Vinculin is required for neuronal mechanosensing but not for axon outgrowth.

Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10-12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.

Distinct focal adhesion protein modules control different aspects of mechanotransduction.

Focal adhesions (FAs) are macromolecular complexes that regulate cell adhesion and mechanotransduction. By performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoactivation (FLAP) experiments, we found that the mobility of core FA proteins correlates with their function. Structural proteins such as tensin, talin and vinculin are significantly less mobile in FAs than signaling proteins such as FAK (also known as PTK2) and paxillin. The mobilities of the structural proteins are directly influenced by substrate stiffness, suggesting that they are involved in sensing the rigidity of the extracellular environment. The turnover rates of FAK and paxillin, as well as kindlin2 (also known as FERMT2), are not influenced by substrate stiffness. By using specific Src and FAK inhibitors, we reveal that force-sensing by vinculin occurs independently of FAK and paxillin phosphorylation. However, their phosphorylation is required for downstream Rac1-driven cellular processes, such as protrusion and cell migration. Overall, we show that the FA is composed of different functional modules that separately control mechanosensing and the cellular mechano-response.

Vinculin controls talin engagement with the actomyosin machinery.

The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.

[Effects of sludge compost used as lawn medium on lawn growth and soil and water environment].

To address effect of the sludge compost-containing medium on the growth of Manila lawn and environment quality, a pot experiment was conducted using six treatments based on contrasting sludge compost addition volume ratios in the soil system (i. e., 0% , 10% , 25% , 50% , 75% and 100%). The results indicated that the growth potential of Manila lawn was increased with increasing sludge compost addition volume ratio. The content of Hg in Manila plant was significantly positively correlated with that in the lawn medium. Although the contents of Cr, Cd and Hg in the lawn medium were synchronously increased with increasing sludge compost addition volume ratio in the soil system, their contents were all lower than the critical levels of third-class standard in the National Soil Environmental Quality Standard. The heavy metal and nitrate concentrations detected in percolating water were significantly positively correlated with those in the lawn medium, respectively. When the sludge compost addition volume ratio was more than 50% in this study, both heavy metal and nitrate concentrations in percolating water would exceed the maximum allowable levels of the National Groundwater Environment Quality Standard.

Evaluation of transdifferentiation from mesenchymal stem cells to neuron-like cells using microfluidic patterned co-culture system.

We design a microfluidic patterned co-culture system for mouse mesenchymal stem cells (mMSCs) and neural cells to demonstrate the paracrine effects produced by the neural cells in facilitating the transdifferentiation from mMSCs to neuron-like cells. Neural cells and mMSC are orderly patterned in the microfluidic co-culturing system without direct cell contact. This configuration provides us to calculate the percentage of neural marker transdifferentiated by mMSCs easily. We obtain higher transdifferentiated ratio of mMSC in the microfluidic co-culturing system (beta III tubulin: 67%; glial fibrillary acidic protein (GFAP): 86.2%) as compared with the traditional transwell co-culturing system (beta III tubulin: 59.8%; GFAP: 52.0%), which is similar to the spontaneous neural marker expression in the undifferentiated MSCs (beta III tubulin: 47.5%; GFAP: 60.1%). Furthermore, mMSCs expressing green fluorescent protein and neural cells expressing red fluorescent protein were also used in our co-culture system to demonstrate the rarely occurring or observed cell fusion phenomenon. The results show that the co-cultured neural cells increased the transdifferentiation efficiency of mMSCs from soluble factors secreted by neural cells.

Fabricating microparticles/nanofibers composite and nanofiber scaffold with controllable pore size by rotating multichannel electrospinning.

Polymeric nanofibers fabricated via electrospinning are regarded as promising scaffolds for biomimicking a native extracellular matrix. However, electrospun scaffolds have poor porosity, resulting in cells being unable to infiltrate into the scaffolds but grow only on its surface. In this study, we modified regular electrospinning into rotating multichannel electrospinning (RM-ELSP) to produce microparticles and nanofibers simultaneously. Gelatin nanofibers (0.1-1 microm) and polycaprolactone (PCL) microparticles (0.5-10 microm) were formed and well-mixed. Adjusting the concentration of PCL and/or gelatin, we can fabricate various microparticles/nanofibers composites with different sizes of PCL particles and different diameters of gelatin nanofibers depending on their concentrations (2-10%) during electrospinning. Using PCL particles as a pore generator, we obtained gelatin nanofiber scaffolds with controllable pore size and porosity. Cells adhere and grow into the scaffold easily during in vitro cell culture.

Fabricate coaxial stacked nerve conduits through soft lithography and molding processes.

In this article we present a new way to fabricate nerve conduits with various multi-channels patterns by microfabrication. Soft lithography was used to manufacture silicon-based structures and replicate them with PDMS for producing nerve conduit subunit molds. After that, 3% chitosan/acetic acid solution was filled into PDMS molds and then hardened and peeled off. Nerve conduit subunits were fabricated repeatedly by a set of methods for mass production. Afterward, a plurality of conduit subunits stacked coaxially and coated with outer membrane to form the whole nerve conduit. Because of the precise capability of soft lithography, it is well-suited for nerve conduits with complex designs, such as a combination of multiple degradation control and drug delivery system. Besides, the miniaturization and batch processes are serviceable to the economic effect and the utilization in industry.

Microcontact printing of laminin on oxygen plasma activated substrates for the alignment and growth of Schwann cells.

Microenvironment mimicking biological situation is a vital issue in tissue regeneration. With much progress being made, one of the major challenges remains to develop a convenient method to fabricate the scaffold microenvironment suitable for cell attachment and proliferation. This article demonstrates the efficacy of microcontact printed laminin, an extracellular matrix protein, on three different oxygen plasma treatment substrates-tissue culture polystyrene, poly(methyl methacrylate) films, and chitosan films-for alignment and growth of the Schwann cells in in vitro culturing. Replica molding of polydimethylsiloxane elastomeric stamps, fabricated from patterned SU-8 structure on silicon master, was used to print laminin on the three substrates. Pattern and growth of Schwann cells for low (10(3) cells/cm(2)) and increased cell density (2 x 10(4) cells/cm(2)) on the varied substrates with and without microcontact printed laminin were characterized. Results of in vitro cell culture of Schwann cells showed a high degree of cell orientation on the laminin-micropatterned substrates for both cell densities. However, different cell seeding densities will strongly impact the morphology and orientation of Schwann cells. Microcontact printing proves to be a convenient means to pattern cell-recognition molecules on scaffold for cell-guilded growth in tissue regeneration.