A thermosensor FUST1 primes heat-induced stress granule formation via biomolecular condensation in Arabidopsis

Abstract

The ability to sense cellular temperature and induce physiological changes is pivotal for plants to cope with warming climate. Biomolecular condensation is emerging as a thermo-sensing mechanism, but the underlying molecular basis remains elusive. Here we show that an intrinsically disordered protein FUST1 senses heat via its condensation in Arabidopsis thaliana. Heat-dependent condensation of FUST1 is primarily determined by its prion-like domain (PrLD). All-atom molecular dynamics simulation and experimental validation reveal that PrLD encodes a thermo-switch, experiencing lock-to-open conformational changes that control the intermolecular contacts. FUST1 interacts with integral stress granule (SG) components and localizes in the SGs. Importantly, FUST1 condensation is autonomous and precedes condensation of several known SG markers and is indispensable for SG assembly. Loss of FUST1 significantly delays SG assembly and impairs both basal and acquired heat tolerance. These findings illuminate the molecular basis for thermo-sensing by biomolecular condensation and shed light on the molecular mechanism of heat stress granule assembly.

Fig. 1. FUST1 undergoes heat-dependent condensation in vivo and in vitro.

a Time-lapse imaging of FUST1-mVenus condensation in Arabidopsis root tip cells. Scale bar, 5 μm. b FRAP of FUST1 condensates in Arabidopsis root tip cells. Time 0 s indicates the time of the photobleaching pulse. White dashed circles indicate the bleached condensates. Scale bar, 1 μm. c Plot showing the fluorescence recovery of FUST1 condensates after photobleaching in Arabidopsis root tip cells. Error bars indicate mean ± SD (n = 13). d In vitro phase separation of 2.5 μM FUST1-GFP at varying temperatures in 40 mM Tris-HCl, pH 7.4 and 100 mM NaCl. Scale bars, 5 μm. e Time-lapse imaging of 2.5 μM FUST1-GFP phase separation at 4 °C and 37 °C in 40 mM Tris-HCl, pH 7.4 and 100 mM NaCl in vitro. Scale bars, 5 μm. f FRAP of FUST1-GFP droplet. Time 0 s indicates the time of the photobleaching pulse. Scale bar, 2 μm. g Plot showing the recovery after photobleaching of FUST1-GFP droplets. Error bars indicate mean ± SD (n = 6). h Relaxation of two FUST1-GFP droplets into one droplet. Time points are indicated above. Scale bar, 2 μm.

Fig. 2. PrLD of FUST1 is the main driver for heat sensing and condensation.

a Top, illustration of the amino acid composition of PrLD. Middle, protein domain structure of FUST1. Bottom, prediction of the IDRs and prion-like domain by PONDR and PLACC algorithms. b Representative confocal microscopic images of tobacco epidermal cells expressing FUST1 and its variants. The cells were treated at 37 °C for 30 min and 22 °C as control. Scale bars, 10 μm. c In vitro phase separation assay of Cy5-labeled PrLD^WT at different temperatures and concentrations. Scale bar, 5 μm. d Phase diagram of PrLD^WT as a function of temperature. Red dots, with detectable condensates. Empty circles, without detectable condensates. e, f Temperature-dependent turbidity of indicated proteins with indicated protein concentrations. In vitro phase separation assay in c and turbidity measurements in e, f were conducted in 40 mM Tris-HCl pH, 7.4 and 500 mM NaCl. Error bars indicate mean ± SD (n = 3). g Representative confocal microscopic images of Arabidopsis root tip cells expressing FUST1, FUST1^∆PrLD and FUST1^{PrLD Y>S}. The roots were treated at 35 °C for 30 min. Scale bars, 10 μm. h Statistical analysis of number of condensates per cell in each genotype in g at 35 °C. Error bars indicate mean ± SD (n = 15). P values were calculated using two-sided Student’s t-test. ****P < 0.0001.

Fig. 3. The mechanism of temperature sensing by PrLD.

a–c The gyration of radius (Rg, a), the number of intramolecular H-bonds (b), and the side chain-side chain contacts between amino acid residues (c) of PrLD^WT or PrLD^Y>S at elevating temperatures as revealed by MD simulation. d The conformation of two PrLD molecules at 310 K or 280 K as revealed by MD simulation. e The structure prediction shows the intermolecular contacts between two PrLD molecules increase at 310 K or 280 K as revealed by MD simulation. f Diagram showing the change of contacts for each amino acid with other residues in PrLD at 310 K compared to 280 K. Positive and negative values indicate gain and loss of contacts, respectively. g Heat map showing the strength of contacts between indicated residues at 280 K or 310 K. h Diagram showing the β-strand probability across PrLD at 280 K or 310 K. i Schematic diagram showing the lock-to-open conformational switch of PrLD at elevating temperatures. The orange region indicates the β-strand lock. j Illustration of the FRET assay. k–m Quantification of mGFP-lifetime in mGFP-PrLD^WT or mGFP-PrLD^WT-mCherry (k) mGFP-PrLD^M1 or mGFP-PrLD^M1-mCherry (l) and mGFP-PrLD^M2 or mGFP-PrLD^M2-mCherry (m). Error bars indicate mean ± SD (n ≥ 20).

Fig. 4. FUST1 interacts with and localizes in the SGs.

a The integral SG components enriched by FUST1-TurboID proximity labeling. Error bars indicate mean ± SD (n = 4). b Volcano plot showing the enrichment of proteins by IP-MS of FUST1 in Arabidopsis. The values were calculated from 4 biological replicates. c Co-immunoprecipitation of FUST1 with indicated proteins expressed in tobacco epidermal cells. Cells were treated at 37 °C for 1 h and crosslinked with UV. Empty vector was included as a negative control. d Representative confocal microscopic images of Arabidopsis root tip cells co-expressing FUST1-mScarlet with G3BP5-mVenus or PAB2-GFP under their native promoters. Roots were treated at 37 °C for 30 min. Scale bars, 10 μm. e FISH with oligodT probes in pFUST1::FUST1-mVenus/fust1-1 roots treated at 37 °C for 30 min. Scale bar, 10 μm. f Partitioning of 2.5 μM indicated proteins by 2.5 μM FUST1 droplets in 40 mM Tris-HCl pH 7.4 and 100 mM NaCl in vitro. Scale bars, 5 μm.

Fig. 5. FUST1 condensation is required for SG formation.

a Confocal microscopy images of Arabidopsis root tip cells expressing indicated proteins under the corresponding native promoters. The roots were treated as indicated. Scale bars, 10 μm. b Representative confocal microscopic images of Arabidopsis root tip cells co-expressing FUST1-mScarlet with G3BP5-mVenus or PAB2-GFP under their native promoters. Roots were treated at 33 °C for 90 min. Scale bars, 10 μm. c Time-lapse imaging of Arabidopsis root tip cells co-expressing G3BP5-mVenus or PAB2-GFP with FUST1-mScarlet under their corresponding native promoters. The plants were treated at 37 °C for the indicated time. Scale bars, 1 μm. d, e Left, Confocal microscopic images of Arabidopsis root tip cells expressing indicated proteins under corresponding native promoters in FUST1 wild type background or fust1-2 mutants. The roots were treated at 37 °C for the indicated time. Scale bars, 10 μm. Right, Statistical analysis of the number of condensates per cell in the indicated genotypes. Error bars indicate mean ± SD (n = 15). P values were calculated using two-sided Student’s t-test. ****P < 0.0001. f FISH with Cy5-oligodT probes in Col-0 and fust1 mutants treated at 37 °C for 30 min. Scale bar, 5 μm. P values were calculated using two-sided Student’s t-test. ****P < 0.0001.

Fig. 6. FUST1 is required for thermotolerance.

a Scheme of heat exposures in basal and acquired tolerance assays. b Left, cell death staining of Arabidopsis roots upon heat stress treatment at 42 °C for 2 h. Scale bars, 100 μm. Right, the percentage of seedlings containing cell death. Error bars indicate mean ± SD (n = 3). Twenty seedlings were assayed in each replicate. P values were calculated using two-sided Student’s t-test. ***P < 0.001, ****P < 0.0001. ns no significance. c Germination rate of Arabidopsis seeds after heat treatment at 50 °C for 60 min. Error bars indicate mean ± SD (n = 4). Each replicate contains 49 seeds. P values were calculated using two-sided Student’s t-test. **P < 0.01. d, f Phenotypes of indicated seedlings assayed for basal (d) or acquired (f) heat tolerance. Pictures were taken at seven days (d) or four days (f) of recovery after treatment as indicated. Scale bars, 0.5 cm. e, g Quantification of the damage rate and fresh weight of seedlings shown in d, f. Error bars indicate mean ± SD (n = 3). At least 50 seedlings were assayed for each replicate. P values were calculated using two-sided Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7. A proposed model for FUST1 in heat sensing and response.

In the cytoplasm, FUST1 is dispersed. When the temperature increases, FUST1 senses heat via conformational switch and condenses with RNA. FUST1 condensates provide primers to recruit stress granule components and drive stress granule assembly, which is required for cell survival.