Abstract
The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage-step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.—Ferrera, D., Canale, C., Marotta, R., Mazzaro, N., Gritti, M., Mazzanti, M., Capellari, S., Cortelli, P., Gasparini, L. Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts.
Highlights
The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins
We found that lamin B1 (LB1) is correctly localized to the nuclear lamina and the nucleoplasm in both autosomal dominant leukodystrophy (ADLD) and CTR nuclei (Fig. 1C)
Consistent with the EM data (Fig. 1D, E), the sequential biochemical extraction of nuclear proteins with increasing salt concentration revealed that the majority of LB1 was in the insoluble fraction of both CTR and ADLD fibroblasts, indicating that LB1 primarily associates with the nuclear lamina (Supplemental Fig. S3A)
Summary
The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). We report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and enhances nuclear stiffness. Changes in other lamins alter the nuclear lamina and affect the structural integrity of the nucleus and its sensitivity to mechanical stress. Lamins A and C (LA/C) play a key role in defining the nuclear lamina’s elastic properties: LA/C-deficient cells have increased numbers of misshapen nuclei and reduced nuclear stiffness [14]. 0892-6638/14/0028-3906 © The Author(s) pressed [16], the stiffness of Xenopus oocyte nuclei increases proportionally to the LA concentration
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