Figure 2.
Figure 2. Chemically induced internalization is via CCVs. (A) Bright green spots formed by chemically induced endocytosis colocalize with clathrin and contain anti-CD8–Alexa 647. Cells coexpressing mCherry–LCa, CD8–dCherry–FRB, and either FKBP–β2–GFP or GFP–FKBP were incubated with anti-CD8–Alexa 647 to label the membrane anchor extracellularly, and rapamycin (200 nM) was applied. Insets show a 2× zoom of the boxed region; intense orange bar indicates rapamycin application. Bars: (main) 10 µm; (insets) 2 µm. (B) Live-cell confocal immunolabeling experiments of cells expressing CD8–dCherry–FRB (dark mCherry variant) with FKBP–β2–GFP. Cells were also transfected with siRNAs against GL2 (control) or CHC. Inhibition of uptake of transferrin–Alexa 647 (A647-Tf; blue) was used as a functional test of knockdown efficacy. Bar, 10 µm. (C–G) CLEM to study the uptake of immunolabeled CD8–dCherry–FRB in cells coexpressing either FKBP–β2–GFP or GFP–FKBP. Results from a single experiment are shown, although CLEM experiments were performed three times. (C) Stills from live-cell wide-field imaging showing cells sequentially labeled with anti-CD8 and Alexa 546 FluoroNanoGold-conjugated secondary antibody (FNG; red), before and after the addition of rapamycin (200 nM). The same cell was processed for electron microscopy and imaged. Bars: (main) 10 µm; (zoom) 2 µm. (D) Electron micrographs of the cells shown in A. Clear uptake of NanoGold into CCVs was seen in cells coexpressing FKBP–β2–GFP but not GFP–FKBP. Bars, 100 nm. (E) Segmentation of CCV profiles in rapamycin-treated FKBP–β2–GFP versus GFP–FKBP samples. Membrane (gray) and coat (purple) for multiple vesicles are shown overlaid. Note that CCVs in FKBP–β2–GFP contained NanoGold, whereas GFP–FKBP CCVs did not. (F) Scatterplot of CCV diameters in FKBP–β2–GFP versus GFP–FKBP samples. Diameter refers to the mean of the major and minor axes. (G) Scatterplot of ellipticity of CCV profiles in FKBP–β2–GFP versus GFP–FKBP samples. Ellipticity is the ratio of semimajor and semiminor axis. The p-values from Student’s t test with Welch’s correction are shown. Dots indicate CCVs and bars indicate mean ± SD.

Chemically induced internalization is via CCVs. (A) Bright green spots formed by chemically induced endocytosis colocalize with clathrin and contain anti-CD8–Alexa 647. Cells coexpressing mCherry–LCa, CD8–dCherry–FRB, and either FKBP–β2–GFP or GFP–FKBP were incubated with anti-CD8–Alexa 647 to label the membrane anchor extracellularly, and rapamycin (200 nM) was applied. Insets show a 2× zoom of the boxed region; intense orange bar indicates rapamycin application. Bars: (main) 10 µm; (insets) 2 µm. (B) Live-cell confocal immunolabeling experiments of cells expressing CD8–dCherry–FRB (dark mCherry variant) with FKBP–β2–GFP. Cells were also transfected with siRNAs against GL2 (control) or CHC. Inhibition of uptake of transferrin–Alexa 647 (A647-Tf; blue) was used as a functional test of knockdown efficacy. Bar, 10 µm. (C–G) CLEM to study the uptake of immunolabeled CD8–dCherry–FRB in cells coexpressing either FKBP–β2–GFP or GFP–FKBP. Results from a single experiment are shown, although CLEM experiments were performed three times. (C) Stills from live-cell wide-field imaging showing cells sequentially labeled with anti-CD8 and Alexa 546 FluoroNanoGold-conjugated secondary antibody (FNG; red), before and after the addition of rapamycin (200 nM). The same cell was processed for electron microscopy and imaged. Bars: (main) 10 µm; (zoom) 2 µm. (D) Electron micrographs of the cells shown in A. Clear uptake of NanoGold into CCVs was seen in cells coexpressing FKBP–β2–GFP but not GFP–FKBP. Bars, 100 nm. (E) Segmentation of CCV profiles in rapamycin-treated FKBP–β2–GFP versus GFP–FKBP samples. Membrane (gray) and coat (purple) for multiple vesicles are shown overlaid. Note that CCVs in FKBP–β2–GFP contained NanoGold, whereas GFP–FKBP CCVs did not. (F) Scatterplot of CCV diameters in FKBP–β2–GFP versus GFP–FKBP samples. Diameter refers to the mean of the major and minor axes. (G) Scatterplot of ellipticity of CCV profiles in FKBP–β2–GFP versus GFP–FKBP samples. Ellipticity is the ratio of semimajor and semiminor axis. The p-values from Student’s t test with Welch’s correction are shown. Dots indicate CCVs and bars indicate mean ± SD.

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