The weakness of senescent dermal fibroblasts.

Skin is the largest human organ with easily noticeable biophysical manifestations of aging. As human tissues age, there is chronological accumulation of biophysical changes due to internal and environmental factors. Skin aging leads to decreased elasticity and the loss of dermal matrix integrity via degradation. The mechanical properties of the dermal matrix are maintained by fibroblasts, which undergo replicative aging and may reach senescence. While the secretory phenotype of senescent fibroblasts is well studied, little is known about changes in the fibroblasts biophysical phenotype. Therefore, we compare biophysical properties of young versus proliferatively aged primary fibroblasts via fluorescence and traction force microscopy, single-cell atomic force spectroscopy, microfluidics, and microrheology of the cytoskeleton. Results show senescent fibroblasts have decreased cytoskeletal tension and myosin II regulatory light chain phosphorylation, in addition to significant loss of traction force. The alteration of cellular forces is harmful to extracellular matrix homeostasis, while decreased cytoskeletal tension can amplify epigenetic changes involved in senescence. Further exploration and detection of these mechanical phenomena provide possibilities for previously unexplored pharmaceutical targets against aging.

Cell shape and tension alter focal adhesion structure.

Cells are not only anchored to the extracellular matrix via the focal adhesion complex, the focal adhesion complex also serves as a sensor for force transduction. How tension influences the structure of focal adhesions is not well understood. Here, we analyse the effect of tension on the location of key focal adhesion proteins, namely vinculin, paxillin and actin. We use micropatterning on gold surfaces to manipulate the cell shape, to create focal adhesions at specific cell areas, and to perform metal-induced energy transfer (MIET) measurements on the patterned cells. MIET resolves the different protein locations with respect to the gold surface with nanometer accuracy. Further, we use drugs influencing the cellular motor protein myosin or mechanosensitive ion channels to get deeper insight into focal adhesions at different tension states. We show here that in particular actin is affected by the rationally tuned force balance. Blocking mechanosensitive ion channels has a particularly high influence on the actin and focal adhesion architecture, resulting in larger focal adhesions with elevated paxillin and vinculin and strongly lowered actin stress fibres. Our results can be explained by a balance of adhesion tension with cellular tension together with ion channel-controlled focal adhesion homeostasis, where high cellular tension leads to an elevation of vinculin and actin, while high adhesion tension lowers these proteins.

Remodeling of the focal adhesion complex by hydrogen-peroxide-induced senescence.

Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We found that H2O2-induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H2O2 treatment.

Influence of ROCK Pathway Manipulation on the Actin Cytoskeleton Height.

The actin cytoskeleton with its dynamic properties serves as the driving force for the movement and division of cells and gives the cell shape and structure. Disorders in the actin cytoskeleton occur in many diseases. Deeper understanding of its regulation is essential in order to better understand these biochemical processes. In our study, we use metal-induced energy transfer (MIET) as a tool to quantitatively examine the rarely considered third dimension of the actin cytoskeleton with nanometer accuracy. In particular, we investigate the influence of different drugs acting on the ROCK pathway on the three-dimensional actin organization. We find that cells treated with inhibitors have a lower actin height to the substrate while treatment with a stimulator for the ROCK pathway increases the actin height to the substrate, while the height of the membrane remains unchanged. This reveals the precise tuning of adhesion and cytoskeleton tension, which leads to a rich three-dimensional structural behaviour of the actin cytoskeleton. This finetuning is differentially affected by either inhibition or stimulation. The high axial resolution shows the importance of the precise finetuning of the actin cytoskeleton and the disturbed regulation of the ROCK pathway has a significant impact on the actin behavior in the z dimension.