Molecular mechanisms behind the macropinocytosis-like engulfment in astrocytes.
(A) TEM image of an astrocyte in the process of engulfing a dead cell (white star) show interaction points (black arrowheads) indicative of receptor/ligand interaction. Dead cells were added to the culture and incubated for 22 h before fixation and prepared for TEM. (B) Phagocytic genes are expressed in the neural cultures. qRT-PCR data showing the expression of GFAP, Megf10, Crk, Rac1 and Mfge8. The average of 5 independent injured and 5 uninjured neural cultures are presented. Megf10, Crk, Rac1 and Megf8 were expressed in all the cultures. Error bars represent SEM. (C) The protein levels of MEGF10 is up-regulated after addition of dead cells. Fresh medium with or without dead cells were added to cell cultures, differentiated for 8 days. The cells were incubated for an additional 1 day (9 days in graph) or 3 days (12 days in graph). Cell lysates were analyzed for MEGF10 expression by Western blot and β Tubulin served as a loading control. (D–E) Engulfed cells are found in macropinocytotic-like vacuoles. Twenty-two hours after scratch injury, the cultures were fixed and astrocytes were identified with specific antibodies to GFAP. Phalloidin and DAPI labeling was used in order to visualize the actin cytoskeleton and cell nuclei, respectively. To be counted as engulfed, the dead cells (condensed nuclei) had to be situated in cytoplasmic vacuoles (white arrowheads) within the astrocytes. (F–H) The highly vacuolized astrocytes appear most active in cell corpse clearing. (F–G) The micrographs show a highly vacuolized astrocyte before engulfment (F) and approximately 28 h later the same astrocyte have ingested several dead cells (G). First picture is taken 25 minutes after injury and time indicated in the pictures is time lapsed after the first image. The dashed line represents the cut. (H) The vacuoles (black arrowheads) can be seen in transmission electron microscope as round vesicles, some apparently empty whereas others contain debris. An astrocyte-ingested dead cell is marked by a white star and the nucleus by Nu. Scale bars: 200 nm (A), 10 µm (D–G), 2 µm (H).
