Fig. 3

Extracellular MBs are internalized by phagocytosis-like mechanism. a HeLa cells stably expressing mCherry-CAAX were incubated with GFP-MBs for 3 h followed by washing and incubation with media at 37 ^oC for another hour. Cells were then fixed and analyzed by microscopy. Arrows point to membranous protrusions surrounding MB during internalization. b, c HeLa cells were incubated with GFP-MBs for 3 h. Cells were then washed and incubated for another 3 h, followed by tomography analysis. Arrow in b (left image) point to the MB. Arrowheads in b point to electron dense coat associated with sites where MB binds to internalizing cell plasma membrane. Right panel in b shows plasma membrane and MB surface model rendered based on tomography. Panel in c shows MB-only surface model. Red color marks MB and plasma membrane interaction sites. Scale bars are equivalent to 500 nm. d–g HeLa cells were incubated with GFP-MBs for 3 h. Cells were then washed and incubated for another 24 h, followed by CLEM/thin section EM analysis. Panel e shows higher magnification image of MB boxed in image d. Panels f and g shows MBs from other cells. N-marks nucleus. Black arrow points to outside membrane. White arrow points to internal membrane. Asterisks mark actin-like electron-dense patches associated with internalized MBs. Scale bar is equivalent to 1 μm. Scale bars in inset and magnified images is equivalent to 250 nm