Myopodin has a Nucleolar Localization in Several Mammalian Cell Lines

Abstract

Synaptopodin 2 is a natively unfolded actin binding protein that stimulates actin polymerization in a Ca2+-calmodulin dependent manner, has multiple binding partners and is rich in smooth muscle dense bodies. Various synaptopodin 2 isoforms have cytoplasmic and nuclear localizations in non-muscle cells and the distribution changes in certain cancers. We prepared an antibody against the C-terminus that recognizes isoform B and myopodin, the truncated form. Immunological staining showed a nuclear location in several cell lines. In further studies with the colon cancer cell line, HT-29 and African green monkey kidney cells, CV1, we observed intense staining in irregular areas within the nucleus. Staining was also observed at the nuclear periphery of HT-29 cells. The strong synaptopodin 2 staining pattern was 90% correlated with markers of nucleoli including a commercial antibody against nucleolin and a counter stain with DAPI. Furthermore, when Actinomycin D was used to inhibit transcription the nuclear stain disappeared. When HT-29 cells were forced to differentiate with sodium butyrate, the synaptopodin 2 antibody revealed a re-localization of the protein into the cytoplasm. RT-PCR and Western blot analysis showed that HT-29 cells lacked Synaptopodin2B, but contained the smaller isoform, myopodin. These studies were performed using cytoplasmic and nuclear fractions. Further analysis showed that the nuclear fraction contained a smaller cross-reactive protein with an apparent mass of 55-60 kDa. Both myopodin and the 60 kDa protein were immunoprecipitated along with proteins tentatively identified as actin and α-actinin. These results show that myopodin and a smaller synaptopodin-like protein are present in nucleoli along with actin and other actin binding proteins.