S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry.

S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly.

Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

Modelling cell shape in 3D structured environments: A quantitative comparison with experiments.

Cell shape plays a fundamental role in many biological processes, including adhesion, migration, division and development, but it is not clear which shape model best predicts three-dimensional cell shape in structured environments. Here, we compare different modelling approaches with experimental data. The shapes of single mesenchymal cells cultured in custom-made 3D scaffolds were compared by a Fourier method with surfaces that minimize area under the given adhesion and volume constraints. For the minimized surface model, we found marked differences to the experimentally observed cell shapes, which necessitated the use of more advanced shape models. We used different variants of the cellular Potts model, which effectively includes both surface and bulk contributions. The simulations revealed that the Hamiltonian with linear area energy outperformed the elastic area constraint in accurately modelling the 3D shapes of cells in structured environments. Explicit modelling the nucleus did not improve the accuracy of the simulated cell shapes. Overall, our work identifies effective methods for accurately modelling cellular shapes in complex environments.

Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions.

Integrin-mediated adhesions are convergence points for multiple signaling pathways. Their inner structure and diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesions by analyzing several adhesion proteins with structured illumination microscopy (SIM). Paxillin (Pax) serves as a scaffold protein and signaling hub in focal adhesions, and focal adhesion kinase (FAK, also known as PTK2) regulates the dynamics of adhesions. We found that their phosphorylated forms, pPax and pFAK, form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared with more randomly distributed signals for FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions.

Biphasic reinforcement of nascent adhesions by vinculin.

Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin(-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin(-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin(-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.

Membrane remodelling triggers maturation of excitation-contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Induction of ligand promiscuity of αVß3 integrin by mechanical force.

αVß3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVß3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVß3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVß3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVß3-Fn binding. Thus, force-dependent conformational changes in αVß3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin.This article has an associated First Person interview with the first author of the paper.

ß1D integrin splice variant stabilizes integrin dynamics and reduces integrin signaling by limiting paxillin recruitment.

Heterodimeric integrin receptors control cell adhesion, migration and extracellular matrix assembly. While the α integrin subunit determines extracellular ligand specificity, the ß integrin chain binds to an acidic residue of the ligand, and cytoplasmic adapter protein families such as talins, kindlins and paxillin, to form mechanosensing cell matrix adhesions. Alternative splicing of the ß1 integrin cytoplasmic tail creates ubiquitously expressed ß1A, and the heart and skeletal muscle-specific ß1D form. To study the physiological difference between these forms, we developed fluorescent ß1 integrins and analyzed their dynamics, localization, and cytoplasmic adapter recruitment and effects on cell proliferation. On fibronectin, GFP-tagged ß1A integrin showed dynamic exchange in peripheral focal adhesions, and long, central fibrillar adhesions. In contrast, GFP-ß1D integrins exchanged slowly, forming immobile and short central adhesions. While adhesion recruitment of GFP-ß1A integrin was sensitive to C-terminal tail mutagenesis, GFP-ß1D integrin was recruited independently of the distal NPXY motif. In addition, a P786A mutation in the proximal, talin-binding NPXY783 motif switched ß1D to a highly dynamic integrin. In contrast, the inverse A786P mutation in ß1A integrin interfered with paxillin recruitment and proliferation. Thus, differential ß1 integrin splicing controls integrin-dependent adhesion signaling, to adapt to the specific physiological needs of differentiated muscle cells.

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.

Tension and Elasticity Contribute to Fibroblast Cell Shape in Three Dimensions.

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone.

3D Laser Micro- and Nanoprinting: Challenges for Chemistry.

3D printing is a powerful emerging technology for the tailored fabrication of advanced functional materials. This Review summarizes the state-of-the art with regard to 3D laser micro- and nanoprinting and explores the chemical challenges limiting its full exploitation: from the development of advanced functional materials for applications in cell biology and electronics to the chemical barriers that need to be overcome to enable fast writing velocities with resolution below the diffraction limit. We further explore chemical means to enable direct laser writing of multiple materials in one resist by highly wavelength selective (λ-orthogonal) photochemical processes. Finally, chemical processes to construct adaptive 3D written structures that are able to respond to external stimuli, such as light, heat, pH value, or specific molecules, are highlighted, and advanced concepts for degradable scaffolds are explored.

Chemoaffinity in topographic mapping revisited--is it more about fiber-fiber than fiber-target interactions?

Axonal projections between two populations of neurons, which preserve neighborhood relationships, are called topographic. They are ubiquitous in the brain. The development of the retinotectal projection, mapping the retinal output onto the roof of the midbrain, has been studied for decades as a model system. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. In addition, however, abundant evidence has been gathered mainly in the 1970s and 80s that the mapping can adjust to variegated targets with stunning flexibility demonstrating the extraordinary robustness of the guidance process. The identification of ephrins and Eph-receptors as the underlying molecular cues has mostly been interpreted as supporting the fiber-target chemoaffinity hypothesis, while the evidence on mapping robustness has largely been neglected. By having a fresh look on the old data, we expound that they indicate, in addition to fiber-target chemoaffinity, the existence of a second autonomous guidance influence, which we call fiber-fiber chemoaffinity. Classical in vitro observations suggest both influences be composed of opposing monofunctional guidance activities. Based on the molecular evidence, we propose that those might be ephrin/Eph forward and reverse signaling, not only in fiber-target but also in fiber-fiber interactions. In fact, computational models based on this assumption can reconcile the seemingly conflicting findings on rigid and flexible topographic mapping. Supporting the suggested parsimonious and powerful mechanism, they contribute to an understanding of the evolutionary success of robust topographic mass wiring of axons.

Single-Molecule Encapsulation: A Straightforward Route to Highly Stable and Printable Enzymes.

A mild, fast, and sequence-independent method for controlled enzyme immobilization is presented. This novel approach involves the encapsulation of single-enzyme molecules and the covalent attachment of these nanobiocatalysts onto surfaces. Fast and mild immobilization conditions, combined with low nonspecific adsorption on hydrophobic substrates, enables well-defined surface patterns via microcontact printing. The biohybrid materials show enhanced activity in organic solvents.

Practical limitations of superresolution imaging due to conventional sample preparation revealed by a direct comparison of CLSM, SIM and dSTORM.

We evaluate the suitability of conventional sample preparation and labelling methods for two superresolution techniques, structured illumination microscopy and direct stochastic optical reconstruction microscopy, by a comparison to established confocal laser scanning microscopy. We show that SIM is compatible with standard fixation procedures and immunofluorescence labelling protocols and improves resolution by a factor of two compared to confocal laser scanning microscopy. With direct stochastic optical reconstruction microscopy, fluorophores can theoretically be localized with much higher precision. However, in practice, with indirect immunofluorescence labelling density can be insufficient due to the bulky probes to reveal biological structures with high resolution. Fine structures like single actin fibres are in fact resolved with direct stochastic optical reconstruction microscopy when using small affinity probes, but require proper adjustment of the fixation protocol. Finally, by a direct comparison of immunofluorescent and genetic labelling with fluorescent proteins, we show that target morphology in direct stochastic optical reconstruction microscopy data sets can differ significantly depending on the labelling method and the molecular environment of the target.

Hierarchical Micro-Nano Surface Topography Promotes Long-Term Maintenance of Undifferentiated Mouse Embryonic Stem Cells.

Understanding of stem cell-surface interactions and, in particular, long-term maintenance of stem cell pluripotency on well-defined synthetic surfaces is crucial for fundamental research and biomedical applications of stem cells. Here, we show that synthetic surfaces possessing hierarchical micro-nano roughness (MN-surfaces) promote long-term self-renewal (>3 weeks) of mouse embryonic stem cells (mESCs) as monitored by the expression levels of the pluripotency markers octamer-binding transcription factor 4 (Oct4), Nanog, and alkaline phosphatase. On the contrary, culturing of mESCs on either smooth (S-) or nanorough polymer surfaces (N-surfaces) leads to their fast differentiation. Moreover, we show that regular passaging of mESCs on the hierarchical MN-polymer surface leads to an increased homogeneity and percentage of Oct4-positive stem cell colonies as compared to mESCs grown on fibroblast feeder cells. Immunostaining revealed the absence of focal adhesion markers on all polymer substrates studied. However, only the MN-surfaces elicited the formation of actin-positive cell protrusions, indicating an alternative anchorage mechanism involved in the maintenance of mESC stemness.

Simultaneous Dual Encoding of Three-Dimensional Structures by Light-Induced Modular Ligation.

A highly efficient strategy for the simultaneous dual surface encoding of 2D and 3D microscaffolds is reported. The combination of an oligo(ethylene glycol)-based network with two novel and readily synthesized monomers with photoreactive side chains yields two new photoresists, which can be used for the fabrication of microstructures (by two-photon polymerization) that exhibit a dual-photoreactive surface. By combining both functional photoresists into one scaffold, a dual functionalization pattern can be obtained by a single irradiation step in the presence of adequate reaction partners based on a self-sorting mechanism. The versatility of the approach is shown by the dual patterning of halogenated and fluorescent markers as well as proteins. Furthermore, we introduce a new ToF-SIMS mode ("delayed extraction") for the characterization of the obtained microstructures that combines high mass resolution with improved lateral resolution.

FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons.

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping.

The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. We argue that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, we demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. We therefore propose to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, we show that our model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling.

Atypical protein kinase C and Par3 are required for proteoglycan-induced axon growth inhibition.

When the CNS is injured, damaged axons do not regenerate. This failure is due in part to the growth-inhibitory environment that forms at the injury site. Myelin-associated molecules, repulsive axon guidance molecules, and extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs) found within the glial scar inhibit axon regeneration but the intracellular signaling mechanisms triggered by these diverse molecules remain largely unknown. Here we provide biochemical and functional evidence that atypical protein kinase C (PKCζ) and polarity (Par) complex proteins mediate axon growth inhibition. Treatment of postnatal rat neurons in vitro with the NG2 CSPG, a major component of the glial scar, activates PKCζ, and this activation is both necessary and sufficient to inhibit axonal growth. NG2 treatment also activates Cdc42, increases the association of Par6 with PKCζ, and leads to a Par3-dependent activation of Rac1. Transfection of neurons with kinase-dead forms of PKCζ, dominant-negative forms of Cdc42, or mutant forms of Par6 that do not bind to Cdc42 prevent NG2-induced growth inhibition. Similarly, transfection with either a phosphomutant Par3 (S824A) or dominant-negative Rac1 prevent inhibition, whereas expression of constitutively active Rac1 inhibits axon growth on control surfaces. These results suggest a model in which NG2 binding to neurons activates PKCζ and modifies Par complex function. They also identify the Par complex as a novel therapeutic target for promoting axon regeneration after CNS injury.