The uniformity of phagosome maturation in macrophages

Abstract

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of beta-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of phagosome labeling. Some phagosomes increased labeling quickly after phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of phagosome maturation are integrated over the entire organelle.

Figure 1.

Distribution of YFP-actin during phagocytosis. (a) Time series showing phase-contrast, the component fluorescence images of (b) CFP, (c) YFP-actin, and (d) ratio images representing YFP-actin/CFP. Each column contains component images of one time point, separated from the adjacent columns by the time, in minutes, indicated in the phase-contrast image panel. The YFP-actin/CFP ratios were high in the forming phagocytic cup but declined quickly after closure of the cup. (e) Time series of phase-contrast images from a single phagocytic event and (f) the corresponding ratio images in a cell expressing YFP and CFP. The ratio was uniform at each time point.

Figure 2.

The particle-tracking image analysis routine. The phagosome was marked in the phase-contrast image by two squares. The inner square defined the center of the object, and the outer square defined the search region. Regions of interest were positioned in the corresponding YFP, CFP, and CFP-thresholded images from the centroid of the phagosome determined above. From time 1 to time 2, the tracking routine followed the phagosome and adjusted the positions of the region of interest accordingly. For each frame, the mean fluorescence intensities, standard deviation, or fractional thresholded area were recorded for the phagosomal region of interest.

Figure 3.

Quantitative analysis of YFP-actin dynamics during phagocytosis. (a) Rate of phagosome extension in cells expressing YFP-actin and CFP was measured as the rate at which the binary mask filled the region of interest. The time point for maximal threshold was set to 5 min, and all the image stacks and corresponding data were realigned accordingly; this synchronized the measurements (e.g., the tracings shown in panel a were used to align the ratios for panel b). (b) Synchronized data from the tracking routine outputs for phagocytic events in cells expressing YFP-actin and CFP. The YFP-actin/CFP ratio in the phagosome (R_p) was normalized with respect to the YFP-actin/CFP ratio for the entire cell, yielding R_p/R_c, (diamonds). R_p/R_c and fraction thresholded area of the region of interest (circles) were plotted relative to time during pseudopod extension around the particle. Before and after extension, R_p/R_c was near 1, but this ratio increased sharply during pseudopod extension (n = 11). (c) R_p/R_c for cells expressing nonchimeric YFP and CFP (n = 5). The bars indicate SEM.

Figure 4.

Distributions of YFP-Rab5a, -Rab7, –LAMP-1, -Golgi, and -ER during phagocytosis. Each column contains component images of one time point, separated from the adjacent columns by the time, in minutes, indicated in panel a, with the exception of panel e. (a) Time series of YFP-Rab5a/CFP ratio images. The ratio increased after phagosome formation and began to decrease after 10 min. (b) Time series of YFP-Rab7/CFP ratio images. The ratio began to rise 5 min after phagosome closure and remained high throughout the observation. (c) Time series of YFP-Golgi/CFP and (d) YFP-ER/CFP ratio images. The ratios were flat at all time points. (e) Time series of YFP–LAMP-1/CFP ratio images. The ratio increased 20 min after internalization and remained high throughout the observation.

Figure 5.

Rab5a, Rab7, and LAMP-1 dynamics were consistent for all phagosomes. Synchronized data from the tracking routine outputs for phagocytic events in cells expressing (a) YFP-Rab5a and CFP (n = 10), (b) YFP-Rab7 and CFP (n = 14), (c) YFP-Rab5a and CFP-Rab7 (n = 11), and (d) YFP–LAMP-1 and CFP (n = 4). The ratio data for each set were aligned as described in Fig. 3. The R_p/R_c values for each marker were similar at all time points. The gray curves in a, b, and d indicate the rates of pseudopod extension. The gray curve in panel c was obtained by dividing the R_p/R_c values for Rab5a (a) by the R_p/R_c values for Rab7 (b).

Figure 6.

ER and Golgi markers were absent from erythrocyte phagosomes. Synchronized data from the tracking routine outputs for phagocytic events in cells expressing (a) YFP-Golgi and CFP (n = 6) and (b) YFP-ER and CFP (n = 9). The unchanging R_p/R_c values in cells indicated absence of these markers on phagosomes. (c) Compiled YFP-ER/CFP ratios for opsonized erythrocytes (n = 9) as compared with YFP-ER/CFP ratios for 3.5-μm latex beads (n = 15). P < 0.0001.

Figure 7.

PI(3)P exhibited two distinct kinetic profiles on phagosomes. (a) Time series of ratio images in a cell expressing YFP-2xFYVE and CFP. YFP-2xFYVE was removed from the phagosome by 10 min. (b) Time series of ratio images in a cell expressing YFP-2xFYVE and CFP. YFP-2xFYVE remained on the phagosome for the length of the observation (20 min). (c) Temporally aligned data from the tracking routine outputs for phagocytic events in cells expressing YFP-2xFYVE and CFP. R_p/R_c increased immediately after internalization for all phagosomes, but after 9 min, R_p/R_c varied (n = 10). (d) R_p/R_c values for phagosomes that lost YFP-2xFYVE at early time points (filled diamonds) (n = 5) as compared with R_p/R_c values for phagosomes that retained YFP-2xFYVE (open diamonds) (n = 5). (e) Quantitative analysis of dynamics in macrophages cotransfected with YFP-2xFYVE and CFP-Rab7 (black circles) (n = 15) as compared with R_p/R_c values for YFP-2xFYVE plus CFP (a) divided by the R_p/R_c values for YFP-Rab7 plus CFP (Fig. 5 b) (gray circles). R_p/R_c was high initially due to high concentrations of PI(3)P on phagosomes and decreased as CFP-Rab7 accumulated on phagosomes. The higher ratio of PI(3)P to Rab7 in the cotransfected group reflects a higher proportion of phagosomes that retained YFP-2xFYVE. 12 of 15 phagosomes retained the marker.

Figure 7.

PI(3)P exhibited two distinct kinetic profiles on phagosomes. (a) Time series of ratio images in a cell expressing YFP-2xFYVE and CFP. YFP-2xFYVE was removed from the phagosome by 10 min. (b) Time series of ratio images in a cell expressing YFP-2xFYVE and CFP. YFP-2xFYVE remained on the phagosome for the length of the observation (20 min). (c) Temporally aligned data from the tracking routine outputs for phagocytic events in cells expressing YFP-2xFYVE and CFP. R_p/R_c increased immediately after internalization for all phagosomes, but after 9 min, R_p/R_c varied (n = 10). (d) R_p/R_c values for phagosomes that lost YFP-2xFYVE at early time points (filled diamonds) (n = 5) as compared with R_p/R_c values for phagosomes that retained YFP-2xFYVE (open diamonds) (n = 5). (e) Quantitative analysis of dynamics in macrophages cotransfected with YFP-2xFYVE and CFP-Rab7 (black circles) (n = 15) as compared with R_p/R_c values for YFP-2xFYVE plus CFP (a) divided by the R_p/R_c values for YFP-Rab7 plus CFP (Fig. 5 b) (gray circles). R_p/R_c was high initially due to high concentrations of PI(3)P on phagosomes and decreased as CFP-Rab7 accumulated on phagosomes. The higher ratio of PI(3)P to Rab7 in the cotransfected group reflects a higher proportion of phagosomes that retained YFP-2xFYVE. 12 of 15 phagosomes retained the marker.