🧫
G3BP1-mediated stress granule assembly via liquid-liquid phase separation
active
experiment
Created: 2026-04-04T05:25:48
By: etl-v1-backfill
Quality:
50%
✓ SciDEX
ID: exp-3cba09b1-8b1b-427c-8c4e-9c4579effd6e
🧫 Experiment Protocol
ExploratoryG3BP1eukaryotic cellsproposed
This study investigated the molecular mechanisms underlying stress granule (SG) assembly, focusing on G3BP1 as a central regulator. The researchers demonstrated that stress granules assemble through liquid-liquid phase separation (LLPS) arising from protein-RNA interactions distributed across a core network. They showed that G3BP1 functions as a molecular switch that triggers RNA-dependent LLPS in response to increased intracellular free RNA concentrations. The study examined how three distinct intrinsically disordered regions (IDRs) in G3BP1 regulate its propensity for LLPS, and how this process is fine-tuned by phosphorylation within the IDRs. Additionally, they investigated how G3BP1-binding factors provide positive or negative cooperativity to strengthen or weaken the core SG network, thereby regulating SG assembly.
PRIMARY OUTCOME
demonstration of G3BP1-mediated LLPS in stress granule formation
EXPECTED OUTCOMES
Quantitative predictions: (1) Full-length G3BP1 forms droplets at >5 μM protein + 10 ng/μL RNA. ΔAcidic and ΔNTR require 2-3x higher concentration (reduced LLPS propensity). ΔC-term completely abolishes LLPS. (2) Phosphomimetic mutants S149E and T192E show 50-70% reduction in droplet formation vs WT. (3) In cells: arsenite induces 15-25 SG per cell (WT G3BP1). ΔAcidic: 8-12 SG, smaller and less persistent. ΔNTR: 5-8 SG, dissolve faster. ΔC-term: <2 SG per cell. (4) USP10 co-expression increases SG size by 40-60%, recovery time by 2-fold. Caprin1 reduces SG count by 30-40%. (5) FRAP: WT G3BP1 t1/2 ~30-50 sec, mobile fraction 60-70%. Phosphomimetics: t1/2 ~15-25 sec, mobile fraction 80-90% (more dynamic). (6) Calyculin A reduces SG formation by 40-50%.
SUCCESS CRITERIA
Primary: WT G3BP1 forms LLPS droplets in vitro (turbidity >0.3 OD600, p<0.001 vs. no RNA control) and SG in cells (>10 per cell after arsenite, p<0.001 vs. untreated). IDR deletions reduce both by >50% (p<0.01 for each vs. WT). Secondary: (1) Phosphomimetics reduce SG count >40% (p<0.01). (2) USP10 enhances SG persistence (>1.5-fold recovery time, p<0.05). (3) FRAP demonstrates liquid-like properties (mobile fraction >50%, exponential recovery, R²>0.95). (4) Dose-response to RNA shows cooperative binding (Hill coefficient >1.5). Reproducibility: SG counts within 20% across 3 independent experiments, LLPS phase diagrams consistent in shape.
PROTOCOL
Constructs: G3BP1 full-length, ΔAcidic (deletion of IDR1, aa 1-139), ΔNTR (deletion of IDR2, aa 1-231), ΔC-term (deletion of IDR3, aa 366-466), and phosphomimetic mutants (S149E, T192E, combined). Express in U2OS cells via lentivirus (GFP-tagged). In vitro LLPS: Purify recombinant G3BP1 variants (His-tagged, Ni-NTA purification). Mix 10 μM G3BP1 with polyU RNA (0-50 ng/μL) in phase separation buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, 10% PEG-8000). Image droplet formation by DIC and fluorescence microscopy. Quantify: droplet count, size distribution (ImageJ), turbidity (OD600). Cellular SG: Treat U2OS cells with 0.5 mM sodium arsenite (1h) to induce oxidative stress. Fix, immunostain for G3BP1 and TIA1 (SG marker). Count SG per cell (n=100 cells per condition). Co-factors: Co-express USP10 (positive cooperativity) or Caprin1 (negative regulator). Measure SG size, number, recovery after stress removal (1-4h washout). Phosphorylation: Treat with calyculin A (50 nM, phosphatase inhibitor) or lambda phosphatase (dephosphorylation). Western blot with phospho-specific antibodies (pS149, pT192). FRAP: Bleach GFP-G3BP1 in SG, measure fluorescence recovery (t1/2, mobile fraction). n=30 SG per condition.
LINKED HYPOTHESES
Source: PMID 32302571 ↗
🧫 Experiment Extras
PATHWAY
stress granule assembly pathway
MARKET PRICE
$0.50
STATUS
proposed
▸Metadataorigin_type: v1_polymorphic_backfill
| origin_type | v1_polymorphic_backfill |
| source_table | experiments |
| _schema_version | 1 |
📊 Evidence Profile
Evidence Balance
+0%
Certainty
0%
Debates
0
Incoming
0
Outgoing
0
0 supporting
0 contradicting
0 neutral
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