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. 2014 Dec 15;127(24):5228–5239. doi: 10.1242/jcs.154393

Fig. 1.

Fig. 1.

Internalized CSF-1 and the CSF-1R are delivered to the lumen of newly formed macropinosomes at CSF-1 concentrations that stimulate BMM proliferation. (A) Macropinocytosis was stimulated by a 5-minute exposure to CSF-1, as observed by the uptake of Texas-Red–dextran (TXR–Dex, 70 kDa). White arrows indicate macropinosomes, which contain TXR–Dex and appear as large round objects. Unsti., unstimulated. Scale bar: 5 µm. (B) A histogram of the volume of TXR–Dex-labeled macropinosomes induced by CSF-1. n = 50 cells; data are representative of three experiments. (C) Imaging of BMMs exposed to a 1-minute pulse of TXR–CSF-1 showed that TXR–CSF-1 was internalized in small endosomes that underwent fusion to produce brighter slightly larger endosomes (punctate structures at 2 and 5 minutes). Macropinosomes were visible as displaced cytoplasm (5 minutes, arrow) to which endosomes containing TXR–CSF-1 were trafficked (8 min, arrow). As the macropinosome matured, TXR–CSF-1 was delivered into the lumen (11 min, arrow). Scale bar: 2 µm. (D) The CSF-1R localized to punctate structures within macropinosomes after a 15-minute exposure to CSF-1. Macropinosomes marked by TXR–Dex (70 kDa) were fixed and permeabilized, causing the dextran to leak out. Samples were subsequently stained for the CSF-1R. The dashed line indicates the outline of the cell. IF, immunofluorescence. Scale bar: 1 µm. (E) CSF-1R localization to macropinosomes after a 15-minute exposure to CSF-1 was quantified from the data shown in D as the percentage of TXR–Dex-positive macropinosomes per cell that are CSF-1R positive (CSF-1R +) and contain a punctate distribution of CSF-1R (punc. CSF-1R). n = 50 cells; data are representative of three experiments and show the mean±s.e.m. (F) BMM growth and macropinocytosis were measured as a function of constant CSF-1 concentrations (conc.) after 7 days in culture. Norm. MP, normalized macropinosome; FOV, field of view. Data are representative of three experiments and show the mean±s.e.m. (G) Immunofluorescence against the CSF-1R in BMMs exposed to 5 and 10 ng/ml CSF-1 for 15 minutes. Arrows indicate macropinosomes. Scale bar: 5 µm. (H) TXR–CSF-1 localized to macropinosomes in human monocyte-derived macrophages following an 8-minute exposure to TXR–CSF-1. Macropinosomes are shown in green (DIC) and TXR-CSF-1 is in magenta. Scale bar: 10 µm. (I) Zoom of the indicated region in H.