MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs.

Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.

Mechanical Stabilization of a Bacterial Adhesion Complex.

The adhesions between Gram-positive bacteria and their hosts are exposed to varying magnitudes of tensile forces. Here, using an ultrastable magnetic tweezer-based single-molecule approach, we show the catch-bond kinetics of the prototypical adhesion complex of SD-repeat protein G (SdrG) to a peptide from fibrinogen ß (Fgß) over a physiologically important force range from piconewton (pN) to tens of pN, which was not technologically accessible to previous studies. At 37 °C, the lifetime of the complex exponentially increases from seconds at several pN to ∼1000 s as the force reaches 30 pN, leading to mechanical stabilization of the adhesion. The dissociation transition pathway is determined as the unbinding of a critical ß-strand peptide ("latch" strand of SdrG that secures the entire adhesion complex) away from its binding cleft, leading to the dissociation of the Fgß ligand. Similar mechanical stabilization behavior is also observed in several homologous adhesions, suggesting the generality of catch-bond kinetics in such bacterial adhesions. We reason that such mechanical stabilization confers multiple advantages in the pathogenesis and adaptation of bacteria.

Mechanochemical Lithography.

Surfaces with patterned biomolecules have wide applications in biochips and biomedical diagnostics. However, most patterning methods are inapplicable to physiological conditions and incapable of creating complex structures. Here, we develop a mechanochemical lithography (MCL) method based on compressive force-triggered reactions. In this method, biomolecules containing a bioaffinity ligand and a mechanoactive group are used as mechanochemical inks (MCIs). The bioaffinity ligand facilitates concentrating MCIs from surrounding solutions to a molded surface, enabling direct and continuous printing in an aqueous environment. The mechanoactive group facilitates covalent immobilization of MCIs through force-triggered reactions, thus avoiding the broadening of printed features due to the diffusion of inks. We discovered that the ubiquitously presented amino groups in biomolecules can react with maleimide through a force-triggered Michael addition. The resulting covalent linkage is mechanically and chemically stable. As a proof-of-concept, we fabricate patterned surfaces of biotin and His-tagged proteins at nanoscale spatial resolution by MCL and verify the resulting patterns by fluorescence imaging. We further demonstrated the creation of multiplex protein patterns using this technique.

Aponeurosis discission, a low-detergent method for tissue-engineered acellular ligament scaffolds.

Detergent treatment is the most commonly used method for the decellularization of ligaments and tendon grafts. However, it is well recognized that detergent treatment can also adversely affect the extracellular matrix. This study found that discission into the aponeurosis layer of the patellar tendon (PT) before decellularization is conducive to extracting cells from the PT using a low quantity of detergent in a short time period. The acellular aponeurosis discission ligament (AADL) retains its native collagen fibril structure and mechanical properties. Moreover, the PT retained cell and tissue compatibility in vitro and in vivo. After implantation into a defective allogeneic PT, we found that the AADL healed well in the host, and its collagen structure exhibited gradual improvement 12 months after implantation with satisfactory reconstruction. IMPACT: The aponeurosis of tendons/ligaments is the main barrier to achieving complete decellularization, and it thus prevents complete recellularization for applications in tissue engineering. Aponeurosis can obstruct the removal of cell components. We found that excising the aponeurosis before decellularization allows for the removal of cellular components with a reduced amount of detergent, thus improving the biological properties of the acellular ligament. To the best of our knowledge, no similar studies have been performed. Graphical abstract.

Principles Governing Catalytic Activity of Self-Assembled Short Peptides.

Molecular self-assembly provides a chemical strategy for the synthesis of nanostructures by using the principles of nature, and peptides serve as the promising building blocks to construct adaptable molecular architectures. Recently, a series of heptapeptides with alternative hydrophobic and hydrophilic residues were reported to form amyloid-like structures, which are capable of catalyzing acyl ester hydrolysis with remarkable efficiency. However, information remains elusive about the atomic structures of the fibrils. What is the origin of the sequence-dependent catalytic activity? How is the ester hydrolysis catalyzed by the fibrils? In this work, the atomic structures of the aggregates were determined by using molecular modeling and further validated by solid-state NMR experiments, where the fibril with high activity adopts twisted parallel configuration within each layer, and the one with low activity is in flat antiparallel configuration. The polymorphism originates from the interactions between different regions of the building block peptides, where the delicate balance between rigidity and flexibility plays an important role. We further show that the p-nitrophenylacetate ( pNPA) hydrolysis reactions catalyzed by two different fibrils follow a similar mechanism, and the difference in microenvironment at the active site between the natural enzyme and the present self-assembled fibrils should account for the discrepancy in catalytic activities. The present work provides understanding of the structure and function of self-assembled fibrils formed with short peptides at an atomic level and thus sheds new insight on designing aggregates with better functions.

Direct Measurement of Length Scale Dependence of the Hydrophobic Free Energy of a Single Collapsed Polymer Nanosphere.

The physics underlying hydrophobicity at macroscopic and microscopic levels is fundamentally distinct. However, experimentally quantifying the length scale dependence of hydrophobicity is challenging. Here we show that the size-dependent hydrophobic free energy of a collapsed polymer nanosphere can be continuously monitored from its single-molecule force-extension curve using a novel theoretical framework. The hydrophobic free energy shows a change from cubic to square dependence of the radius of the polymer nanosphere at a radius of ∼1 nm-this is consistent with Lum-Chandler-Weeks theory and simulations. We can also observe a large variation of the hydrophobic free energy of each polymer nanosphere implying the heterogeneity of the self-assembled structures and/or the fluctuation of the water-polymer interface. We expect that our approach can be used to address many fundamental questions about hydrophobic hydration, which are otherwise inaccessible by ensemble measurements.

Dimerization of Cell-Adhesion Molecules Can Increase Their Binding Strength.

Cell-adhesion molecules (CAMs) often exist as homodimers under physiological conditions. However, owing to steric hindrance, simultaneous binding of two ligands to the homodimers at the same location can hardly be satisfied, and the molecular mechanism underlying this natural design is still unknown. Here, we present a theoretical model to understand the rupture behavior of cell-adhesion bonds formed by multiple binding ligands with a single receptor. We found that the dissociation forces for the cell-adhesion bond could be greatly enhanced in comparison with the monomer case through a ligand rebinding and exchange mechanism. We also confirmed this prediction by measuring dimeric cRGD (cyclic Arg-Gly-Asp) unbinding from integrin (αvß3) using atomic force microscopy-based single-molecule force spectroscopy. Our finding addresses the mechanism of increasing the binding strength of cell-adhesion bonds through dimerization at the single-molecule level, representing a key step toward the understanding of complicated cell-adhesion behaviors. Moreover, our results also highlight a wealth of opportunities to design mechanically stronger bioconjunctions for drug delivery, biolabeling, and surface modification.

Mg2+ -Dependent High Mechanical Anisotropy of Three-Way-Junction pRNA as Revealed by Single-Molecule Force Spectroscopy.

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three-way-junction (3WJ) pRNA, derived from ϕ29 DNA packaging motor, shows strong mechanical anisotropy upon Mg2+ binding. In the absence of Mg2+ , 3WJ-pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg2+ , the unfolding forces can differ by more than 4-fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ-pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg2+ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ-pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.

In situ single-molecule investigations of the impacts of biochemical perturbations on conformational intermediates of monomeric α-synuclein.

α-Synuclein aggregation is a common trait in synucleinopathies, including Parkinson's disease. Being an unstructured protein, α-synuclein exists in several distinct conformational intermediates, contributing to both its function and pathogenesis. However, the regulation of these monomer conformations by biochemical factors and potential drugs has remained elusive. In this study, we devised an in situ single-molecule manipulation approach to pinpoint kinetically stable conformational intermediates of monomeric α-synuclein and explore the effects of various biochemical factors and drugs. We uncovered a partially folded conformation located in the non-amyloid-ß component (NAC) region of monomeric α-synuclein, which is regulated by a preNAC region. This conformational intermediate is sensitive to biochemical perturbations and small-molecule drugs that influencing α-synuclein's aggregation tendency. Our findings reveal that this partially folded intermediate may play a role in α-synuclein aggregation, offering fresh perspectives for potential treatments aimed at the initial stage of higher-order α-synuclein aggregation. The single-molecule approach developed here can be broadly applied to the study of disease-related intrinsically disordered proteins.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Squalene monooxygenase facilitates bladder cancer development in part by regulating PCNA.

Bladder cancer (BC) is one of the most common cancers worldwide. Although the treatment and survival rate of BC are being improved, the risk factors and the underlying mechanisms causing BC are incompletely understood. Squalene monooxygenase (SQLE) has been associated with the occurrence and development of multiple cancers but whether it contributes to BC development is unclear. In this study, we performed bioinformatics analysis on paired BC and adjacent non-cancerous tissues and found that SQLE expression is significantly upregulated in BC samples. Knockdown of SQLE impairs viability, induces apoptosis, and inhibits the migration and invasion of BC cells. RNA-seq data reveals that SQLE deficiency leads to dysregulated expression of genes regulating proliferation, migration, and apoptosis. Mass spectrometry-directed interactome screening identifies proliferating cell nuclear antigen (PCNA) as an SQLE-interacting protein and overexpression of PCNA partially rescues the impaired viability, migration, and invasion of BC cells caused by SQLE knockdown. In addition, we performed xenograft assays and confirmed that SQLE deficiency inhibits BC growth in vivo. In conclusion, these data suggest that SQLE promotes BC development and SQLE inhibition may be therapeutically useful in BC treatment.

Single-Cell Quantification of the Mechanical Stability of Cell-Cell Adherens Junction Using Glass Micropipettes.

Micropipette-based methods have been widely used for the manipulation of cells and characterization of the mechanical properties at the cell or tissue level. Here, we introduce the glass micropipette-based mechanical assays for the stability of cell-cell adhesion. A probing microbead coated with specific adhesion ligands, captured by a glass micropipette, is manipulated to form the adhesion complexes with the corresponding receptors on a single cell. Once the cell is moving away from the micropipette, forces are generated from 20 pN to 100 nN to the adhesion complexes, which are quantified in real-time based on the bending of the glass micropipette. We specifically emphasize the principle and method to probe the rupturing forces of the adhesion complexes at controlled force loading rates, the ligand coating on the probe microbeads, the force calibration of the glass micropipette, and the applications of the method to probe the E-cadherin-based cell-cell adhesions. The principles can be broadly applied to other cell adhesions such as cell-matrix adhesions, neuronal synapses, and bacterial-cell adhesions.

Tension Gauge Tethers as Tension Threshold and Duration Sensors.

Mechanotransduction, the process by which cells respond to tension transmitted through various supramolecular linkages, is important for understanding cellular behavior. Tension gauge tethers (TGTs), short fragments of double-stranded DNA that irreversibly break under shear-stretch conditions, have been used in live cell experiments to study mechanotransduction. However, our current understanding of TGTs' mechanical responses is limited, which limits the information that can be gleaned from experimental observations. In this study, we quantified the tension-dependent lifetime of TGTs to better understand their mechanical stability under various physiologically relevant stretching conditions. This work has broad applications for using TGTs as tension threshold and duration sensors and also suggests the need to revisit previous interpretations of experimental observations.

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

Modulation of E-Cadherin Function through the AmotL2 Isoforms Promotes Ameboid Cell Invasion.

The spread of tumor cells and the formation of distant metastasis remain the main causes of mortality in cancer patients. However, the mechanisms governing the release of cells from micro-environmental constraints remain unclear. E-cadherin negatively controls the invasion of epithelial cells by maintaining cell-cell contacts. Furthermore, the inactivation of E-cadherin triggers invasion in vitro. However, the role of E-cadherin is complex, as metastasizing cells maintain E-cadherin expression, which appears to have a positive role in the survival of tumor cells. In this report, we present a novel mechanism delineating how E-cadherin function is modulated to promote invasion. We have previously shown that E-cadherin is associated with p100AmotL2, which is required for radial actin formation and the transmission of mechanical force. Here, we present evidence that p60AmotL2, which is expressed in invading tumor cells, binds to the p100AmotL2 isoform and uncouples the mechanical constraint of radial actin filaments. We show for the first time that the coupling of E-cadherin to the actin cytoskeleton via p100AmotL2 is directly connected to the nuclear membrane. The expression of p60AmotL2 inactivates this connection and alters the properties of the nuclear lamina, potentiating the invasion of cells into micropores of the extracellular matrix. In summary, we propose that the balance of the two AmotL2 isoforms is important in the modulation of E-cadherin function and that an imbalance of this axis promotes ameboid cell invasion.

Actin polymerisation and crosslinking drive left-right asymmetry in single cell and cell collectives.

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures. Screening based on deep-learning analysis of actin patterns identified actin polymerisation regulators, depletion of which suppresses chirality (mDia1) or reverses chirality direction (profilin1 and CapZß). The reversed chirality  is mDia1-independent but requires the function of actin-crosslinker α-actinin1. A robust correlation between the effects of a variety of actin assembly regulators on chirality of individual cells and cell collectives is revealed. Thus, actin-driven cell chirality may underlie tissue and organ asymmetry.

Preparation and biological characteristics of a bovine acellular tendon fiber material.

Acellular tendon matrix is an ideal substitute for constructing tissue engineering ligaments, but using detergents causes damage to collagen and fibrin during the process of decellularization. In this study, fresh tendons were lyophilized and separated into fresh tendon fiber (FTF) bundles, and then the cellular components in FTF were removed to prepare acellular tendon fiber (ATF) without adding chemical detergent. H&E staining and DAPI fluorescence microscopy showed no nucleus and DNA residue. Compared with FTFs, the DNA content of ATFs was significantly lower without the collagen content change before and after decellularization. The microstructure of collagen fibrils in ATFs was intact under scanning electron microscopy (SEM), and the maximum tensile load and elastic modulus between FTFs and ATFs were not statistically different. The ATF bundles were cultured with SD rat tenocytes for 72 hr and cells attachment to fiber surfaces were observed under SEM. ATF bundles were then implanted into paraspinal muscles, and histological analysis showed fibroblast-like cells within the ATFs and was similar to the control group (fresh tendon autograft) in morphology. H&E staining showed that the number of lymphocytes and plasma cells in ATF was less than that in fresh tendon autograft. ATF bundles were twisted into linear fiber materials by hand, of which the maximum breaking strength was similar to silk with same diameter. These findings demonstrated that ATFs retain their original fibril structure and mechanical properties after decellularization by trypsin and pancreatic deoxyribonuclease without detergent. Lyophilized ATFs linear fiber material provides the possibility of preparing personalized ligament and other tissue engineering scaffolds.

Electronic metal-support interaction modulates single-atom platinum catalysis for hydrogen evolution reaction.

Tuning metal-support interaction has been considered as an effective approach to modulate the electronic structure and catalytic activity of supported metal catalysts. At the atomic level, the understanding of the structure-activity relationship still remains obscure in heterogeneous catalysis, such as the conversion of water (alkaline) or hydronium ions (acid) to hydrogen (hydrogen evolution reaction, HER). Here, we reveal that the fine control over the oxidation states of single-atom Pt catalysts through electronic metal-support interaction significantly modulates the catalytic activities in either acidic or alkaline HER. Combined with detailed spectroscopic and electrochemical characterizations, the structure-activity relationship is established by correlating the acidic/alkaline HER activity with the average oxidation state of single-atom Pt and the Pt-H/Pt-OH interaction. This study sheds light on the atomic-level mechanistic understanding of acidic and alkaline HER, and further provides guidelines for the rational design of high-performance single-atom catalysts.