| Summary: | <p>Many primary cells, cell lines and cells <em>in situ</em> in tissues may have under certain circumstances a network of invaginations of the nuclear envelope. This network called the nucleoplasmic reticulum (NR) brings deep within the nucleus the conventional functions of the nuclear envelope such as anchorage and regulation of genetic material and transport of ions and macromolecular cargo. Abundant NR is a hallmark of cancer cells and senescence.</p>
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<p>We hypothesised that the NR may be involved in the exchange of material across the nuclear boundary. To investigate that, we had a tripartite approach where we (i) study NR assembly and disassembly; (ii) investigate the NR structure and explore whether there is bidirectional nucleocytoplasmic exchange of material; (iii) explore the changes in gene expression leading to or resulting from the induction of NR formation.</p>
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<p>We tagged endogenous Lamin A and Lamin B1 with fluorophores and found that this enables the acquisition of long time-courses, in live cells, with minimal phototoxicity. We demonstrated that the addition of a fluorophore to the N-termini of both lamins does not affect the toxicity of saquinavir. We observed that the assembly and disassembly of NR can occur in interphase without the nuclear envelope reassembly step of mitosis. Moreover, we established that the membrane curvature-associated reticulon, RTN4, can be found on NR and NE, but that it is not required for NR assembly.</p>
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<p>To address whether the NR is involved in the exchange of material across the nuclear envelope we explored its structure. We found that the invaginations of the nuclear envelope may contain a cytoplasmic core and nuclear pore complexes. Using photocleavable proteins we detected import of fluorophores across the NR. Finally, we explored NR abundance relative to the cell cycle and nuclear volume. We found that immediately after mitosis nuclei have abundant NR but that, although some are disassembled, there is a net increase in NR abundance if the nuclei become more voluminous or polyploid. We also found that when colonies are derived by clonal expansion from single cells there is a correlation between the slowest growing colonies, with the fewest cells, and the highest abundance of NR per cell.</p>
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<p>Finally, we investigated the changes in gene expression leading to or resulting from inducing the NR physiologically or pathologically, and identified five genes that are significantly changed in both conditions. Changes in specific pathways have also been identified and include a reduction in some lipid related pathways. In parallel we observed that cells from patients with dysfunctional CCTα have increased NR abundance indicating that a reduction in the canonical lipid pathways may lead to an increase in NR abundance caused by the scavenger pathways.</p>
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<p>Overall, we have demonstrated that the NR is a structure that is formed by a highly regulated and dynamic machinery that incorporates newly synthesised elements and that it is involved in the exchange of material across the nuclear envelope.</p>
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