Fig. 2.

Activated CSF-1R is sequestered within the lumen of macropinosomes. (A) Images of CSF-1-stimulated BMMs containing TXR–Dex-labeled macropinosomes before and after selective membrane permeabilization with digitonin. Scale bar: 10 µm. (B–D) BMMs were co-stained with CSF-1R antibodies recognizing either the cytosolic tail (Cyto tail, green) or extracellular domain (EC domain, red) to determine to the topology of trafficking CSF-1R. (B) Resting (unstimulated, Unsti.) BMMs were either fixed or fixed and permeabilized with digitonin (dig). Note the absence of cytoplasmic tail staining in the unpermeabilized cells. Following digitonin treatment, both the extracellular and intracellular epitopes of the CSF-1R could be stained. PM, plasma membrane. (C) Images of digitonin- and Triton X-100 (TX100)-permeabilized BMMs 5 minutes after exposure to CSF-1. CSF-1R was internalized and only the cytosolic tail stained following permeabilization with digitonin, whereas cells that underwent total membrane permeabilization with Triton X-100 showed staining for both the cytosolic tail and the extracellular domain, suggesting the extracellular domain was protected by the membrane of the early endosome (EE). (D) Digitonin- and Triton-X-100-permeabilized BMMs 15 minutes after exposure to CSF-1. Neither epitope was accessible with digitonin permeabilization, indicating full enclosure of the CSF-1R in macropinosomes (MP). Triton X-100 exposed both epitopes within macropinosomes. Arrows indicate receptor clustering within macropinosomes. Boxes in the merged images indicated regions that are enlarged to the right. Scale bar: 3.3 µm.