MACROPINOSOME MATURATION AND FUSION WITH TUBULAR LYSOSOMES IN MACROPHAGES

Macropinosomes formed by addition of recombinant macrophage colony-sti mulating factor (rM-CSF) to mouse macrophages migrate centripetally an d shrink, remaining detectable by phase microscopy for up to 15 min. T his longevity allowed us to study how macropinosomes age. Macropinosom es were pulse labeled for 1 min with fixable fluorescein dextran (FDx1 0f), a probe for fluid phase pinocytosis, and chased for various times . To quantify changes in their antigenic profile, pulse-labeled macrop inosomes of different ages were fixed and stained for immunofluorescen ce with a panel of antibodies specific for the transferrin receptor (T fR), the late endosome-specific, GTP-binding protein rab 7 or lysosoma l glycoprotein A (lgp-A), and the percentage of antibody positive, FDx 10f-labeled macropinosomes was scored. Some newly formed macropinosome s were positive for TfR, but few were rab 7 or lgp-A-positive. With in termediate chase times (2-4 min), staining for rab 7 and lgp-A increas ed 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 acquisiti on and loss of characteristic endocytic vesicle markers. However, unli ke a maturation process, their merger with the tubular lysosomal compa rtment 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 de xtran (TRDx10)-labeled tubular lysosomes. This occurred in two steps: macropinosomes acquired lgp-A first, and then several minutes later th e cation-independent mannose-6-phosphate receptor (CI-MPR) and markers of lysosomal content (cathepsin L or pre-loaded TRDx10), all apparent ly derived from tubular lysosomes. Thus, macropinosome progress throug h macrophages showed features of both the maturation and vesicle shutt le models of endocytosis, beginning with a maturation process and endi ng by merger into a stable, resident lysosomal compartment.