Figure 1. Characterization of mutant FUS proteins transiently expressed in neuroblastoma SH-SY5Y cells. (A) Scheme of FUS domain organization and GFP-fusion constructs. (B) All GFP-labeled FUS variants utilized in the study are expressed at a comparable level in neuroblastoma cells as revealed with anti-GFP antibody. Total protein extracts for western blot analysis were prepared from cells lysed 24 h after transfection with corresponding expression plasmid. C is an unrelated cytosolic GFP-fusion protein used as a control. The size of proteins in kDa is shown. (C) Diffuse distribution of cytoplasmically localized FUS variants FUS R522G, FUS 1–513, and FUS ΔRRMcyt is detected in transfected cells at early stages of the ectopic protein accumulation (4–12 h post-transfection) or in cells with relatively low levels of its expression. In contrast, in most cells expressing FUS 1–359, small aggregates are formed very early after transfection. Scale bar: 20 µm. (D) Intracellular distribution of GFP-FUS fusion proteins and morphology of aggregates formed in the cytoplasm 36 h post-transfection. See also Videos S1 and S2. (E) Twenty-four hours after transfection, the majority of cells expressing FUS 1–359 possess aggregates of some form, and more than half of them display large juxtanuclear structures. (F) Juxtanuclear aggregates formed by FUS 1–359 protein are negative for endosomal/lysosmal compartment markers LAMP-1 and LAMP-2. Scale bars: (D), 15 µm; (C and F), 20 µm.