Mutations in different domains of lamin A change the mechanical properties of the nucleus

摘要

The rigid skeleton of cell nucleus, nuclear lamina (NL) consists mostly of A- and B-type lamins. Such structure maintains the nuclear shape and size and also provides mechanical link between nucleoplasm and cytoplasm. Mutations of structural components of NL are the cause of a wide group of diseases - laminopathies. According to “mechanical” hypothesis, this may be due to a disturbance of the mutant protein polymerization, as shown by the in vitro experiments. How NL assembly occurs in living cells is still unclear. Our aims were to visualize the structure of the nucleoskeleton in normal and pathological states and to estimate the mechanical properties of the nuclear envelope (NE) in living cells. In our work we used different types of cells: smooth muscle myocytes, cardiac progenitor cells, rat cardiomyocyte and human fibrosarcoma cells expressing wt lamin A or its mutant forms (G465D, R471C, R482L, R527C) fused to GFP. The resistance of NE of such cells to mechanical stress was studied by treating them with 15/30 Hanks' solution. The effect of a hypo-osmotic shock causes a mechanical stress inside the nucleus that leads to the formation of stable protrusions of NE - induced nuclear buds allowing us to indirectly estimate determined changes in the NE mechanical properties. Mechanical properties of NE were also measured with a scanning ion-conductance microscope (SICM), which allows to obtain stiffness index with high spatial resolution. In parallel, structural organization of NL was analyzed by structured illumination microscopy. We found the expansion of the distance between the NL microdomains are increased in nuclei containing mutant proteins. Furthermore, before treatment with a hypotonic solution, local disarrangement of the NL and nuclear asymmetry were observed in some cells. The use of SICM showed the dependence of the nucleus mechanical properties on the amount of lamin A and its isoforms. Therefore, the mechanical properties of the NE have tight relationship with molecular composition of NL. Thus, the cell nuclei expressing the additional lamin A gene were found to be 1.3 times more rigid than the wild-type cell nuclei, and changes in the structure of lamin A lead to improper organization of the nuclear skeleton. These may be due to the polymerization abnormality of dimers and decreased network stability and/or sequestration of normal protein. The research was supported by Russian Science Fund № 17-15-01290.