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Atomic force microscopy (AFM) has been widely used to acquire surface topography upon different scanning modes and to quantify mechanical properties of a cell using single-point ramp force mode. However, these traditional measurements need massive force curves originating from multiple points of a cell to exclude the potential errors resulted from limited and factitious selections of testing points, making the measurements time-consuming and highly localized. PeakForce Quantitative NanoMechanics (PF QNM) is a high-speed (faster by 3–4 order of magnitude) and global surface mechanical property mapping method with high spatial resolution, overcoming the drawbacks of traditional ramp mode especially used for a live cell with high heterogeneity. In this protocol, we elaborated how to run PF QNM measurements for live cells and relevant modification may be needed when extending this method to other cell-like soft materials.

Synthetic signaling receptors enable programmable cellular responses coupling with customized inputs. However, engineering a designer force-sensing receptor to rewire mechanotransduction remains largely unexplored. Herein, we introduce nongenetically engineered artificial mechanoreceptors (AMRs) capable of reprogramming non-mechanoresponsive receptor tyrosine kinases (RTKs) to sense user-defined force cues, enabling de novo-designed mechanotransduction. AMR is a modular DNA–protein chimera comprising a mechanosensing-and-transmitting DNA nanodevice grafted on natural RTKs via aptameric anchors. AMR senses intercellular tensile force via an allosteric DNA mechano-switch with tunable piconewton-sensitive force tolerance, actuating a force-triggered dynamic DNA assembly to manipulate RTK dimerization and activate intracellular signaling. By swapping the force-reception ligands, we demonstrate the AMR-mediated activation of c-Met, a representative RTK, in response to the cellular tensile forces mediated by cell-adhesion proteins (integrin, E-cadherin) or membrane protein endocytosis (CI-M6PR). Moreover, AMR also allows the reprogramming of FGFR1, another RTK, to customize mechanobiological function, for example, adhesion-mediated neural stem cell maintenance.

Migrating neutrophils are found to leave behind subcellular trails in vivo, but the underlying mechanisms remain unclear. Here, an in vitrocell migration test plus an in vivoobservation was applied to monitor neutrophil migration on intercellular cell adhesion molecule-1 (ICAM-1) presenting surfaces. Results indicated that migrating neutrophils left behind long-lasting, chemokine-containing trails. Trail formation tended to alleviate excessive cell adhesion enhanced by the trans-binding antibody and maintain efficient cell migration, which was associated with differential instantaneous edge velocity between the cell front and rear. CD11a and CD11b worked differently in inducing trail formation with polarized distributions on the cell body and uropod. Trail release at the cell rear was attributed to membrane ripping, in which β2-integrin was disrupted from the cell membrane through myosin-mediated rear contraction and integrin–cytoskeleton dissociation, potentiating a specialized strategy of integrin loss and cell deadhesion to maintain efficient migration. Moreover, neutrophil trails left on the substrate served as immune forerunners to recruit dendritic cells. These results provided an insight in elucidating the mechanisms of neutrophil trail formation and deciphering the roles of trail formation in efficient neutrophil migration.

Double-layered channels of sinusoid lumen and Disse space separated by fenestrated liver sinusoidal endothelial cells (LSECs) endow the unique mechanical environment of the liver sinusoid network, which further guarantees its biological function. It is also known that this mechanical environment changes dramatically under liver fibrosis and cirrhosis, including the reduced plasma penetration and metabolite exchange between the two flow channels and the reduced Disse space deformability. The squeezing of leukocytes through narrow sinusoid lumen also affects the mechanical environment of liver sinusoid. To date, the detailed flow-field profile of liver sinusoid is still far from clear due to experimental limitations. It also remains elusive whether and how the varied physical properties of the pathological liver sinusoid regulate the fluid flow characteristics. Here a numerical model based on the immersed boundary method was established, and the effects of Disse space and leukocyte elasticities, endothelium permeability, and sinusoidal stenosis degree on fluid flow as well as leukocyte trafficking were specified upon a mimic liver sinusoid structure. Results showed that endothelium permeability dominantly controlled the plasma penetration velocity across the endothelium, whereas leukocyte squeezing promoted local penetration and significantly regulated wall shear stress on hepatocytes, which was strongly related to the Disse space and leukocyte deformability. Permeability and elasticity cooperatively regulated the process of leukocytes trafficking through the liver sinusoid, especially for stiffer leukocytes. This study will offer new insights into deeper understanding of the elaborate mechanical features of liver sinusoid and corresponding biological function.

Mechanical or physical cues are associated with the growth and differentiation of embryonic stem cells (ESCs). While the substrate stiffness or topography independently affects the differentiation of ESCs, their cooperative regulation on lineage-specific differentiation remains largely unknown. Here, four topographical configurations on stiff or soft polyacrylamide hydrogel were combined to direct hepatic differentiation of human H1 cells viaa four-stage protocol, and the coupled impacts of stiffness and topography were quantified at distinct stages. Data indicated that the substrate stiffness is dominant in stemness maintenance on stiff gel and hepatic differentiation on soft gel while substrate topography assists the differentiation of hepatocyte-like cells in positive correlation with the circularity of H1 clones initially formed on the substrate. The differentiated cells exhibited liver-specific functions such as maintaining the capacities of CYP450 metabolism, glycogen synthesis, ICG engulfment, and repairing liver injury in CCl4-treated mice. These results implied that the coupling of substrate stiffness and topography, combined with the biochemical signals, is favorable to improve the efficiency and functionality of hepatic differentiation of human ESCs.

Integrins are heterodimeric transmembrane proteins that mediate cellular adhesion and bidirectional mechanotransductions through their conformational allostery. The allosteric pathway of an I-domain-containing integrin remains unclear because of its complexity and lack of effective experiments. For a typical I-domain-containing integrin αXβ2, molecular dynamics simulations were employed here to investigate the conformational dynamics in the first two steps of outside-in activation, the bindings of both the external and internal ligands. Results showed that the internal ligand binding is a prerequisite to the allosteric transmission from the α- to β-subunits and the exertion of external force to integrin-ligand complex. The opening state of αI domain with downward movement and lower half unfolding of α7-helix ensures the stable intersubunit conformational transmission through external ligand binding first and internal ligand binding later. Reverse binding order induces a, to our knowledge, novel but unstable swingout of β-subunit Hybrid domain with the retained close states of both αI and βI domains. Prebinding of external ligand greatly facilitates the following internal ligand binding and vice versa. These simulations furthered the understanding in the outside-in activation of I-domain-containing integrins from the viewpoint of internal allosteric pathways.

Global cytoskeleton reorganization is well-recognized when cells are exposed to distinct mechanical stimuli, but the localized responses at a specified region of a cell are still unclear. In this work, we mapped the cell-surface mechanical property of single cells in situ before and after static point loading these cells using atomic force microscopy in PeakForce-Quantitative Nano Mechanics mode. Cell-surface stiffness was elevated at a maximum of 1.35-fold at the vicinity of loading site, indicating an enhanced structural protection of the cortex to the cell. Mechanical modeling also elucidated the structural protection from the stiffened cell cortex, in which 9–15% and 10–19% decrease of maximum stress and strain of the nucleus were obtained. Furthermore, the flat-ended atomic force microscopy probes were used to capture cytoskeleton reorganization after point loading quantitatively, revealing that the larger the applied force and the longer the loading time are, the more pronounced cytoskeleton reorganization is. Also, point loading using a microneedle combined with real-time confocal microscopy uncovered the fast dynamics of actin cytoskeleton reorganization for actin-stained live cells after point loading (<10 s). These results furthered the understandings in the transmission of localized mechanical forces into an adherent cell.

The force spectra for various LFA-1/Mac-1–ligand bonds were compared and their functions tested in mediating PMN recruitment under flow. Multiple endothelial ligands present distinct bond rupture forces and lifetimes, which correlate well with their biological phenotypes. In addition to ICAM, JAM or RAGE is also crucial in cell adhesion and crawling.

How appendages, such as plant leaves or animal limbs, develop asymmetric shapes remains a fundamental question in biology. Although ongoing research has revealed the genetic regulation of organ pattern formation, how gene activity ultimately directs organ shape remains unclear. Here, we show that leaf dorsoventral (adaxial-abaxial) polarity signals lead to mechanical heterogeneity of the cell wall, related to the methyl-esterification of cell-wall pectins in tomato and Arabidopsis. Numerical simulations predicate that mechanical heterogeneity is sufficient to produce the asymmetry seen in planar leaves. Experimental tests that alter pectin methyl-esterification, and therefore cell wall mechanical properties, support this model and lead to polar changes in gene expression, suggesting the existence of a feedback mechanism for mechanical signals in morphogenesis. Thus, mechanical heterogeneity within tissue may underlie organ shape asymmetry. Mechanical heterogeneity due to differences in methyl-esterification of cell wall pectins is sufficient to produce asymmetric leaf morphology, as predicted by a mathematical model and validated in Arabidopsisand tomato.

Blood cell aggregation and adhesion to endothelial cells under shear flow are crucial to many biological processes such as thrombi formation, inflammatory cascade, and tumor metastasis, in which these cellular interactions are mainly mediated by the underlying receptor–ligand bindings. While theoretical modeling of aggregation dynamics and adhesion kinetics of interacting cells have been well studied separately, how to couple these two processes remains unclear. Here we develop a combined model that couples cellular aggregation dynamics and adhesion kinetics under shear flow. The impacts of shear rate (or shear stress) and molecular binding affinity were elucidated. This study provides a unified model where the action of a fluid flow drives cell aggregation and adhesion under the modulations of the mechanical shear flow and receptor–ligand interaction kinetics. It offers an insight into understanding the relevant biological processes and functions.

•Membrane fusion plays key roles on cellular or subcellular activities and membrane fusion dynamics are crucial for understanding corresponding functions.•Here we reviewed the advances in dynamic features of membrane fusion, the regulation mechanism of lipid composition, mechanical microenvironment, protein and ion on the fusion dynamics, as well as the theoretical model for describing the process.•Future perspectives were also proposed.

Binding of selectins to P-selectin glycoprotein ligand-1 (PSGL-1) mediates tethering and rolling of leukocytes on the endothelium during inflammation. Previous measurements obtained with a flow-chamber assay have shown that mutations of three tyrosines at the PSGL-1 N-terminus (Y46, Y48, and Y51) increase the reverse rates and their sensitivity to the force of bonds with P- and L-selectin. However, the effects of these mutations on the binding affinities and forward rates have not been studied. We quantified these effects by using an adhesion frequency assay to measure two-dimensional affinity and kinetic rates at zero force. Wild-type PSGL-1 has 2.2- to 8.5-fold higher binding affinities for P- and L-selectin than PSGL-1 mutants with two of three tyrosines substituted by phenylalanines, and 9.6- to 49-fold higher affinities than the PSGL-1 mutant with all three tyrosines replaced. In descending order, the affinity decreased from wild-type to Y48/51F, Y46/51F, Y46/48F, and Y46/48/51F. The affinity differences were attributed to major changes in the forward rate and minor changes in the reverse rate, suggesting that these tyrosines regulate the accessibility of PSGL-1 to P- and L-selectin viaelectrostatic interactions, which is supported by molecular-dynamics simulations. Our results provide insights into the structure-function relationship of receptor-ligand binding at a single-residue level.

By mediating the tethering and rolling of leukocytes on vascular surfaces, the interactions between P-selectin and the P-selectin glycoprotein ligand 1 (PSGL-1) play crucial roles during inflammation cascade. Tensile stretch produced by rolling leukocytes and shear stress exerted by blood flow constitute the two types of mechanical forces that act on the P-selectin/PSGL-1 bond. These forces modulate not only dissociation kinetics of this bond, but also the leukocyte adhesion dynamics. However, the respective contribution of the two forces to bond dissociation and to the corresponding microstructural bases remains unclear. To mimic the mechanical microenvironment, we developed two molecular dynamics approaches; namely, an approach involving the shear flow field with a controlled velocity gradient, and the track dragging approach with a defined trajectory. With each approach or with both combined, we investigate the microstructural evolution and dissociation kinetics of the P-LE/SGP-3 construct, which is the smallest functional unit of the P-selectin/PSGL-1 complex. The results demonstrate that both shear flow and tensile stretch play important roles in the collapse of the construct and that, before bond dissociation, the former causes more destruction of domains within the construct than the latter. Dissociation of the P-LE/SGP-3 construct features intramolecular destruction of the epidermal-growth-factor (EGF) domain and the breaking of hydrogen-bond clusters at the P-selectin-lectin/EGF interface. Thus, to better understand how mechanics impacts the dissociation kinetics of the P-selectin/PSGL-1 complex, we propose herein two approaches to mimic its physiological mechanical environment.

Abstract: Receptor-ligand bond dissociation under applied force is crucial to elucidate its biological functionality when the molecular bond is usually connected to a mechanical probe. While the impact of probe stiffness, k, on bond rupture force has recently attracted more and more attention, the mechanism of how it affects the bond lifetime, however, remains unclear. Here we quantified the dissociation lifetime of selectin-ligand bond using an optical trap assay with low stiffness ranging from 3.5×10−3 to 4.7×10−2 pN/nm. Our results indicated that bond lifetime yielded distinct distributions with different probe stiffness, implying the stochastic feature of bond dissociation. It was also found that the mean lifetime varied with probe stiffness and that the catch bond nature was visualized at k⩾3.0×10−2 pN/nm. This work furthered the understanding of the forced dissociation of selectin-ligand bond at varied probe stiffness, which is physiologically relevant to the tethered rolling of leukocytes under blood flow.

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate rf (≥102pN/s), defined as the product of spring constant k and retract velocity v, how the low rf (<102pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at rf≤20pN/s with low k (∼10−3–10−2pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when rf increased up to 20pN/s. It was also found that f varied with different combinations of k and v even at the same rf. The most probable force, f*, was enhanced with the spring constant when k<47.0×10−3pN/nm, indicating that the bond dissociation at low rf was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low rf or k.

Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitrohepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high, but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations are not only consistent with experimental measurements using in vitroliver sinusoidal chip, but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.