Macropinosome maturation and fusion with tubular lysosomes in macrophages

Abstract

Macropinosomes formed by addition of recombinant macrophage colony-stimulating factor (rM-CSF) to mouse macrophages migrate centripetally and shrink, remaining detectable by phase microscopy for up to 15 min. This longevity allowed us to study how macropinosomes age. Macropinosomes were pulse labeled for 1 min with fixable fluorescein dextran (FDx10f), a probe for fluid phase pinocytosis, and chased for various times. To quantify changes in their antigenic profile, pulse-labeled macropinosomes of different ages were fixed and stained for immunofluorescence with a panel of antibodies specific for the transferrin receptor (TfR), the late endosome-specific, GTP-binding protein rab 7 or lysosomal glycoprotein A (lgp-A), and the percentage of antibody positive, FDx10f-labeled macropinosomes was scored. Some newly formed macropinosomes were positive for TfR, but few were rab 7 or lgp-A-positive. With intermediate chase times (2-4 min), staining for rab 7 and lgp-A increased to > 60%, while TfR staining declined. After a long chase (9-12 min), rab 7 staining returned to low levels while lgp-A staining remained at a high level. Thus, macropinosomes matured by progressive acquisition and loss of characteristic endocytic vesicle markers. However, unlike a maturation process, their merger with the tubular lysosomal compartment more nearly resembled the incorporation of a transient vesicle into a pre-existing, stable compartment. Shortly after their formation, FDx10f-labeled macropinosomes contacted and merged with Texas red dextran (TRDx10)-labeled tubular lysosomes. This occurred in two steps: macropinosomes acquired lgp-A first, and then several minutes later the cation-independent mannose-6-phosphate receptor (CI-MPR) and markers of lysosomal content (cathepsin L or pre-loaded TRDx10), all apparently derived from tubular lysosomes. Thus, macropinosome progress through macrophages showed features of both the maturation and vesicle shuttle models of endocytosis, beginning with a maturation process and ending by merger into a stable, resident lysosomal compartment.