In this study, the small heat shock protein (sHSP) chaperones, aB-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for aB-crystallin that could involve transcriptional splicing effects. Both aB-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While aB-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of aB-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous aB-crystallin as well as some cells with ectopic aB-crystallin expression. All consistently showed aB-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear aB-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of aB-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective aB-crystallin. In contrast, the transfection of R120G aB-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of aB-crystallin. These data suggest that the cardiomyopathy-causing mutation for aB-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and aB-crystallin transgenic models of their respective cardiomyopathies.
- Nuclear speckles
- Interchromatin granules