Fig. 5.

Macropinosomes are Rab5 positive during CSF-1 endosome delivery and transition to Rab7 positive during CSF-1 delivery to their lumen. (A) Movies of BMMs expressing mCit–Rab5a (green) and exposed to TXR–CSF-1 (red) for 1 minute were recorded (supplementary material Movie 5). Images shown here highlight the trafficking of TXR–CSF-1 in endosomes (5 minutes) and their trafficking to macropinosomes (7.7 minutes). Scale bar: 5 µm. (B) Zoom of the rectangular region in A (5 minutes), showing colocalization of TRX–CSF-1 (red) and mCit–Rab5a (yellow). (C) Normalized (Norm.) Pearson correlation coefficient between mCit–Rab5a and TXR–CSF-1 fluorescence intensities. The peak correlation occurs around the time at which TXR–CSF-1 was delivered to the macropinosomes (∼6 minutes, average of ten cells). (D) A zoom of the square boxed region in A shows mCit–Rab5a-negative TXR–CSF-1-positive endosomes fused with mCit–Rab5a-positive macropinosomes. (E) BMMs transduced with YFP–Rab7 (green) and exposed to TXR–CSF-1 for 1 minute were imaged. Frames at the indicated times show macropinosomes acquiring YFP–Rab7 when TXR–CSF-1 was transported into their lumen. Scale bar: 2 µm. (F) BMMs were stained for endogenous Rab7 (green) and total CSF-1R (red) after exposure to 100 ng/ml CSF-1 for the times indicated. Arrows highlight Rab7-positive macropinosomes containing TXR–CSF-1. Scale bar: 5 µm.