Cell size and shape affect cellular processes such as cell survival, growth and differentiation, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosis is a process that stands diametrically opposed to functional tissue regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. Wound healing is guided by precise spatiotemporal deposition and remodelling of the extracellular matrix (ECM). The ECM, comprising the non-cellular component of tissues, is a signalling depot that is differentially regulated in scarring and regenerative healing. This Review focuses on the importance of the native matrix components during mammalian wound healing alongside a comparison to scar-free healing and then presents an overview of matrix-based strategies that attempt to exploit the role of the ECM to improve wound healing outcomes.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain-derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs-key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9. L aminin-111, together with laminin-511, is among the first extracellular matrix (ECM) proteins expressed during early embryogenesis. Members of the laminin family are highly conserved between different species and are critical constituents of basement membranes in the early blastocyst as well as a variety of other tissues (1). The expression of the three laminin-111 chains-α1, β1, and γ1-is initiated as early as at the two-cell stage (2-4). Laminin-111-together with collagen IV, nidogen, perlecan, and other proteins-is assembled into the basement membrane (5), where it provides not only physical support, but also the capacity to modulate ECM function when modified by other proteins (6-8).The interactions between matrix metalloproteinases (MMPs) and ECM proteins have been extensively studied over the past decades (9, 10). The cellular expression of MMPs is precisely regulated such that ECM molecules are processed at different cell stages (11). It has been shown that laminin-111 can be processed by different MMPs and a variety of other enzymes and that such modifications are mainly related to cell migration because cleavage of laminin results in a loosened basement membrane (6,8,12,13). Recent insights into the existence of "cryptic" ECM interaction sites-sequences usually hidden within the tertiary structure of the protein or within the assembly of the ECM-have enabled a greater understanding of how cells interact with the ECM and of the astonishingly complex interaction between cells and ECM proteins (14). The ECM can no longer be thought of as just a passive scaffold and physical support for cells in vivo or as a more-or-less adequate cell culture substrate in vitro; the ECM and its modification is now known to act as one of the key constituents in cell regulation. Therefore, to successfully model development in vitro by using pluripotent stem cells, it is imperative to give careful consideration to cell-ECM interactions.In this work, we address the question of whether MMP2 (15-17) modifies laminin-111 and whether s...

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The concept of self-assembly is one of the most promising strategies for the creation of defined nanostructures and therefore became an essential part of nanotechnology for the controlled bottom-up design of nanoscale structures. Surface layers (S-layers), which represent the cell envelope of a great variety of prokaryotic cells, show outstanding self-assembly features in vitro and have been successfully used as the basic matrix for molecular construction kits. Here we present the three-dimensional structure of an S-layer lattice based on tetrameric unit cells, which will help to facilitate the directed binding of various molecules on the S-layer lattice, thereby creating functional nanoarrays for applications in nanobiotechnology. Our work demonstrates the successful combination of computer simulations, electron microscopy (TEM), and small-angle X-ray scattering (SAXS) as a tool for the investigation of the structure of self-assembling or aggregating proteins, which cannot be determined by X-ray crystallography. To the best of our knowledge, this is the first structural model at an amino acid level of an S-layer unit cell that exhibits p4 lattice symmetry.

S-layer proteins have a wide range of application potential due to their characteristic features concerning self-assembling, assembling on various surfaces, and forming of isoporous structures with functional groups located on the surface in an identical position and orientation. Although considerable knowledge has been experimentally accumulated on the structure, biochemistry, assemble characteristics, and genetics of S-layer proteins, no structural model at atomic resolution has been available so far. Therefore, neither the overall folding of the S-layer proteins-their tertiary structure-nor the exact amino acid or domain allocations in the lattices are known. In this paper, we describe the tertiary structure prediction for the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2. This calculation was based on its amino acid sequence using the mean force method (MF method) achieved by performing molecular dynamic simulations. This method includes mainly the thermodynamic aspects of protein folding as well as steric constraints of the amino acids and is therefore independent of experimental structure analysis problems resulting from biochemical properties of the S-layer proteins. Molecular dynamic simulations were performed in vacuum using the simulation software NAMD. The obtained tertiary structure of SbsB was systematically analyzed by using the mean force method, whereas the verification of the structure is based on calculating the global free energy minimum of the whole system. This corresponds to the potential of mean force, which is the thermodynamically most favorable conformation of the protein. Finally, an S-layer lattice was modeled graphically using CINEMA4D and compared with scanning force microscopy data down to a resolution of 1 nm. The results show that this approach leads to a thermodynamically favorable atomic model of the tertiary structure of the protein, which could be verified by both the MF Method and the lattice model.