The Analysis on the Dynamic Properties of HuR Granules in Living Cells via FLIP Technology

Abstract: HuR (human antigen R)， a kind of multifunctional RNA-binding protein， is involved in the SGs (stress granules) assembly. SGs are a type of cytoplasmic RNA foci that accumulate in response to environmental stress stimuli. Here， the fluorescence loss in photobleaching (FLIP) technology was used to study the dynamic nature of HuR granules in living cells. Firstly， the plasmid encoding RFP-HuR fusion protein was transfected into HeLa cells via the liposome reagent. Western blot and immunofluorescence assays were then performed to verify the expression of RFP-HuR protein. For FLIP assay， one HuR granule region of interest (ROI) was repeatedly photo-bleached using 405 nm laser beam， other granule or nuclear region were monitored， and the adjacent non-bleached cell was used as a control. The results indicated that RFP-HuR fusion protein was expressed efficiently and co-localized with the G3BP (Ras-GAP SH3 domain-binding protein) (one SGs marker protein) in HeLa cells. In FLIP assay， fluorescence density in the cytosolic pulse bleach region was reduced from 2 500 a.u. to 0 a.u. after the first bleaching. The fluorescence density of the adjacent HuR granule region reduced from 1 800 a.u. and maintained at about 200 a.u. after 12 bleaching cycles (240 s)， suggesting that HuR-containing SGs structure is highly dynamic. The nuclear fluorescence density also reduced from 4 400 a.u. to 2 000 a.u.， suggesting that HuR is a nucleocytoplasmic shuttling protein. The nuclear pool of HuR protein is likely to be in equilibrium with cytosolic SGs-routed pool. The FLIP technology can be used to analyze the dynamic properties of different stress associated proteins， helping to investigate the molecular mechanisms underlying the SGs-related diseases.

CSTR: 32200.14.cjcb.2014.09.0004