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. 2018 May 10;9:1861. doi: 10.1038/s41467-018-04220-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Single-molecule tracking of HIV-1 Env trimers reveals that the Env-CT does not significantly alter diffusivity on the nanoscale, but determines the fraction of Env that is confined/immobile. a, d Single-particle tracking of WT-Env (left), CTΔ144-Env (middle), and d8-Env (right) labeled with Fab b12-Atto565 in a CEM-A cells and d COS7 cells. Scale bars are 2 μm (top) and 500 nm (red inset region, bottom). Tracks were classified as immobile and confined (dark green) versus mobile (light green). b, e Mean diffusion coefficients were computed based on individual molecular tracks in CEM-A (WT-Env, n = 1514; CTΔ144-Env, n = 9694; and d8-Env, n = 2139, each from four biological replicates) and COS7 (WT-Env, n = 274; CTΔ144-Env, n = 2238; and d8-Env, n = 1019 each from ≥4 biological replicates). b In CEM-A cells, the mean diffusion coefficients of tracks classified as mobile for the CTΔ144-Env (D_mobile = 0.149 ± 0.054 μm² s⁻¹) and d8-Env (D_mobile = 0.107 ± 0.067 μm² s⁻¹) are not significantly different when compared to WT-Env (D_mobile = 0.097 ± 0.049 μm² s⁻¹). e For COS7 cells, mean diffusion coefficients of tracks classified as mobile indicate that the CTΔ144 (D_mobile = 0.108 ± 0.053 μm² s⁻¹) and d8 mutations (D_mobile = 0.144 ± 0.087 μm² s⁻¹) do not significantly alter the diffusion rates of Env when compared to WT-Env (D_mobile = 0.085 ± 0.056 μm² s⁻¹). c, f The Env-CT dictates the confined/immobile fraction of Env on the plasma membrane. c In CEM-A cells, the confined/immobile fractions of the CTΔ144-Env and (27 ± 5%) and d8-Env (56 ± 15%) were both found to be significantly smaller relative to WT-Env (80 ± 6%). f In COS7 cells, the confined/immobile fractions of the CTΔ144-Env (57 ± 5%) and d8-Env (72 ± 13%) were significantly lower relative to WT-Env (92 ± 4%), suggesting that steric trapping of Env is dependent on the Env-CT. Error bars indicate s.d. ***P < 0.0001 and n.s. indicates not significant using one-way ANOVA with Tukey’s post-test. CEM-A (WT-Env, n = 7862; CTΔ144-Env, n = 13,338; and d8-Env, n = 5434) and COS7 (WT-Env, n = 3790; CTΔ144-Env, n = 5283; and d8-Env, n = 4384)