FUS mislocalization and accumulation in assembled SGs demonstrated here is consistent with previous studies of mutant human FUS sub-Ro 19-4603 cellular localization in mammalian cell lines and supports the use of the zebrafish model for investigating the cellular physiology of FUS in motor neuron disease. The R521C mutation is one of the most common fALS mutations and has been reported to cause relatively less aggressive forms of the disease compared to other mutations like P525L and R522G. Our results contribute insight into the subcellular distribution of FUS-R521C and illustrate that it may not be just mildly mislocalized as previously reported. Interestingly, mislocalization of human FUS-R21C-GFP in zebrafish cells was more severe than a previous study in HeLa cells where transient expression of HA-tagged FUS-R521C or FUS-R521H showed only 5�C10% HA-immunolabelled mutant FUS in the cytosol. The transgenic model of stable expression has the advantage over transient expression in cell lines in that the gene of interest is expressed during the normal development of the organism and in primary differentiated cells, including motor neurons, in comparison with immortalized cell lines where the cellular physiology may be more artificial. This has implications for hypothesized correlation to severity of fALS disease – Dormann et al found by comparison that mutations FUS-P525L and FUS-R522G that cause aggressive and early onset fALS were severely mislocalized in their transfected cells with 50�C65% found in the cytosol, similar to R521C reported here in zebrafish cells. We conclude that factors other than relative mislocalization are likely also to play important roles in disease severity. Nevertheless, an increase in Ro 67-7476 cytosolic FUS caused by mis-localization of the mutant protein out of the nucleus, appeared to significantly affect zebrafish cell susceptibility to SG assembly with the mutant FUS-R521C-GFP showing the greater vulnerability to accumulate in SGs and the lower propensity for reversal on recovery. FUSWT- GFP cells expressing similar or even higher levels of exogenous protein, maintained a largely nuclear distribution of the protein and exhibited a lower propensity to generate human FUS containing SGs in the cytosol. We never observed FUS inclusions in the nucleus. Three other studies have shown that FUS mutants, but not wild-type FUS, form SGs under similar conditions. By contrast, our results show that FUS-WT-GFP can also be induced to form cytosolic SGs, albeit to a lesser extent compared to FUSR521C- GFP.