Figure 1
Figure 1. HIV-1 Nef targets Lck to the TGN and RE and prevents IS recruitment of the kinase. Scale bars indicate 10 μm. (A) Shown are representative confocal micrographs of Jurkat T lymphocytes transiently expressing GFP or Nef.GFP after staining for endogenous Lck. (B) 3D deconvolution of confocal micrographs scanning through Jurkat T lymphocytes expressing Lck.GFP in the presence of RFP (control) or Nef.RFP (+ Nef.RFP). (C) Quantification of Lck distribution in single cells. Depicted are the percentages of the total per-cell Lck signal detected in intracellular accumulation (see also supplemental Figure 1A). Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analyzed. (D) Representative confocal micrographs of Jurkat T lymphocytes expressing Lck.GFP in the absence (control) or presence of Nef.myc (+ Nef.myc). Lck is shown in green and subcellular markers in red. Subcellular markers were detected with staining by the respective Ab for endogenous (end.) proteins or by the fluorescent tag of coexpressed marker proteins. GalT.CFP is a Golgi marker; PDI, ER marker; EEA1,EE marker; Rab11.GFP and TfR, RE marker; TGN38.GFP, TGN marker; and LAMP1.GFP, lysosome marker. (E) Quantification of Lck colocalization with subcellular markers. Depicted are Manders coefficients of Lck overlapping with the indicated subcellular markers. Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analyzed. (F) Shown are representative merged micrographs of Jurkat T lymphocytes expressing GFP or the indicated Nef.GFP fusion proteins (green) in conjugates with SEE-pulsed Raji B cells (blue). Endogenous Lck is depicted in red. Note that expression of Nef prevents polarization of Lck to the IS and instead induces targeting of Lck to RE/TGN compartments. (G) Frequencies of RE/TGN accumulation versus IS recruitment of Lck on the expression of the indicated GFP fusion proteins. Values are the arithmetic means of at least 3 independent experiments ± SD, in which more than 100 conjugates were analyzed for the predominant localization of Lck per condition. Micrographs for those Nef proteins not shown in panel F are depicted in supplemental Figure 3A. (H) Micrographs of primary human T lymphocytes infected with WT ΔNef HIV-1 IRES.GFP reporter viruses (infected cells are shown in green). (I) Frequencies of RE/TGN accumulation and IS recruitment of Lck in HIV-1–infected primary human T lymphocytes from 2 donors.

HIV-1 Nef targets Lck to the TGN and RE and prevents IS recruitment of the kinase. Scale bars indicate 10 μm. (A) Shown are representative confocal micrographs of Jurkat T lymphocytes transiently expressing GFP or Nef.GFP after staining for endogenous Lck. (B) 3D deconvolution of confocal micrographs scanning through Jurkat T lymphocytes expressing Lck.GFP in the presence of RFP (control) or Nef.RFP (+ Nef.RFP). (C) Quantification of Lck distribution in single cells. Depicted are the percentages of the total per-cell Lck signal detected in intracellular accumulation (see also supplemental Figure 1A). Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analyzed. (D) Representative confocal micrographs of Jurkat T lymphocytes expressing Lck.GFP in the absence (control) or presence of Nef.myc (+ Nef.myc). Lck is shown in green and subcellular markers in red. Subcellular markers were detected with staining by the respective Ab for endogenous (end.) proteins or by the fluorescent tag of coexpressed marker proteins. GalT.CFP is a Golgi marker; PDI, ER marker; EEA1,EE marker; Rab11.GFP and TfR, RE marker; TGN38.GFP, TGN marker; and LAMP1.GFP, lysosome marker. (E) Quantification of Lck colocalization with subcellular markers. Depicted are Manders coefficients of Lck overlapping with the indicated subcellular markers. Each symbol designates a value for an individual cell. Bars indicate the mean values of all cells analyzed. (F) Shown are representative merged micrographs of Jurkat T lymphocytes expressing GFP or the indicated Nef.GFP fusion proteins (green) in conjugates with SEE-pulsed Raji B cells (blue). Endogenous Lck is depicted in red. Note that expression of Nef prevents polarization of Lck to the IS and instead induces targeting of Lck to RE/TGN compartments. (G) Frequencies of RE/TGN accumulation versus IS recruitment of Lck on the expression of the indicated GFP fusion proteins. Values are the arithmetic means of at least 3 independent experiments ± SD, in which more than 100 conjugates were analyzed for the predominant localization of Lck per condition. Micrographs for those Nef proteins not shown in panel F are depicted in supplemental Figure 3A. (H) Micrographs of primary human T lymphocytes infected with WT ΔNef HIV-1 IRES.GFP reporter viruses (infected cells are shown in green). (I) Frequencies of RE/TGN accumulation and IS recruitment of Lck in HIV-1–infected primary human T lymphocytes from 2 donors.

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