Dynamics of the Interphase Nucleus (funded by The Wellcome Trust)

The interphase nucleus is a dynamic, yet surprisingly tightly-packed organelle. As a model for intranuclear mobility, we are studying the dynamics of the Cajal body. Cajal bodies (CBs) are subnuclear organelles that contain factors required for splicing, ribosome biogenesis, and transcription. CBs are localised in the nucleoplasm and are often found at the nucleolar periphery. In a collaboration with the Lamond lab, we used a stable HeLa cell line, HeLaGFP-coilin, that expresses the CB marker protein, p80 coilin, fused to the Green Fluorescent Protein (GFP-coilin) to study CB dynamics in living cells. Time lapse recordings on 63 nuclei of HeLaGFP-coilin cells showed that all CBs move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures and separation of smaller bodies from larger CBs. The GFP-coilin protein is dynamically associated with CBs as shown by changes in their fluorescence intensity over time (Platani et al, 2001).

To determine the mechanism of CB mobility, we have tracked > 350 CBs in the nuclei of 50 living cells and calculated mean square displacements (MSD) of individual CBs. Brownian diffusion is the primary mode of CB mobility, although some examples of diffusion with flow were noted. Analysis of the MSDs demonstrated that most CBs diffuse within a confined volume. To identify the nature of this confinement, HeLaGFP-coilin cells were transfected with a plasmid encoding YFP-histone H2B and the dynamics of CBs and chromatin recorded by time-lapse fluorescence imaging. CBs that diffused within confined volumes were either immediately next to chromatin or surrounded by chromatin, suggesting that CBs can either be tethered to or confined by chromatin. Furthermore, calculation of the diffusion constant (D) of individual CBs through a time-lapse sequence showed large increases in D when chromatin constraints were relieved. ATP depletion and inhibition of transcription increase Cajal body mobility and decease stable association of Cajal bodies and chromatin. This behaviour is fundamentally different than the ATP-dependent mobility observed for chromatin and suggests a novel mechanism governing Cajal body and possibly other nuclear body dynamics (Platani et al, 2002; PDF available here, Supplemental Info here).