PROCEEDINGS |
Green fluorescent protein (GFP) and its variants have become powerful tools with which to study the dynamics of many fundamental processes in living cells. I will discuss the advantages of GFP and important parameters that one must consider in performing live-cell microscopy experiments. We have used GFP to study the movements of pre-mRNA splicing factors in the nuclei of baby hamster kidney cells. Splicing factors localize in 20-30 nuclear clusters, termed speckles, that each measure approximately 0.5 - 0.75 um across their longest length. In addition, these factors are diffusely distributed throughout the nucleoplasm. At the electron microscopic level speckles correspond to interchromatin granule clusters (IGCs), first described by Hewson Swift in 1959. Transcription sites are not found within these speckles but instead occur at their periphery or at some distance from a speckle. We have fused GFP to the splicing factor SF2/ASF and have followed its dynamics in living cells. We find that each speckle is restricted to its neighborhood, however, extensive dynamics are observed on the periphery of the speckles. Upon activation of a gene we have observed splicing factors to leave one or more speckles and to be recruited to the site of active transcription. Using a series of mutants we have been able to demonstrate that the recruitment process is dependent upon phosphorylation of serine residues at the C-terminus of the splicing factor. In addition, we have found that the recruitment of splicing factors to the site of transcription is mediated by the C-terminal domain of the large subunit of RNA polymerase II.