|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Functional analysis of Cdc42 signaling and microtubules in polarized protrusion formation of directionally migrating neuronal precursors. (A) Dominant-negative mutant of Cdc42 (N17-Cdc42) was co-expressed with EGFP in rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 3), and images from 0, 40, 80, 120 minutes time points are displayed. N17-Cdc42-expressing cells showed a bipolar morphology and some were rounded up. (B) Function-interfering mutant of Par6 (Par6-NT) altered orientation of TLPs and frequently induced branching (arrowheads) (see supplementary material Movie 4). (C) Neuronal precursors expressing a constitutively active mutant of GSK3ß (S9A-GSK) showed a branching phenotype that was somewhat weaker than that seen in Par6-NT-expressing cells (arrowheads). (D) Microtubule binding-deficient mutant of APC (APC
CT) produced branched TLPs (arrowheads). (E) Cerebellar explants electroporated with EGFP were pre-cultured for 24 hours before treatment with 5 µM nocodazole (microtubule-depolymerization drug), for 15 minutes, followed by in situ fluorescent imaging. The same drug-containing medium was used during the time-lapse imaging. Disruption of microtubule structure by nocodazole induced fragmentation of leading processes (see supplementary material Movie 5). In some cases, altered orientation of TLPs were observed (arrows). (F) Fluorescent images of leading processes visualized by microtubule plus-end binding protein EB3-GFP. EB3-GFP showed a broad localization that increased toward the TLPs. These processes displayed non-directional extension of the polarized process (see supplementary material Movie 6). Bar, 10 µm.
|
