Table 1.
Constructs used in experiments and their amino acid composition.
Figure 1.
UNC93B1 is localized in the ER and in the lysosomes.
(A–D) HEK293T cells were transfected with a UNC93B1-mCherry-Myc (cyan). (A) ER was dyed with ER-Tracker Blue-White DPX. (B) Lysosomes were marked with LysoTracker Green DND-26. (C) Endosomes were stained with Transferrin AlexaFluor 633 conjugate. (D) Plasma membrane was dyed with CTB-Alexa 647. All dyes are shown in magenta. White arrows indicate colocalization. (E) HEK293T cells were transfected with a UNC93B1-mCherry-Myc (magenta) and TLR3-mCer (cyan). Plasma membrane was dyed with CTB-Alexa 647 (red). Membrane localization was evaluated from plots of normalized fluorescence intensities of UNC93B1-mCherry-Myc and TLR3-mCer and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.
Figure 2.
Poly(I:C) and TLR3 colocalize at the surface of plasma membrane.
HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and Renilla reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of cytochalasin D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.
Figure 3.
Response of chimeric constructs between TLR3 and TLR9.
(A) Schematic representation of chimeric constructs where the transmembrane segments or cytoplasmic domains of human TLR3 and TLR9 receptors have been exchanged. (B, C) HEK293T cells were transfected with TLR3, TLR3-9-3 and TLR3-3-9 (B) or TLR9, TLR9-3-9 and TLR9-9-3 (C). Western blot was performed using anti-TLR3 or anti-HA antibodies. Anti–β-actin antibodies were used as a loading control. The representative data from three experiments are shown. (D-E) HEK293 cells were transiently transfected with TLR3, TLR9 or chimeric constructs TLR3-9-3, TLR3-3-9, TLR9-3-9, and TLR9-9-3 alone or with UNC93B1. Cells were transfected with IFN-β (D, F) or NF-κB (E, G) promoter reporter plasmids and Renilla reporter plasmid. After 18 h of stimulation with poly(I:C) (10 μg/ml) (D, E) or ODN10104 (10 μg/ml) (F, G), luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.
Table 2.
Responsiveness and fold induction after UNC93B1 overexpression of constructs containing TLR3 and TLR9 domains.
Figure 4.
Localization of chimeric receptors with exchanged TM segments of TLR3 and TLR9.
HEK293T cells were transiently transfected TLR3-9-3-mCer (A and B - cyan), TLR9-3-9-YFP (C and D - yellow), and with UNC93B1. Plasma membrane markers SynaptoRed and CTB-Alexa 555 are shown in magenta. (A) Localization of TLR3-9-3-mCer on plasma membrane in cells overexpressing UNC93B1. (B) Intracellular localization of TLR3-9-3-mCer in HEK293T without overexpression of UNC93B1. (C) Intracellular localization of TLR9-3-9-YFP in cells overexpressing UNC93B1. (D) Intracellular localization of TLR9-3-9-YFP in HEK293T without overexpression of UNC93B1. (A-D) Data are representative of three experiments. TLR membrane localization was evaluated from plots (bottom) of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.
Figure 5.
Localization of chimeric receptors with exchanged cytosolic domains of TLR3 and TLR9.
HEK293T cells were transiently transfected TLR3-3-9-YFP (A and B - yellow) or TLR9-9-3-YFP (C and D - yellow) and with UNC93B1. Plasma membrane markers SynaptoRed and CTB-Alexa 555 are shown in magenta. (A) Localization of TLR3-3-9-YFP on plasma membrane in cells overexpressing UNC93B1. (B) Intracellular localization of TLR3-3-9-YFP in HEK293T without overexpression of UNC93B1. (C) Intracellular localization of TLR9-9-3-YFP in cells overexpressing UNC93B1. (D) Intracellular localization of TLR9-9-3-YFP in HEK293T without overexpression of UNC93B1. (A–D) Data are representative of three experiments. TLR membrane localization was evaluated from plots (bottom) of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.
Figure 6.
Structural alterations do not affect translocation of chimeric receptors from ER to endosomes and lysosomes.
HEK293T cells were transiently transfected with TLR3-mCer, TLR9-YFP, TLR3-9-3-mCer, TLR9-3-9-YFP, TLR3-3-9-YFP or TLR9-9-3-YFP. ER was dyed with ER-Tracker Red, lysosomes were marked with LysoTracker Red DND-99. To stain endosomes, cells were cotransfected with EEA1-Tomato. All dyes are shown in magenta. White arrows indicate colocalization. Images are selected from three independent experiments. Scale bars, 10 μm.