Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine CC muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca regulatory channels, including sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca release-activated Ca channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.