Figure 4

Live-cell imaging of polymerized tubulin distribution, and ultrastructural analysis of microtubules in NPC-derived neural progeny with CDK5 activation. Differentiating NPCs were infected on day 2 with an adenoviral vector expressing p35. For live imaging of tubulin distribution, on day 4, cells on coverslips were incubated with Tubulin Tracker Green to stain polymerized tubulin. Coverslips were imaged using a fluorescent microscope. For electron microscopy analysis, NPCs were grown on coverslips embedded in 35 mm dishes, fixed in glutaraldehyde and processed for electron microscopic analysis. (A,B) Live-cell imaging of polymerized tubulin stained with Tubulin Tracker in cell bodies and processes of NPC-derived neural progeny infected with p35-adv. (A) Under control conditions, polymerized tubulin is apparent in bundled microtubule structures in cell processes (arrows). (B) Under conditions of abnormal CDK5 activation, polymerized tubulin staining was primarily detected in diffuse, cytoplasmic fibers (arrows). (C,D) Electron microscopy images showing organized cytoskeletal structure in control NPC-derived neural progeny with long, parallel tubule structures (arrows) along the processes of cells. (E,F) NPC-derived neural progeny expressing high levels of p35 displayed a disorganized cytoskeleton with less abundant, short and web-like filamentous structures (arrows) in the processes of cells. Scale bar = 30 μm (A,B); 0.5 μm (C,E); 0.25 μm (D,F).