Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level

Abstract

IgG transport within and across cells is essential for effective humoral immunity. Through a combination of biochemical and in vivo analyses, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a central role in delivering IgGs within and across cells. However, little is known about the molecular and cellular mechanisms that are involved in the exocytosis of IgG from cells that express FcRn. Here, we use single-molecule fluorescence microscopy to analyze exocytic processes in FcRn-GFP-transfected human endothelial cells. We show that exocytosis can occur by means of multiple modes that range from complete fusion of the exocytic vesicle with the plasma membrane to a slower-release mode ("prolonged release") that only involves partial mixing of membrane contents. Even for prolonged release, diffusion of FcRn into the plasma membrane can occur, indicating that FcRn is directly involved in IgG exocytosis. The slower-release mode is characterized by periodic, stepwise release of IgG, rather than the rapid burst that is observed for complete-fusion events. Analyses of single-molecule tracks suggest that IgG may be bound to FcRn for several seconds after exocytosis. Unexpectedly, after diffusion out of the exocytic site, IgG and FcRn molecules can also migrate back into the epicenter of the release site. Such retrograde movement may represent a mechanism for FcRn retrieval. Our studies provide insight into the events that lead to IgG exocytosis.

Fig. 1.

Exocytic events in FcRn-GFP-transfected HMEC-1 cells. (A and B) Cells were pulsed with 1 mg/ml Alexa 546-labeled IgG for 60 min, were washed, and were then imaged by using dual-color TIRFM. (C) FcRn-GFP-transfected cells were imaged by using single-color TIRFM. Individual frames showing areas of interest of the cell surface are shown, with the time (in seconds) at which each frame was taken indicated (first frame is arbitrarily set to time 0, except for B, where time 0 corresponds to start of intensity plot). The exocytic sites are indicated by boxes within these images. The complete cell images from which the imagesin A and B are derived are shown in Fig. 4, which is published as supporting information on the PNAS web site). (A) Complete fusion of exocytic compartment with plasma membrane, resulting in a single release of three IgG molecules. (B) Prolonged release, with IgG-release events at 14.2, 15.8, 22.2, 48.4, 77.3, and 86.1 s. For A and B, arrows indicate individual IgG molecules (Alexa 546-labeled, red) and FcRn-GFP is green. Plots in A and B show GFP (FcRn, green) and Alexa 546 (IgG, red) fluorescence intensities at the exocytic site as a function of time for each type of release event. Vertical dashed lines indicate times at which IgG molecules emerged from the exocytic site (for B, not all IgG-release events are shown in the individual frames, but these results can be seen in Movie 2). (C) Pulses of FcRn-GFP release from an FcRn-GFP-positive compartment (release events at 10.6, 12.5 and 14.45 s). Arrows indicate single FcRn-GFP molecules. (Bars, 1 μm.) Movies 1, 2, and 3 correspond to Fig. 1 A, B, and C, respectively.

Fig. 2.

Exocytic events involving partial release of FcRn and IgG can occur. Individual frames from areas of interest are shown, with the time in seconds at which each frame was taken (first frame is arbitrarily set to time 0). The boxes show the exocytic sites. (A) Dual-color TIRFM analyses of FcRn-GFP-transfected HMEC-1 cells pulsed with 1 mg/ml Alexa 546-labeled IgG for 60 min, followed by washing. A partial release event starts at 3.4 s. Arrows indicate a red (Alexa 546) and green (GFP) fluorescent compartment that remains after exocytosis and subsequently moves out of the focal plane. (B) Single-color TIRFM of FcRn-GFP-transfected HMEC-1 cells showing release of a single FcRn-GFP molecule (7.25 s), followed by two sequential fusion events (starting at 11.75 and 17.6 s). Arrows indicate single FcRn-GFP molecules. (Bars, 1 μm.) Movies 4 and 5 correspond to Fig. 2 A and B, respectively.

Fig. 3.

Tracks of individual IgG and FcRn-GFP molecules after exocytic events. (A and B) FcRn-GFP-transfected HMEC-1 cells were pulsed in 1 mg/ml Alexa 546-labeled IgG, were washed, and were then imaged by using dual-color TIRFM. (C and D) FcRn-GFP-transfected cells were imaged by using single-color TIRFM. (A) Three tracks of single IgG molecules during a complete-fusion event superimposed on the image of the event at the moment of fusion. (B Left) Four tracks of single IgG molecules during a prolonged-release event superimposed on an image of the exocytic site. (Center and Right) The movement of IgG molecules back toward the exocytic site is shown, and the data are taken from a later stage of the prolonged-release event shown at Left. (C) Three tracks of FcRn-GFP molecules during a complete-fusion event superimposed on the image of the event at the moment of fusion. (Right) Movement of an FcRn-GFP molecule back toward the exocytic site. (D) Three tracks of single FcRn-GFP molecules during a prolonged-release event superimposed on an image of the exocytic site. (Bars, 1 μm.)