The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear-cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell-cell adhesion and barrier function, as well as altered cell-matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH-expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.

Characterization of 3D Printed Stretching Devices for Imaging Force Transmission in Live-Cells.

INTRODUCTION: Cell stretch is a method which can rapidly apply mechanical force through cell-matrix and cell-cell adhesions and can be utilized to better understand underlying biophysical questions related to intracellular force transmission and mechanotransduction. METHODS: 3D printable stretching devices suitable for live-cell fluorescent imaging were designed using finite element modeling and validated experimentally. These devices were then used along with FRET based nesprin-2G force sensitive biosensors as well as live cell fluorescent staining to understand how the nucleus responds to externally applied mechanical force in cells with both intact LINC (linker of nucleoskeleton and cytoskeleton) complex and cells with the LINC complex disrupted using expression of dominant negative KASH protein. RESULTS: The devices were shown to provide a larger strain ranges (300% uniaxial and 60% biaxial) than currently available commercial or academic designs we are aware of. Under uniaxial deformation, the deformation of the nucleus of NIH 3T3 cells per unit of imposed cell strain was shown to be approximately 50% higher in control cells compared to cells with a disrupted LINC complex. Under biaxial deformation, MDCK II cells showed permanent changes in the nuclear morphology as well as actin organization upon unloading, indicating that failure, plastic deformation, or remodeling of the cytoskeleton is occurring in response to the applied stretch. CONCLUSION: Development and open distribution of low-cost, 3D-printable uniaxial and biaxial cell stretching devices compatible with live-cell fluorescent imaging allows a wider range of researchers to investigate mechanical influences on biological questions with only a minimal investment of resources.

Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex.

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.