Y cells for 1 hour and thapsigargin – for 4 hours. (B) In SH-SY5Y cells subjected to oxidative stress (SA for 1 hour) CREST-GFP undergoes significant shift to the cytoplasm. (C, D) CREST deletion mutant lacking autoregulatory domain (CR_dNT) is readily recruited to stress granules (C) and shows higher enrichment in these structures compared to full-length protein (D, left graph). This phenomenon is related to higher cytoplasmic levels of CR_dNT since the fluorescence intensity ratio stress granules/cytoplasm is similar for full-length and truncated protein (D). (E) Cytoplasmic aggregates of CREST-GFP do not overlap with SA-induced stress granules but are found in their immediate vicinity. (F) Cytoplasmic aggregates of CREST-GFP do not overlap with P-bodies (visualized by anti-Dcp1a staining, arrowheads in the enlarged panel). (G) Aggresomes formed by GFP-tagged CREST lacking MFD domain are negative for a SG marker G3BP1. In B and D, fluorescence was measured in stress granules and/or cytoplasm of GFP-positive cells as described in Materials and methods, and cytoplasmic intensity for non-stressed cells (B) or full-length CREST-GFP (D) (mean ± s.e.m.) was taken as equal 1 (***p < 0.001). Scale bars, 10 μm.

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