Universal cytosolic mislocalization of mutant FUS-GFP protein expressed in zebrafish cells.

<p><b>(A)</b> A band at ∼100 kDa was seen uniquely in transgenic lysates corresponding to full length human FUS (75 kDa) conjugated to GFP (25 kDa) but not non-transgenic lysates. Lower MW bands (∼30–40 kDa) consistent with endogenous FUS were detected in all including non-transgenic zebrafish lysates. Multiple bands for endogenous FUS may indicate some degradation. N.S. denotes non-specific bands. Alpha-tubulin was used as a loading control. (<b>B</b>) Flow cytometric analysis demonstrated GFP expression in dissociated transgenic larvae. Y-axis units are normalized to the number of cells analyzed (5000–10000 cells per sample) and GFP intensity readings are presented in a log scale on the x-axis. (<b>i</b>) Cell suspensions of GFP positive vs. GFP negative larvae siblings derived from human FUS-WT-GFP transgenic fish. Clear separation of cells positive and negative for GFP is shown by the GFP +ve peak on the right and the GFP –ve peak on the left. (<b>ii</b>) Mean GFP fluorescence per cell in fresh cell suspensions from FUS-R521C-GFP and FUS-WT-GFP lines. (<b>C</b>) There was no significant difference in total GFP intensity per cell between lines after plating and culturing cells. Error bars represent SE. (<b>D</b>) Quantification of GFP fluorescence (A.U.) in nucleus and cytosol in individual cells in cultures from each fish line (n = 50–100 cells) demonstrated that there was a significant elevation in cytosolic and reduction in nuclear GFP fluorescence in FUS-R521C-GFP compared to FUS-WT-GFP cells (post-hoc Tukey HSD. *** = P<0.001). (<b>E</b>) To address the question whether elevated levels of FUS fusion protein expression could in itself cause mislocalization, we measured the total GFP fluorescence intensity versus the % nuclear GFP fluorescence in 50 to 100 individual cells of each genotype. Data were collected from 3 independent experiments for each line. There was no significant correlation between level of total GFP expressed in a cell and its subcellular distribution based on the R² values for each line (FUS-WT-GFP: R² = 0.0145 and FUS-R521C-GFP: R² = 0.3458) (n = 79 and 41 respectively).