Figure 8.

Movements of HDAC4-GFP in unstimulated fibers in the presence of leptomycin B, KN-62, and staurosporine. (A) A typical living fiber expressing HDAC4-GFP is shown in Ringer's solution without any treatment for 30 min (−30 and 0) and after 30 and 60 min in the presence of 40 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1. In the presence of leptomycin B, nuclear HDAC4-GFP fluorescence was significantly increased. No changes in cytoplasmic fluorescence were detected. Bar, 50 μm. (B) Time course of nuclear and cytoplasmic HDAC4-GFP fluorescence before and during exposure to 20 nM leptomycin B. Leptomycin B block of nuclear export of HDAC4-GFP caused an increase of HDAC4-GFP in the nucleus. (C) KN-62 was first added to culture dishes with fibers expressing HDAC4-GFP, and the fluorescence remained constant for 60 min. 1 μM staurosporine, a general kinase inhibitor, was then added to the same dishes without washing out of KN-62. The fluorescence signal was recorded for another 60 min. The same group of fibers that had no response to KN-62 subsequently responded to staurosporine, with a significant increase in nuclear HDAC4-GFP, indicating that staurosporine inhibition of a KN-62–insensitive kinase underlies the HDAC4 nuclear accumulation produced by staurosporine.