human cell expression constructs  (Addgene inc)

Journal: Methods in Enzymology

Article Title: Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples

doi: 10.1016/bs.mie.2022.07.015

Figure Lengend Snippet: Cell fusion assay based on high-affinity NanoLuc® complementation (HiBiT) (A) Schematic representation of the assay set-up. LgBiT and spike-expressing HEK293T cells are mixed with HiBiT- and ACE2-expressing Vero E6 cells and co-incubated overnight. Upon spike interaction with ACE2, cell membranes merge, forming a multinucleated syncytium and enabling complementation of both NanoLuc® fragments, eventually leading to light emission upon substrate addition. Pre-incubation of spike-expressing cells with neutralizing antibodies will prevent the spike–ACE2 interaction and limit syncytia formation. Multiple sera can be tested simultaneously in 96- or 384-well format, enabling high-throughput screening of samples. (B) Representative microscopy pictures, illustrating syncytium formation between Vero E6 cells expressing an mKOrange-tagged membrane marker and HEK293T cells expressing a NeonGreen cytoplasmic marker. DAPI-stained nuclei are depicted in blue. (C) Representative picture of a 96-well plate after substrate addition taken with an ordinary mobile phone camera (Huawei p20 Pro). The intense blue bioluminescent signal emitted by the NanoLuc® is well visible even with the naked eye. No light is emitted from wells where cells did not efficiently form syncytia, indicative of the presence of neutralizing antibodies.

Article Snippet: • Cell culture flasks (Greiner Bio-One) • Dulbecco's modified Eagle medium (DMEM, GIBCO) supplemented with 0.04 mM phenol red, 1 mM sodium pyruvate, 4 mM l -glutamine, 4.5 g/L glucose, supplemented with 10% fetal bovine serum (FBS, Merck) and 100 units/mL of penicillin and 100 μg/mL of streptomycin (GIBCO) • 10-cm cell culture dishes (Corning) • Opti-MEM, Reduced Serum Medium (GIBCO) • Polyethylenimine (PEI, 1 mg/mL, Merck) • LgBiT expression construct • HiBiT expression construct • SARS-CoV-2 spike protein expression construct • Human embryonic kidney 293T (HEK293T) cells (for example CRL-3216, ATCC) • Vero E6 cells (for example CoronaGrow-VeroE6, CL0001, VectorBuilder) See section below for amino acid sequences of protein expression constructs.

Techniques: Cell Fusion Assay, Expressing, Incubation, High Throughput Screening Assay, Microscopy, Membrane, Marker, Staining

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Identification of genes enriched in dry Arabidopsis seeds. (B-C) The seed proteome is enriched for specific amino acids (B) and intrinsic disorder (C). Mann-Whitney. (D) The seed proteome is enriched for prion-like proteins. Binomial test. AT4G28300 is an uncharacterized prion-like protein, which we name here FLOE1. (E) FLOE1p:FLOE1-GFP is expressed during embryonic development and forms condensates. (F) FLOE1-GFP forms condensates in embryos dissected from dry seed in a hydration-dependent and reversible manner. Embryonic cotyledons are shown. PSV denotes autofluorescent protein storage vacuoles that are more prominent in the dry state than in the hydrated state (see also ). (G) Cell-to-cell variation in subcellular FLOE1-GFP heterogeneity in response to salt. Radicles are shown. * denotes nuclear localization. (H) Quantification of cellular FLOE1 heterogeneity as a function of salt concentration. Black line denotes the 95 th percentile of the 2M NaCl heterogeneity distribution. (I) Quantification of the percentage of cells per radicle that show FLOE1 condensation as a function of salt concentration. Mean ± SEM. Four-parameter dose-response fit. (J) Quantification of the percentage of cells per radicle that show FLOE1 nuclear localization as a function of salt concentration. Mean ± SEM. Gaussian fit. (K) FLOE1-GFP condensation exhibits reversibility between high and no salt treatment. Radicles are shown. (L) Scheme highlighting different FLOE1 behaviors upon imbibition.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: MANN-WHITNEY, Concentration Assay

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Tissue-specific expression of FLOE1 derived from ePlant . (B) RT-qPCR analysis of different developmental stages shows peak expression in mature dry seeds, and a decrease in expression upon imbibition. “Dark”, “green” and “yellow” refer to the maturation stages of the siliques (from younger to older), which roughly correspond to 4-7, 8-10 and 11-13 days post-anthesis , and “imbibed” corresponds to seeds that were imbibed in sterile double-distilled water for 24 h. Col-0 (WT) plants were used. One-way ANOVA. **** p-value < 0.0001. Mean ± SD shown. (C) Expression of FLOE1 in developing embryos detected by GUS staining in FLOE1p:FLOE1-GUS transgenic lines.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Sterility, Staining, Transgenic Assay

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) YFP-FLAG localizes diffusely with modest nuclear enrichment in Arabidopsis torpedo stage embryos without any condensates forming. (B) GFP localizes diffusely with modest nuclear enrichment in imbibed dry seed-derived embryo radicles without any condensates forming. (C) Autofluorescence of protein storage vacuoles in non-transgenic control plants is much weaker in the hydrated state. (D) Dissection in glycerin does not alter the presence of FLOE1-GFP condensates throughout embryonic development before desiccation. For the mature stage, a zoom in of the radicle is shown. (E) Cycloheximide (CHX) treatment does not prevent FLOE1-GFP condensate formation in imbibed embryo radicles. (F-G) Incubation of FLOE1-GFP embryos in osmolyte solutions prevents FLOE1 condensate formation. Mannitol: Mann-Whitney. Sorbitol: One-way ANOVA Kruskal-Wallis. **** p-value < 0.0001. a.u. = arbitrary units.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Derivative Assay, Transgenic Assay, Control, Dissection, Incubation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Recombinant MBP-FLOE1 phase separates in the test tube upon MBP cleavage with TEV protease. Irregular small aggregates can be seen pre-cleavage highlighting FLOE1 aggregation-propensity. (B) FLOE1 domain structure. CC = predicted coiled coil, DUF = DUF1421. Balloon plots show amino acid composition of the disordered domains. (C-D) Expression of FLOE1 domain deletion mutants in tobacco leaves (C) and human U2OS cells (D). V = vacuole, C = cytoplasm, N = nucleus. (E) Summary of FLOE1 behavior in tobacco leaves and human cells. (F) Chimeric proteins containing both the FLOE1 nucleation domain and the PrLDs from FLOE1 (QPS) or the human FUS protein form cytoplasmic condensates. Percentages display number of cells lacking or containing condensates. Average of three experiments. Arrowheads point at cytoplasmic condensates. (G) The number of QPS tyrosine residues alters FLOE1 phase separation in human cells and tobacco leaves. (H) FLOE1 phase diagram as a function of concentration and number of QPS tyrosines. (I) Number of QPS tyrosines affects intracondensate FLOE1 dynamics. Mobile fraction as assayed by FRAP is shown. One-way ANOVA. (J-K) QPS tyrosine-phenylalanine and tyrosine-tryptophan substitutions alter condensate morphology (J) and intracondensate dynamics compared to WT (K). One-way ANOVA. (L) DS deletion or DS tyrosine/phenylalanine-serine substitutions alter condensate morphology. (M) TEM shows that mutant DS FLOE1 condensates have filamentous substructure that is absent in the WT. U2OS cells. (N) DS tyrosine/phenylalanine-serine substitutions alter intracondensate dynamics. Student’s t-test. (O) DS tyrosine/phenylalanine-serine substitutions alter condensate morphology. Mann-Whitney. (P) Scheme summarizing synergistic and opposing roles of FLOE1 domains on the material property spectrum. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. Purple band denotes WT mean ± SD (I, K, N, O)

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Recombinant, Expressing, Concentration Assay, Mutagenesis, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Size exclusion chromatography of FLOE1-MBP. For more details about Peaks # 1 and 2, see the Materials and Methods section. (B) Differential interference contrast (DIC) images of FLOE1-MBP before (left) and after (right) TEV cleavage. Some irregularly shaped aggregates can be seen in the starting material, but otherwise the sample has no droplets. After removal of the solubilizing protein, MBP, through TEV cleavage, FLOE1 forms spherical droplets indicative of liquid-liquid phase separation (LLPS). Scale bars are 10 μm. (C) SDS-PAGE confirms that TEV cleavage reaction was successful and shows the composition of the droplets observed in (B). Lane 1: FLOE1-MBP uncleaved construct (Peak #2). Lane 2: Sample from Peak #2 following 20 hours of incubation with TEV. To see the composition of the droplets and ensure they are primarily composed of FLOE1, we spun down the sample for 10 minutes under 20,000 g and separated the supernatant from the pellet. Lane 3: Pellet shows strong FLOE1 enrichment and very little MBP. Lane 4: Supernatant shows that both FLOE1 and MBP are present. The existence of a FLOE1 band in the supernatant indicates an equilibrium between the dilute and dense phases of FLOE1, as expected in LLPS. The strong MBP band indicates that these FLOE1 droplets selectively exclude MBP. Lane 5: TEV.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Size-exclusion Chromatography, SDS Page, Construct, Incubation

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Domain architecture of FLOE1 with repetitively spaced aromatic residues highlighted in blue. (B) Sequences of amino acid substitution mutants. Yellow represents serine substitutions and green represents aromatic residue substitutions. Original aromatic residues are shown in blue.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Residue

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A-B) Correlative light-electron microscopy (CLEM) allows us to confirm the presence of GFP-FLOE1 in cytoplasmic condensates in transfected U2OS cells. (A) Shows wildtype FLOE1 condensates, whereas (B) shows mutant DS domain (8xYF-S) condensates. (C-D) Standard transmission electron microscopy of wildtype (C) and mutated DS domain (D) FLOE1 condensates. Zooms of (C-D) can be found in . (D) Highlights filamentous substructure of mutated DS FLOE1 condensates.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Electron Microscopy, Transfection, Mutagenesis, Transmission Assay

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Absence of FLOE1 does not affect seed characteristics. Mann-Whitney. ns = not significant (B) Absence of FLOE1 does not affect germination under standard conditions. Mean ± SEM. Four-parameter dose-response fit. Two-way ANOVA with Šidák correction. Representative of two independent experiments. (C) Increased germination of floe1-1 knockout line under water stress is rescued by WT FLOE1 complementation (+WT). Mean ± SEM. One-way ANOVA. Representative of three independent experiments. (D) Different FLOE1 WT complemented lines with different expression levels, as assayed by RT-qPCR, show dose-dependent effect of FLOE1 function on germination under salt stress. Mean ± SEM. Linear regression. (E) Two CRISPR-Cas9 FLOE1 mutant lines show enhanced germination under varying salt stress conditions. Mean ± SEM. Four-parameter dose-response fit. Two-way ANOVA. Representative of two independent experiments. (F) Four CRISPR-Cas9 FLOE1 mutants lines show enhanced germination under salt stress. Mean ± SEM. One-way ANOVA. Representative of three independent experiments. (G) Both WT and floe1-1 seedlings show developmental defects upon germination under salt stress. floe1-1 picture is the same as in and is shown for comparison. (H) Quantification of FLOE1 condensate formation upon alleviation from salt stress. Horizontal line indicates cut-off for FLOE1 condensation (see Material and Methods). Mann-Whitney. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. MS = Murashige and Skoog medium (standard conditions). a.u. = arbitrary units.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: MANN-WHITNEY, Knock-Out, Expressing, Quantitative RT-PCR, CRISPR, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) floe1-1 seeds show higher germination rates under salt stress. Two-way ANOVA. Four-parameter dose-response fit. Representative of two independent experiments. (B) Seedlings show developmental defects under salt stress. Three-week-old floe1-1 seedlings are shown (wildtype shown in ). (C) Seeds retain full germination potential under standard conditions after a 15-day 230mM salt stress treatment. Representative of two independent experiments. (D) FLOE1 condensates are largely absent in ungerminated seeds after 15 days of incubation under salt stress. FLOE1 condensates appear within two hours after transfer to standard conditions (MS medium). (E) Scheme highlighting position of tested FLOE1 mutants on the material properties spectrum. (F) Representative images of floe1-1 mutants complemented with ΔQPS, ΔDUF and ΔDS forms of FLOE1 upon dissection in water. Embryo radicles are shown. (G) Close-up images of WT and mutant FLOE1 condensates. Embryo radicles are shown. (H) Quantification of FLOE1 condensate size. One-way ANOVA. (I) ΔDS FLOE1 condensates are not dependent on hydration. Embryo radicles are shown. (J) Germination rate of WT, floe1-1 and complemented lines. One-way ANOVA. Representative of three independent experiments. (K) Scheme highlighting FLOE1’s role in regulating germination and the effect of mutants with altered material properties. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Incubation, Dissection, Mutagenesis

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A-B) Since FLOE1 is a dosage-dependent regulator of seed germination under water stress, we wanted to rule out that expression differences in the mutant lines would be responsible for the observed differences in their germination rates. We assayed FLOE1 expression levels in dry seeds via RT-qPCR (A). As shown before, there was a linear correlation between FLOE1 expression level and the germination rate (B). floe1-1 lines complemented with the ΔDUF mutant followed a similar trend, confirming that the DUF domain deletion does not affect germination in our assays (B). floe1-1 lines complemented with the ΔDS mutant showed low levels of transgene expression according to RT-qPCR (A, Right Panel. One-way ANOVA. *** p-value < 0.001. Mean ± SEM.) which was consistent with the sparser localization of the protein in radicles . Yet, despite these low expression levels, the ΔDS complemented lines consistently induced extreme germination rates, which we never observed for floe1-1 or WT complemented lines. floe1-1 lines complemented with the ΔQPS mutant showed high levels of transgene expression according to RT-qPCR (B). Despite these high transgene levels, and robust protein expression in radicles , ΔQPS complemented lines had germination rates similar to the parental floe1-1 line, in stark contrast with WT complemented lines with higher relative expression, supporting the loss-of-function phenotype of this mutant. B: Mean ± SEM. Germination data are representative of three independent experiments. (C) All complemented lines are able to fully germinate under standard conditions (43.5h time point shown) Mean ± SEM. Representative of two independent experiments. (D) ΔDUF and ΔQPS complemented lines have similar germination rates as WT complemented lines. In contrast, ΔDS complemented lines show faster germination rates under standard conditions. Mean ± SEM. Two-way ANOVA. Average of 3-4 independent transgenic lines.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Transgenic Assay

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A-B) Arabidopsis thaliana has two FLOE1 isoforms. FLOE1.2 is missing most of the DS-rich region (A) and has larger condensates than FLOE1.1 in tobacco leaves (B). [446-459] granules over 3 biological replicates. Mann-Whitney. (C) The large FLOE1.2 condensates recruit FLOE1.1. (D) FLOE1 has two A. thaliana paralogs that form larger condensates in tobacco leaves. [35-172] granules over 3 biological replicates. Mann-Whitney. (E) A species tree of the plant kingdom with example species and their number of FLOE homologs. (F) Gene tree of FLOE homologs. Numbers correspond to A. thaliana FLOE1, FLOE2 and FLOE3. (G) Distribution of DS and QPS length differences between the FLOE1-like and FLOE2-like clades among monocots and eudicots. Mann-Whitney. (H) Examples of FLOE homologs from across the plant kingdom. N denotes nuclear localization. For full species names for (E,F) see .

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Hydration-dependent phase separation of a prion-like protein regulates seed germination during water stress

doi: 10.1101/2020.08.07.242172

Figure Lengend Snippet: (A) Venn diagram showing differentially expressed genes (DEGs) between wildtype and floe1-1 seeds under different conditions: dry seed (dry), normal imbibition (water), imbibition in 220 mM NaCl (salt stress). (B) Word cloud showing enrichment of GO or KEGG terms for DEGs under salt stress. Red terms are associated with floe1-1 upregulated DEGs, black terms are associated with wildtype upregulated (or floe1-1 downregulated) DEGs. Font size is proportional to –log10(p-value). The only KEGG pathway enriched for the WT was “ribosome” (p-value = 3.88E-17, not shown). (See also Table S2). (C) floe1-1 seeds show a decreased germination potential upon aging. Average germination rates of seeds from five plants per genotype. Mean ± SEM. Four-parameter dose-response fit. Two-way ANOVA. ** p-value < 0.01.

Article Snippet: FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression (Table S3) and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).

Techniques: