Danielsson, B. E. | bioRxiv Science

bioRxiv2022

Nuclear lamina strain states revealed by intermolecular force biosensor

Nuclear lamins have been considered to be an important structural element of the nucleus. The nuclear lamina is thought both to shield DNA from excessive mechanical forces and to transmit mechanical forces onto the DNA. However, to date there is not yet a technical approach to directly measure mechanical forces on nuclear lamins at the protein level. To overcome this limitation, we developed a nanobody-based intermolecular tension FRET biosensor capable of measuring the mechanical strain of lamin filaments. Using this sensor, we were able to show that the nuclear lamina is subjected to significant force. These forces are dependent on nuclear volume, actomyosin contractility, functional LINC complex, chromatin condensation state, cell cycle, and EMT. Interestingly, large forces were also present on nucleoplasmic lamins, indicating that these lamins may also have an important mechanical role in the nucleus. Overall, we demonstrate that nanobody-based approach allows construction of novel force biosensors for mechanobiology studies.

biophysics↗

bioRxiv2021

Progerin-Expressing Endothelial Cells are Unable to Adapt to Shear Stress

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. HGPS patients experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study we examined how progerin-expressing ECs adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control ECs to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in ECs, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that ECs either expressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). ECs overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing ECs had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, pre-alignment of cells to shear stress prior to progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of EC to properly adapt to shear stress.

biophysics↗