Transferrin receptor recycling in the absence of perinuclear recycling endosomes

Abstract

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Figure 1.

Tfn recycling in CHO-TfnR cell and cytoplasts. (A) Diagram of rate constants and pathways derived from the mathematical model used to interpret recycling data. (B) Fit of model to experimental data. Predicted recycling curves (blue line) were fit to recycling data (blue squares). The expected percentage of Tfn to be in EE (green line) and RE (red line) over time are shown. Note the relative distribution of Tfn at 2 and 25 min (arrows). Values for each data point were averaged and shown as the mean ± SD (n = 9). (C) CHO-TfnR cells bound with Alexa 594–Tfn on ice were chased 25 min at 37°C to label REs (red channel). Overlaid phase and fluorescence images 0, 4, 6, and 8 min into microsurgery are shown and the resulting karyoplast (k) and cytoplast (c) indicated. Panels were extracted from Video 1. (D) After microsurgery, Alexa 594–Tfn was bound to the cell surface on ice, internalized at 37°C for the indicated times before fixation. Cytoplasts (c) and the karyoplasts (k) are shown. Arrows point to the REs in karyoplasts (7 and 25 min). Cells were outlined for clarity. Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.200111048/DC1. Bar, 10 μm.

Figure 2.

Tfn passes through two spatially and temporally distinct endocytic compartments in cytoplasts. Alexa 594–Tfn was bound to the surface of normal and cut cells (on ice) and internalized for 25 min (A, to label REs) before being subjected to a final 2 min pulse of Alexa 488–Tfn (B, to label EEs). The cells were fixed in PFA, mounted, and analyzed by fluorescence microscopy. Typical labeling patterns of uncut cells are shown (u). A cytoplast and accompanying karyoplast (c and k) are shown for comparison. Cytoplasts from three independent experiments are shown in insets. Arrows indicate the compartment labeled at a longer time in cytoplasts. (C) Merged image of Alexa 594–Tfn (red, 25 min) and Alexa 488–Tfn (green, 2 min). Bars, 10 μm.

Figure 3.

Cytoplasts contain bona fide EE and RE. CHO-TfnR cells were submitted to microsurgery to create karyoplasts (k) and cytoplasts (c). Both are outlined in some cases for clarity. (A) Cytoplasts and cells internalized Alexa 594–Tfn (red) for 2 min before fixation, then labeled for EEA-1 (green). Arrows indicate colocalization of Tfn and EEA-1 (yellow). Insets in A are a magnification of the uncut cell periphery. (B) Cytoplasts generated from Rab11-GFP–expressing cells were allowed to internalize Alexa 594–Tfn (red) for 2 min before fixation. (C and D) Cells prepared as in A and B but Alexa 594-Tfn internalized for 25 min (E and F). Cells prepared as in A and B but treated with AlF₄ ⁻ during the chase period and Tfn internalized for the indicated times. Arrows indicate Tfn (red) containing endosomes. Bars, 10 μm.

Figure 4.

Rab11-GFP recruitment in cytoplasts. (A) CHO-TfnR cells expressing Rab11-GFP were submitted to microsurgery. Fluorescence images were acquired either before (Uncut), immediately after (0 min), or 45–60 min after microsurgery to assess the formation of Rab11-positive structures in the cytoplasts. Arrows indicate cytoplasts. The two cells shown are from separate experiments. (B) CHO-TfnR cells expressing Rab11-GFP were submitted to microsurgery before time-lapse video microscopy. Exposures were adjusted to visualize peripheral Rab11 structures, while overexposing perinuclear regions. One phase contrast image of a cut cell is shown. Panels are inverted images of the GFP (black on white) overlain with synchronous contrast enhanced Tfn images (red) at 0:30, 5:00, 10:00, 15:00, and 19:45 (min:s). Cytoplast (c), karyoplast (k), and uncut cell (u) are shown. Insets are a magnified, contrast-enhanced view of the boxed region of the cytoplast. Red arrows indicate RE-like structures that recruit Rab11-GFP throughout the time-course. Green arrows indicate structures in the cytoplast that initially recruit Rab11-GFP but fade to the limit of detection over time. In contrast, Rab11-GFP–positive EE remain stable in position and intensity over time in uncut cells (blue arrows). Images in B were extracted from Video 2. Video 2 is available at http://www.jcb.org/cgi/content/full/jcb.200111048/DC1. Bars, 10 μm.