Fig. 6.

NM ruptures and cell death during the migration of B1KO neurons into a field of silicon pillars (8 μm in diameter; 22 μm in height; spaced 4 μm apart). (A, Left) Scanning electron micrograph of a silicon wafer (with one side flat and the other side patterned with uniformly spaced silicon pillars). (Scale bar, 30 μm.) (A, Middle and Right) Scanning electron micrographs of the uniformly spaced silicon pillars. (Scale bars, 2 μm.) (B) Live-cell fluorescence microscopy images (20-min intervals) of NLS-GFP–expressing WT and B1KO neurons after the cells had migrated into the field of pillars. NM ruptures (escape of the NLS-GFP into the cytoplasm) were observed in B1KO neurons (red arrows; Bottom) but not in WT neurons (Top). (Scale bars, 20 μm.) (C) Immunofluorescence microscopy of WT and B1KO neurons with a caspase 3-specific antibody (a marker of apoptotic cell death). Neurospheres were pipetted onto the smooth portion of the silicon wafer, and individual neurons were allowed to migrate into the field of pillars. The migration of neurons into the field of pillars subjects the cells as well as the cell nucleus to constrictive forces. Cell death, as judged by caspase 3 staining, was observed in B1KO neurons (but not WT neurons) after the cells had migrated into the field of pillars. DNA was stained with DAPI (blue). (Scale bars, 50 μm.) The panels below show caspase 3 staining in black against a white background. The edge of the field of pillars is marked by a yellow dashed line. (D) Fluorescence microscopy with the LIVE/DEAD fluorescent vital dye, revealing cell death in B1KO neurons (but not WT neurons) after the migration of cells into the field of pillars. The LIVE/DEAD fluorescent vital dye fluoresces green in live cells and red in dead cells. (Scale bar, 50 μm.)