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

Abstract

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.

Sarcolipin Signaling Promotes Mitochondrial Biogenesis and Oxidative Metabolism in Skeletal Muscle

“…We show here that SLN interaction with SERCA alters cytosolic Ca 2+ transients, leading to activation of Ca 2+ -dependent signaling pathways, especially CamKII, and increased PGC1α expression. Published studies and results from this study demonstrate that an increase in cytosolic Ca 2+ transients leads to activation of Ca 2+ signaling pathways and recruitment of PGC1α to promote mitochondrial biogenesis ( Chin, 2005 ; Lee et al, 2018 ; Mansueto et al, 2017 ; Michel et al, 2007 ; Wright et al, 2007 ; Wu et al, 2002 ). Another key transcription factor, transcription factor EB (TFEB), because of its calcium-dependent regulation, may also be involved in this SLN-dependent signaling pathway ( Mansueto et al, 2017 ).…”

Uptake and Fate of Extracellular Membrane Vesicles: Nucleoplasmic Reticulum-Associated Late Endosomes as a New Gate to Intercellular Communication

“…Mitochondria remained in most cases at the entrance of the NEI [ 5 , 145 , 146 , 167 ]. Under particular situations, mitochondria might shuttle in NEI and influence Ca 2+ signaling [ 141 , 168 ]. It will be important to further dissect the types of membrane-bound structures that navigate in NEI, particularly in those making tunnels throughout the nucleus.…”

AKAP6 and phospholamban colocalize and interact in HEK‐293T cells and primary murine cardiomyocytes

“…We generated and assessed neonatal cardiomyocyte cultures from CD1 mouse neonates as published previously (Chis et al., ). The isolation of adult rat ventricular cardiomyocyte was performed as previously described (Lee et al., ).…”