The nano-scale mechanical properties of the extracellular matrix regulate dermal fibroblast function.

Changes in the mechanical properties of dermis occur during skin aging or tissue remodeling and affect the activity of resident fibroblasts. With the aim to establish elastic culture substrates that reproduce the variable softness of dermis, we determined Young's elastic modulus E of human dermis at the cell perception level using atomic force microscopy. The E of dermis ranged from 0.1 to 10 kPa, varied depending on body area and dermal layer, and tended to increase with age in 26-55-year-old donors. The activation state of human dermal fibroblasts cultured on "skin-soft" E (5 kPa) silicone culture substrates was compared with stiff plastic culture (GPa), collagen gel cultures (0.1-9 kPa), and fresh human dermal tissue. Fibroblasts cultured on skin-soft silicones displayed low mRNA levels of fibrosis-associated genes and increased expression of the matrix metalloproteinases (MMPs) MMP-1 and MMP-3 as compared with collagen gel and plastic cultures. The activation profile exhibited by fibroblasts on "skin-soft" silicone culture substrates was most comparable with that of human dermis than any other tested culture condition. Hence, providing biomimetic mechanical conditions generates fibroblasts that are more suitable to investigate physiologically relevant cell processes than fibroblasts spontaneously activated by stiff conventional culture surfaces.

The single-molecule mechanics of the latent TGF-ß1 complex.

BACKGROUND: TGF-ß1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-ß1 binds to the latent TGF-ß1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-ß1 from ECM stores. Latent TGF-ß1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism. RESULTS: By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-ß1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-ß1 activation: the α1 helix and the latency lasso, which together have been referred to as the "straitjacket" that keeps TGF-ß1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ~40 pN, was achieved only when TGF-ß1 was bound to the LTBP-1 in the ECM. CONCLUSIONS: Our results directly demonstrate opening of the TGF-ß1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-ß1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-ß1 by reducing integrin affinity, cell contractility, and binding of latent TGF-ß1 to the ECM provides new possibilities to therapeutically modulate TGF-ß1 actions.

Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells.

"A picture is worth a thousand words" goes the proverb. A poor picture however can be worse than saying nothing at all. This is particularly true for immunofluorescence pictures that in addition to their informative character bear an esthetic component. We here provide a panel of straightforward methods to process tissue sections and cultured cells for immunostaining of cytoskeletal elements, primarily those associated with actin filaments. We want to emphasize to the reader the fact that the choice of the processing method will have an important influence on the outcome of the immunostaining and thus on the interpretation of the results. Fixation of cultured cells with cross-linking reagents such as paraformaldehyde efficiently preserves structural elements at the expense of reduced antigenicity. The degree and timing of cell permeabilization with detergents, along with chemical cross-linking, contributes to the clarity and resolution of distinct structures but can also lead to loss of information. Fixation with organic solvents like methanol will, in most cases, better preserve antigens but will produce a higher background and impact on structural integrity. Therefore, it is recommended to test different protocols for a "new" protein or epitope - the results will pay back your investment.