Fig 1: OptoGranules are light-inducible dynamic stress granules.(a) Design of Opto-G3BP1 and Opto-Control constructs. (b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with a single 5-msec pulse of 488 nm blue light (power density ~2.5 MW/cm2) in a defined ROI. Representative images are shown from n = 3 independent experiments. (c) Quantification of data in cells treated as in (b). Five cells with similar expression levels were counted. Granule numbers are shown relative to the granule number at the peak of OptoGranule assembly. Error bars represent s.e.m. (d-f) U2OS cells were stably transfected with Opto-Control or Opto-G3BP1, or stable Opto-G3BP1 cells were transiently transfected with G3BP1-GFP, and stimulated with a blue-light laser (power density ~4.5 W/cm2) for 3 mins. Regions marked with yellow circles were photobleached and monitored for fluorescence recovery. Data are shown as representative images (d), relative fluorescence intensity of photobleached region over time (e), and relative mobile fraction derived from (e) (f). For (e, f) n = 15 cells for Opto-Control; n = 12 for Opto-G3BP1; n = 14 for G3BP1-GFP. Data are representative of n = 3 independent experiments. Data shown as mean + s.d. ns, not significant by one-way ANOVA with Dunnett’s test. (g) U2OS cells transiently transfected with Opto-G3BP1 and the stress granule marker GFP-TIA1 were stimulated with a blue-light laser (power density ~2.5 MW/cm2) for 5 msec. Cells were sequentially imaged by 561 nm and 488 nm channels; we note that the 488 nm channel used for imaging also activates Opto-G3BP1 (power density 2.2 W/cm2). Representative images are shown from n = 3 independent experiments. (h-j) U2OS cells stably expressing Opto-Control or Opto-G3BP1 constructs were stimulated for 6 hr without or with continuous blue light (~2 mW/cm2) using custom-made LED arrays for global activation. Cells were immunostained with PABP antibody (h), TDP-43 antibody (i), or RNA fluorescence in situ hybridization using FAM-labelled oligo (dT)20 as a probe (j). Scale bars, 10 µm in all micrographs.
Fig 2: Persistent OptoGranules are cytotoxic and evolve to pathological inclusions.(a,b) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and viability was assessed by crystal violet staining (a) or CellTiter-Glo 2.0 luminescence (b). Whiskers represent minimum to maximum from n = 9 biological replicates. ****p<0.0001.; ns, not significant by two-way ANOVA with Tukey’s post-test. (c,d) U2OS cells stably expressing Opto-Control or Opto-G3BP1 were exposed to chronic persistent (c) or chronic intermittent (d) blue light (445 nm) stimulation with live-cell imaging (power density ~0.12 W/cm2) as illustrated in the schematic (left) and assessed for cell survival by counting living cells (right). Blue boxes in schematic indicate the timing of light induction; red line is an idealized graph of the cellular response. Chronic persistent paradigm: n = 26 for Opto-Control and n = 28 for Opto-G3BP1. Chronic intermittent paradigm: n = 7 for Opto-Control and n = 10 for Opto-G3BP1. Data are shown from n = 3 independent experiments. ****p<0.0001 by log-rank (Mantel-Cox) test. (e) Timeline of protein accumulation in OptoGranules in U2OS cells. (f-h) U2OS cells stably expressing Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and co-immunostained with p-TDP-43 and A11 antibodies (f), SQSTM1 and ubiquitin antibodies (g), or VCP and TDP-43 antibodies (h). (i) quantification of data from (f-h). Error bars represent s.e.m. Images in f-h are representative of n = 3 independent experiments. ***p=0.0002 (2 hr), ***p=0.0001 (3 hr) for TDP-43, **p=0.0048 (2 hr), ***p=0.0002 (3 hr) for A11, **p=0.0051 (5 hr) for ubiquitin, ****p<0.0001 for SQSTM1, ***p=0.0003 for pTDP-43, and ****p<0.0001 for VCP by one-way ANOVA with Dunnett’s test. Scale bars, 10 µm in all micrographs.
Fig 3: Persistent OptoGranules are cytotoxic and evolve to pathological inclusions in human iPSC-derived neurons.(a) Schematic illustrating generation of iPSC-derived neurons stably expressing Opto-G3BP1. (b) iPSC-derived neurons expressing Opto-Control (mRuby) or Opto-G3BP1 (mRuby) were intermittently exposed to a 488 nm blue-light laser (90% laser power, power density 6.3 W/cm2) followed by image acquisition with a 561 nm channel. Representative images are shown from n = 3 independent experiments. (c) iPSC-derived neurons expressing Opto-Control or Opto-G3BP1 were exposed to chronic persistent stimulation as in Figure 3c and survival was assessed by counting living cells. n = 35 cells for Opto-Control and n = 34 cells for Opto-G3BP1. Data are representative of n = 3 independent experiments. ****p<0.0001 by log-rank (Mantel-Cox) test. (d) Timeline of pathological protein accumulation in OptoGranules in iPSC-derived neurons. (e-h) iPSC-derived neurons expressing Opto-G3BP1 were stimulated with continuous blue light (~2 mW/cm2) for indicated times using custom-made LED arrays and co-immunostained with MAP2 and TDP-43 antibodies (e), MAP2 and A11 antibodies (f), MAP2 and p-TDP-43 (P01) antibodies (g), or ubiquitin and SQSTM1 antibodies (h). See also Figure 4—figure supplement 1e for line scans of images shown in (h).(i) quantification of data from e-h. Error bars represent s.e.m. Images in e-h are representative of n = 3 independent experiments. *p=0.0489 (2 hr), ***p=0.0001 (5 hr) for SQSTM1 and ****p<0.0001 for pTDP-43 by one-way ANOVA with Dunnett’s test. Scale bars, 10 µm in all micrographs.
Fig 4: OptoGranule formation is dependent on the local concentration of activated G3BP1 and dependent on polysome disassembly, but independent of eIF2a phosphorylation.(a) U2OS cells stably expressing Opto-G3BP1 were intermittently exposed to a blue-light laser (488 nm) for activation followed by image acquisition with a 561 nm channel. Blue light intensity was sequentially increased from top to bottom (488 nm power density measurement from top to bottom: 1%, 0.02 W/cm2; 5%, 0.04 W/cm2; 25%, 0.95 W/cm2; 75%, 5.5 W/cm2). Representative images are shown from n = 3 independent experiments. (b) Quantification of data in cells treated as in (a). Error bars represent s.d. (c) U2OS cells with different expression levels of Opto-G3BP1 were intermittently exposed to a 488 nm blue-light laser (90% laser power, power density 6.3 W/cm2) followed by image acquisition with a 561 nm channel. Relative expression levels from top to bottom: 0.19, 0.32, 0.78 and 1 a.u. Representative images are shown from n = 3 independent experiments. (d) Quantification of data in cells treated as in (c). (e) U2OS cells stably expressing Opto-G3BP1 were pre-treated with cycloheximide (CHX) or ISRIB for 30 min and then exposed to 45 min of sodium arsenite (0.5 mM NaAsO2) or 6 hr of continuous blue light (~2 mW/cm2) using custom-made LED arrays for global activation, and immunostained with PABP antibody. (f) Quantification of granule-positive cells from (e). Data are shown as mean ± s.e.m. from n = 3 independent experiments. ****p<0.0001; ns, not significant by one-way ANOVA with Tukey’s post-test. (g) Immunoblot showing phosphorylated eIF2a (P-eIF2a), eIF2a, and actin levels in cells treated with sodium arsenite (0.5 mM NaAsO2) for 45 min, exposed to 42°C heat shock for 1 hr, or activated with blue light for 6 hr. See also Figure 2—figure supplement 1 for sequential probe images. Scale bars, 10 µm in all micrographs.
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