g3bp1 memerald  (TaKaRa)

Journal: bioRxiv

Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

doi: 10.1101/2023.03.22.533832

Figure Lengend Snippet: (A) A Schematic of our microfluidic device used to extract the relative elastic modulus of GUVs. The bright-field images on the right illustrate GUV deformation within the device with a channel size: opening = 15 µm, tapered end = 2 µm. (B) A plot of the GUV deformation (strain) under variable pressure applied across the V-shaped channel (stress). Simple linear regression, R 2 (Forth/Back) – 0.991/0.980. ANCOVA-slopes (p=0.26) and intercepts (p=0.13) are not significantly different. (C) Quantification of the relative elastic modulus of GUVs at 100 µM Ca 2+ with 0.5 µM ANXA11 FL, coincubated with either 2 µM ALG2 or 20 µM CALC. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ****p < 0.0001, n=3. (D) The experimental pipeline for FAPS-based RNP granule isolation from a stable G3BP1-mEmerald U2OS line. (E) Representative fluorescence images of ATTO594 GUVs incubated with 100µM Ca 2+ and 0.5µM ANXA11 co-incubated with either 2µM ALG2 or 20µM CALC. To each condition, purified RNP granules (labelled with mEmerald-G3BP1) were added to a final concentration of 0.2 mg/ml. Scale bar - 5 µm. (F) Quantification of the fluorescence intensity of RNP granules (mEm-G3BP1) recruited to ANXA11-GUV assemblies as displayed in (E). Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, *p < 0.05, ****p < 0.0001, n=7 (21-78 GUV).

Article Snippet: G3BP1-mEmerald was created by subcloning the G3BP1 cassette from the pOPINS-G3BP1 construct into an mEm-N1 backbone derived from the Clontech N1 system.

Techniques: Isolation, Fluorescence, Incubation, Purification, Concentration Assay

Journal: bioRxiv

Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

doi: 10.1101/2023.03.22.533832

Figure Lengend Snippet: (A) fluorescence images of a stable G3BP1-mEmerald (Orange) U2OS line labelled with after stress granule induction using 0.5 mM sodium arsenite for 30 min. SPY650-DNA was used to label the nucleus (white). On the right is an image of FAPS-isolated granules which have been pelleted and resuspended in a small volume of dilution buffer. Scale bar - 1.5 µm. (B) An ATTO594 GUV incubated with 100 µM Ca 2+ and 0.5 µM ANXA11 FL and 0.2 mg/ml of recombinantly purified mEmerald-G3BP1 at a low and high imaging exposure. Scale bar - 5 µm. (C) Fluorescence images of ATTO594 GUVs incubated with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or ARD. 0.2 mg/ml of G3BP1-mEmerald positive FAPS-isolated RNP granules were added to each condition. Scale bar - 5 µm. (D) Quantification of RNP granule recruitment to ANXA11-GUV assemblies as represented in (C). Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (40-106 GUVs).

Article Snippet: G3BP1-mEmerald was created by subcloning the G3BP1 cassette from the pOPINS-G3BP1 construct into an mEm-N1 backbone derived from the Clontech N1 system.

Techniques: Fluorescence, Isolation, Incubation, Purification, Imaging

Journal: Nature Communications

Article Title: Spatially non-uniform condensates emerge from dynamically arrested phase separation

doi: 10.1038/s41467-023-36059-1

Figure Lengend Snippet: a Reconstituted stress granules can form a double-emulsion structure when their composition changes upon the addition of 250 ng/μL Poly-rA RNA. The trapped droplets are poor in the protein G3BP1 and in nucleic acids. The before and after images show different condensates. b The number of trapped droplets in the images of Fig. 4a and Supplementary Fig. as function of the radius of the condensates. Similar to the Poly-rA PEG system, more trapped droplets can be formed in larger condensates. n = 157 and 104 for before and after additional or RNA respectively. The average number of trapped droplets and one standard deviation is shown after additional RNA.

Article Snippet: Cotransfection of the G3BP1-mEmerald-PiggyBac vector and a transposase (TransposagenBio) plasmid into CCL-2 HeLa cells (ATCC, authenticated via STR profiling) was followed by blasticidin selection to isolate a polyclonal G3BP1-mEmerald population.

Techniques: Standard Deviation