Recruitment of CD63 to Cryptococcus neoformans phagosomes requires acidification

Abstract

The subcellular localization of the cluster of differentiation 63 (CD63) tetraspanin and its interaction with the class II MHC antigen presentation pathway were examined in the context of phagocytosis by live cell imaging, by using monomeric red fluorescent protein-tagged mouse CD63 expressed in primary bone marrow-derived cell cultures. Upon phagocytosis of Cryptococcus neoformans and polystyrene beads, CD63 was recruited selectively to C. neoformans-containing phagosomes in a MyD88-independent acidification-dependent manner. Bead-containing phagosomes, within a C. neoformans-containing cell, acidified to a lesser extent and failed to recruit CD63 to a level detectable by microscopy. CD63 recruitment to yeast phagosomes occurred independently of class II MHC and LAMP-1. These observations indicate that the composition of distinct phagosomal compartments within the same cell is determined by phagosomal cargo and may affect the outcome of antigen processing and presentation.

Fig. 1.

CD63 fusion proteins mature normally. (A and B) RAW 264.7 cells were transduced to stably express mouse CD63-mRFP1 or HA-CD63. Cells were pulse-labeled for 15 min with [³⁵S]cysteine and methionine and chased for 0, 90, or 180 min (A) or labeled for 3 h (B). Cells were lysed in 1% Brij58 and proteins immunoprecipitated with anti-mRFP1 or anti-HA antibody. At each time point, half of the sample was treated with endoglycosidase H (A) or F (B) for 1 h at 37°C. Samples were run on a 12% SDS-polyacrylamide gel and polypeptides visualized by autoradiography. The “CHO” labels indicate the number of N-linked glycans and complex-type sugars (C.T.) present. (C) Human astrocytoma U373 cells expressing mouse CD63-mRFP1 were fixed, permeabilized, and stained with anti-human CD63 antibody. Localization of mouse CD63-mRFP1 (Left) and human CD63 (Center) and the merged image (Right).

Fig. 2.

CD63 and class II MHC are recruited selectively to yeast phagosomes. (A) B6-derived bone marrow cultures expressing CD63-mRFP1 were exposed to “dragon green” polystyrene beads. Still images show CD63-mRFP1 distribution in the cell. (B) Bone marrow cells from B6 mice expressing CD63-mRFP1 (Left) or from class II MHC-eGFP-expressing mice (Right) were incubated with CN. The images display the phagosomal distribution of CD63-mRFP1 and class II MHC-eGFP. (C) B6-derived bone marrow cultures were transduced with CD63-mRFP1 and incubated with both CN and dragon green polystyrene beads. An example of CD63-mRFP1 distribution in a cell having taken up two yeast, and one bead is shown. The far-right bright-field image demonstrates the presence of CN within the cell. (Scale bars, 10 μm.)

Fig. 3.

Class II MHC recruitment to the phagosome precedes that of CD63. (A) Within 10 min after addition of CN to bone marrow cultures derived from class II MHC-eGFP mice and transduced to express CD63-mRFP1, phagosomes acquire both CD63-mRFP1 and class II MHC (Left, CD63-mRFP1; Center, class II MHC; and Right, merge). (Scale bar, 10 μm.) (B) Time-lapse images in both red and green channels show that class II MHC-eGFP appears around the phagosomes (indicated by the arrows) slightly before CD63-mRFP1. (Scale bar, 5 μm.) (C) Kinetic analysis of fluorescence acquisition around phagosomes. Each data point represents the average intensity measured at each time point for at least nine independent Cap59−/− CN phagocytic events for CD63-mRFP1 (■) and class II MHC-eGFP (○) recruitment. The linear fits indicate the initial rates of recruitment for each protein to the phagosome.

Fig. 4.

CD63 recruitment and phagosome acidification coincide. Bone marrow cultures derived from B6 mice and transduced to express CD63-mRFP1 were incubated with CN and the pH indicator, Lysosensor. (A) Time-lapse imaging of phagosome acidification as determined by Lysosensor recruitment (Upper) and of CD63-mRFP1 recruitment (Lower) are shown. Representative images have been selected at 0, 200, and 500 sec. (Scale bar, 5 μm.) (B) Colocalization of Lysosensor (Left) and of CD63-mRFP1 (Center) in the CN phagosome after at least 10-min incubation is apparent in the merged image (Right). (Scale bar, 10 μm.)

Fig. 5.

Phagosomal recruitment of CD63, but not class II MHC or LAMP-1, is blocked by inhibition of vacuolar ATPase. Bone marrow-derived cell cultures from B6 mice were lentivirally transduced with CD63-mRFP1 or LAMP1-GFP. Alternatively, bone marrow cultures were derived from class II MHC-eGFP mice. (A) Cells were incubated with CN in the absence or presence of 1 μM bafilomycin and followed for CD63-mRFP1 (Left), class II MHC-eGFP (Center), or LAMP1-GFP (Right) recruitment. Images shown were taken after 30-min incubation. (Scale bars, 5 μm.) (B) B6 bone marrow cells were transduced with CD63-mRFP1 and paraformaldehyde-fixed. Analysis by immunofluorescence with anti-mRFP1 (Left) and LAMP-1 (Center) antibodies shows colocalization of these two molecules (Right). (Scale bars, 10 μm.)