Fig. 3: Efficient FG phase entry of HIV-1 capsid spheres.
From: HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor
a,b, FG phases were assembled by phase-separating a well-characterized, cohesive GLFG repeat domain (ref. 50; Supplementary Fig. 1 for FG-domain sequences) and then probed for entry of indicated fluorescent species. Non-fused GFPs and mCherry remained firmly phase-excluded, whereas 40 nm capsid spheres accumulated to high partition coefficients. Partially assembled (hexameric) capsomere–sinGFP4a fusions showed only faint surface binding (visible in high-sensitivity scans). Phase separation was initiated by rapidly diluting FG domains (1 mM in 2 M guanidinium·HCl) with 25 volumes of buffer (50 mM Tris/HCl pH 7.5, 250 mM NaCl, 1 mM IP6), followed 5 min later by a further fourfold dilution in buffer with indicated fluorescent probes. Confocal scans were taken after another hour, when the FG particles had settled to the slide bottom. Fluorescent probes: mCherry (3 µM), free EGFP or sinGFP4a (3 μM), capsomeres (0.4 μM), 40 nm capsid spheres (10 nM, with 15% EGFP- or sinGFP4a-labelled CA protomers). Scan settings were adjusted individually. c,d, Quantification of a,b but with larger datasets. For each datapoint (representing one FG particle), the integrated intraparticle signal (in) was compared to outside regions (out). Note the log-linear scale and that free GFP derivatives are well excluded. Capsid spheres accumulated to very high partition coefficients (in:out = 300–500) which are still underestimated because the outside signals were as low as the background (background fluorescence + instrumental noise) and this background was not subtracted (to avoid division by zero). The ‘in-value’ for the capsomere partitioning refers to the intraphase signal, excluding the rim. Numbers are means; bars indicate mean ± s.d. N = number of quantified FG particles and outside areas, respectively. e, FG phase entry of free mCherry and 40 nm capsid spheres was probed as in a,b but using SLFG repeats, FSFG repeats or FG repeats from T. brucei Nup158. Experiments were repeated with identical outcomes (a, n = 4; b, n = 13; e, n = 4). Scale bars, 10 μm.
