Figure 5.
Densin-180 induces branching of neuronal dendrites. A, Hippocampal neurons were kept for 7 d in culture. After transfection with vectors coding for EGFP or EGFP-Densin-180, cells were cultivated for another 7 d (7 + 7) and stained for EGFP. B, Costaining for MAP2. Neurons were transfected after 7 d in culture with the Densin-180-myc construct; after 2 d (7 + 2) or 7 d (7 + 7) in culture, cells were fixed, and overexpressed Densin-180 was detected by anti-myc, followed by Cy3-anti-mouse. MAP2 was detected using a polyclonal MAP antiserum (kindly provided by Dr. Stefan Kindler, Institut für Zellbiochemie, Hamburg, Germany), followed by Cy2-anti rabbit. C, Mapping of functional domains of Densin-180. Neurons were transfected with various deletion constructs of Densin-180, as indicated (top panel) and stained for the presence of Densin-180 using the ant-myc antibody as before. In parallel, constructs were transfected into HEK293 and assayed for their ability to target the protein to the plasma membrane. Note that only constructs that contain the leucine-rich repeat region are localized at the plasma membrane in HEK293 cells and have the ability to induce branching of dendrites in transfected neurons. Scale bars: neurons, full view, 20 μm; dendrite enlargements, HEK cells, 5 μm. D, MDCK-II cells stably expressing the Densin-180 LRR region (Densin1-470) were stained for Densin (green) and the tight junction marker ZO-1 (red; vertical sections only). Horizontal xy sections (left) were recorded on the confocal microscope; vertical xz sections (right) were then reconstructed using LSM software. Note the delineation of green (Densin) fluorescence by the red (ZO-1) signal. Scale bar, 5 μm; thickness of xz sections, 7 μm.