Macropinosome maturation and fusion with tubular lysosomes in macrophages

doi:10.1083/jcb.121.5.1011

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Macropinosome maturation and fusion with tubular lysosomes in macrophages

EL Racoosin and JA Swanson
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.

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.
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