Journal: Journal of molecular biology

Article Title: The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

doi: 10.1016/j.jmb.2023.168182

Figure Lengend Snippet: Figure 5. In vivo droplet fusion and fission of WT Gag and Gag.L219A. One characteristic of BMCs is that they are dynamic and can undergo fusion or fission over time. (A) Using a single confocal z-plane, live-cell timelapse imaging was conducted (1 frame/min) in a QT6 cell expressing WT Gag-SNAPTag bound to SNAP-ligand conjugated to JF549. The nucleus was outlined in a white dashed line. Several WT Gag-SNAPTag foci were observed to undergo fusion or fission in different cellular compartments: nucleus (foci indicated by white arrows), cytoplasm (yellow arrows), and at the plasma membrane (magenta arrows). Droplets of interest were also displayed at higher magnification (inset). See also Supplementary Movies 3a, 3b, and 3c for timelapse images. (B) A single optical Z- plane of a QT6 cell expressing the Gag.L219A-YFP nuclear export mutant was imaged using confocal timelapse microscopy at one frame every minute. Over a period of two minutes, three different pairs of droplets underwent fusion. The nucleus is outlined with a white dotted-line. Scale bar = 2 mm. See also Supplementary Movie 4 for timelapse images.

Article Snippet: The FUS.IDR protein sequence used was: MASNDYTQQATQSYGAYPTQPGQGYSQQSS QPYGQQSYSGYSQSTDTSGYGQSSYSSYGQS QNTGYGTQSTPQGYGSTGGYG SSQSSQSSYG QQSSYPGYGQQPAPSSTSGSYGSSSQSSSYG QPQSGSYSQQPSYGGQQQSYGQQQSYNPPQ GYGQQNQYNSSSGGGGGGGGGGNYGQDQS SMSSGGGSGGGYGNQDQSGGGGSGGYGQQ DRGGRGRGGS GGGGGGGGGGYNRSSG The HNRNPA1.IDR protein sequence used was: MASASSSQRGRSGSGNFGGGRG GGFGGND NFGRGGNFSGRGGFGGSRGGGGYGGSG DGY NGFGNDGSNFGGGGSYNDFGNYNNQSSNFGP MKGGNFGGRSSGPYGGGGQYFAK PRNQGGY GGSSSSSSYGSGRRF Cell lines, transfection, fixation Chemically transformed quail fibroblast QT6 cells, which are a natural host of RSV, which infects domesticated fowl,129 were maintained as described.130 HEK293T cells obtained from ATCC (CRL-3216) were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% FBS, 1% sodium pyruvate, penicillin/streptomycin and fungizone at 37 C. Cells were seeded onto coverslips for fixed cells or on MatTek dishes for live-cell imaging.

Techniques: In Vivo, Imaging, Expressing, Clinical Proteomics, Membrane, Mutagenesis, Microscopy

Journal: Journal of molecular biology

Article Title: The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

doi: 10.1016/j.jmb.2023.168182

Figure Lengend Snippet: Figure 10. In vivo and in vitro perturbation of BMCs with 1,6-hexanediol. A characteristic of BMCs is disruption by 1,6-hexandiol. QT6 cells expressing the indicated constructs (A) were incubated with 10% 1,6-hexanediol for 1 min and in vitro droplets (B) were treated for 10 min. (A) Panel a. NONO-YFP foci were disrupted by 1,6-hexanediol to a minor degree in QT6 cells. Panels b, c, and d. WT RU5.Gag-YFP and nuclear-restricted Gag foci became diffuse throughout the entire cell following treatment, indicating that Gag foci were sensitive to disruption in all cellular compartments. Scale bar = 2 mm. (B) The effect of 1,6-hexanediol on in vitro droplets was observed on recombinant WT Gag, MAp2p10-NTD, and NC proteins. 1,6-hexanediol (10%) was mixed with 5 mM of each protein in the presence of Ficoll-400 and NaCl, then incubated for 10 min before imaging. For better display of the NC droplets visualized using DIC, the image was overlaid with spheres generated by the spot function in Imaris (magenta). Scale bar = 5 mm. (C) and (D) 10 low-magnification fields were collected for each condition and subjected to the Imaris spot function to measure the number and size of droplets. In all cases, 1,6-hexanediol treatment induced statistically significant changes.

Article Snippet: The FUS.IDR protein sequence used was: MASNDYTQQATQSYGAYPTQPGQGYSQQSS QPYGQQSYSGYSQSTDTSGYGQSSYSSYGQS QNTGYGTQSTPQGYGSTGGYG SSQSSQSSYG QQSSYPGYGQQPAPSSTSGSYGSSSQSSSYG QPQSGSYSQQPSYGGQQQSYGQQQSYNPPQ GYGQQNQYNSSSGGGGGGGGGGNYGQDQS SMSSGGGSGGGYGNQDQSGGGGSGGYGQQ DRGGRGRGGS GGGGGGGGGGYNRSSG The HNRNPA1.IDR protein sequence used was: MASASSSQRGRSGSGNFGGGRG GGFGGND NFGRGGNFSGRGGFGGSRGGGGYGGSG DGY NGFGNDGSNFGGGGSYNDFGNYNNQSSNFGP MKGGNFGGRSSGPYGGGGQYFAK PRNQGGY GGSSSSSSYGSGRRF Cell lines, transfection, fixation Chemically transformed quail fibroblast QT6 cells, which are a natural host of RSV, which infects domesticated fowl,129 were maintained as described.130 HEK293T cells obtained from ATCC (CRL-3216) were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% FBS, 1% sodium pyruvate, penicillin/streptomycin and fungizone at 37 C. Cells were seeded onto coverslips for fixed cells or on MatTek dishes for live-cell imaging.

Techniques: In Vivo, In Vitro, Disruption, Expressing, Construct, Incubation, Recombinant, Imaging, Generated