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NRF2 (nuclear factor erythroid-2-related factor 2) is a key regulator of genes involved in the cell's protective response to oxidative stress. Upon activation by disturbed redox homeostasis, NRF2 promotes the expression of metabolic enzymes to eliminate reactive oxygen species (ROS). Cell internalization of peroxisome-like artificial organelles that harbor redox-regulating enzymes was previously shown to reduce ROS-induced stress and thus cell death. However, if and to which extent ROS degradation by such nanocompartments interferes with redox signaling pathways is largely unknown. Here, we advance the design of H 2 O 2 -degrading artificial nano-organelles (AnOs) that exposed surface-attached cell penetrating peptides (CPP) for enhanced uptake and were equipped with a fluorescent moiety for rapid visualization within cells. To investigate how such AnOs integrate in cellular redox signaling, we engineered leukemic K562 cells that report on NRF2 activation by increased mCherry expression. Once internalized, ROS-metabolizing AnOs dampen intracellular NRF2 signaling upon oxidative injury by degrading H 2 O 2 . Moreover, intracellular AnOs conferred protection against ROSinduced cell death in conditions when endogenous ROS-protection mechanisms have been compromised by depletion of glutathione or knockdown of NRF2. We demonstrate CPP-facilitated AnO uptake and AnO-mediated protection against ROS insults also in the T lymphocyte population of primary peripheral blood mononuclear cells from healthy donors. Overall, our data suggest that intracellular AnOs alleviated cellular stress by the on-site reduction of ROS., Competing Interests: Competing interests statement:The authors declare no competing interest.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy., Competing Interests: The authors declare no competing interest.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Fibronectin (FN) forms fibrillar networks coupling cells to the extracellular matrix. The formation of FN fibrils, fibrillogenesis, is a tightly regulated process involving the exposure of cryptic binding sites in individual FN type III (FN-III) repeats presumably exposed by mechanical tension. The FN-III1 module has been previously proposed to contain such cryptic sites that promote the assembly of extracellular matrix FN fibrils. We have combined NMR and steered molecular dynamics simulations to study the structure and mechanical unfolding pathway of FN-III1. This study finds that FN-III1 consists of a beta-sandwich structure that unfolds to a mechanically stable intermediate about four times the length of the native folded state. Considering previous experimental findings, our studies provide a structural model by which mechanical stretching of FN-III1 may induce fibrillogenesis through this partially unfolded intermediate.

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII(7) and FnIII(15) with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed.

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa extracellular matrix protein, with functional sites that are regulated by unfolding events. Fn was labeled with multiple donor and acceptor fluorophores such that intramolecular FRET could be used to distinguish a range of Fn conformations. The sensitivity of FRET to unfolding was tested by progressively denaturing labeled Fn using guanidium chloride. To investigate Fn conformation changes during cell binding and matrix assembly, we added labeled Fn to the culture medium of NIH 3T3 fibroblasts. Coexisting conformations of Fn were visualized using fluorescence microscopy, and spectra from specific features were measured with an attached spectrometer. Using FRET as an indicator of Fn conformation, Fn diffusely bound to cells was in a compact state, whereas Fn in matrix fibrils was highly extended. Matrix fibrils exhibited a range of FRET that suggested some degree of unfolding of Fn's globular modules. Fn in cell-associated clusters that preceded fibril formation appeared more extended than diffuse cell-bound Fn but less extended than fibrillar Fn, suggesting that Fn undergoes extension after cell binding and before polymerization. FRET thus provides an approach to gain insight into the integrin-mediated pathway of Fn fibrillogenesis.

The structural changes accompanying stretch-induced early unfolding events were investigated for the four type III fibronectin (FN-III) modules, FN-III(7), FN-III(8), FN-III(9), and FN-III(10) by using steered molecular dynamics. Simulations revealed that two main energy barriers, I and II, have to be overcome to initiate unraveling of FN-III's tertiary structure. In crossing the first barrier, the two opposing beta-sheets of FN-III are rotated against each other such that the beta-strands of both beta-sheets align parallel to the force vector (aligned state). All further events in the unfolding pathway proceed from this intermediate state. A second energy barrier has to be overcome to break the first major cluster of hydrogen bonds between adjacent beta-strands. Simulations revealed that the height of barrier I varied significantly among the four modules studied, being largest for FN-III(7) and lowest for FN-III(10), whereas the height of barrier II showed little variation. Key residues affecting the mechanical stability of FN-III modules were identified. These results suggest that FN-III modules can be prestretched into an intermediate state with only minor changes to their tertiary structures. FN-III(10), for example, extends 12 A from the native "twisted" to the intermediate aligned state, and an additional 10 A from the aligned state to further unfolding where the first beta-strand is peeled away. The implications of the existence of intermediate states regarding the elasticity of fibrillar fibers and the stretch-induced exposure of cryptic sites are discussed.

The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized in situ by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways.

The 10th type III module of fibronectin possesses a beta-sandwich structure consisting of seven beta-strands (A-G) that are arranged in two antiparallel sheets. It mediates cell adhesion to surfaces via its integrin binding motif, Arg78, Gly79, and Asp80 (RGD), which is placed at the apex of the loop connecting beta-strands F and G. Steered molecular dynamics simulations in which tension is applied to the protein's terminal ends reveal that the beta-strand G is the first to break away from the module on forced unfolding whereas the remaining fold maintains its structural integrity. The separation of strand G from the remaining fold results in a gradual shortening of the distance between the apex of the RGD-containing loop and the module surface, which potentially reduces the loop's accessibility to surface-bound integrins. The shortening is followed by a straightening of the RGD-loop from a tight beta-turn into a linear conformation, which suggests a further decrease of affinity and selectivity to integrins. The RGD-loop therefore is located strategically to undergo strong conformational changes in the early stretching stages of the module and thus constitutes a mechanosensitive control of ligand recognition.