Mian Long

Direct observation of catch bonds involving cell-adhesion molecules

Bonds between adhesion molecules are often mechanically stressed. A striking example is the tensile force applied to selectin–ligand bonds, which mediate the tethering and rolling of flowing leukocytes on vascular surfaces. It has been suggested that force could either shorten bond lifetimes, because work done by the force could lower the energy barrier between the bound and free states (‘slip’), or prolong bond lifetimes by deforming the molecules such that they lock more tightly (‘catch’). Whereas slip bonds have been widely observed, catch bonds have not been demonstrated experimentally. Here, using atomic force microscopy and flow-chamber experiments, we show that increasing force first prolonged and then shortened the lifetimes of P-selectin complexes with P-selectin glycoprotein ligand-1, revealing both catch and slip bond behaviour. Transitions between catch and slip bonds might explain why leukocyte rolling on selectins first increases and then decreases as wall shear stress increases. This dual response to force provides a mechanism for regulating cell adhesion under conditions of variable mechanical stress.

Measuring Receptor/Ligand Interaction at the Single-Bond Level: Experimental and Interpretative Issues

AbstractThere is increased interest in measuring kinetic rates, lifetimes, and rupture forces of single receptor/ligand bonds. Valuable insights have been obtained from previous experiments attempting such measurements. However, it remains difficult to know with sufficient certainty that single bonds were indeed measured. Using exemplifying data, evidence supporting single-bond observation is examined and caveats in the experimental design and data interpretation are identified. Critical issues preventing definitive proof and disproof of single-bond observation include complex binding schemes, multimeric interactions, clustering, and heterogeneous surfaces. It is concluded that no single criterion is sufficient to ensure that single bonds are actually observed. However, a cumulative body of evidence may provide reasonable confidence.

Differential regulation of stiffness, topography, and dimension of substrates in rat mesenchymal stem cells

The physiological microenvironment of the stem cell niche, including the three factors of stiffness, topography, and dimension, is crucial to stem cell proliferation and differentiation. Although a growing body of evidence is present to elucidate the importance of these factors individually, the interaction of the biophysical parameters of the factors remains insufficiently characterized, particularly for stem cells. To address this issue fully, we applied a micro-fabricated polyacrylamide hydrogel substrate with two elasticities, two topographies, and three dimensions to systematically test proliferation, morphology and spreading, differentiation, and cytoskeletal re-organization of rat bone marrow mesenchymal stem cells (rBMSCs) on twelve cases. An isolated but not combinatory impact of the factors was found regarding the specific functions. Substrate stiffness or dimension is predominant in regulating cell proliferation by fostering cell growth on stiff, unevenly dimensioned substrate. Topography is a key factor for manipulating cell morphology and spreading via the formation of a large spherical shape in a pillar substrate but not in a grooved substrate. Although stiffness leads to osteogenic or neuronal differentiation of rBMSCs on a stiff or soft substrate, respectively, topography or dimension also plays a lesser role in directing cell differentiation. Neither an isolated effect nor a combinatory effect was found for actin or tubulin expression, whereas a seemingly combinatory effect of topography and dimension was found in manipulating vimentin expression. These results further the understandings of stem cell proliferation, morphology, and differentiation in a physiologically mimicking microenvironment.

Probabilistic modeling of shear-induced formation and breakage of doublets cross-linked by receptor-ligand bonds.

A model was constructed to describe previously published experiments of shear-induced formation and breakage of doublets of red cells and of latexes cross-linked by receptor-ligand bonds (. Biophys. J. 65:1318-1334; Tees and Goldsmith. 1996. Biophys. J. 71:1102-1114;. Biophys. J. 71:1115-1122). The model, based on McQuarries master equations (1963. J. Phys. Chem. 38:433-436), provides unifying treatments for three distinctive time periods in the experiments of particles in a Couette flow in which a doublet undergoes 1) formation upon two-body collision between singlets; 2) evolution of bonds at low shear rate; and 3) break-up at high shear rate. Neglecting the applied force at low shear rate, the probability of forming a doublet per collision as well as the evolution of probability distribution of bonds in a preformed doublet were solved analytically and found to be in quite good agreement with measurements. At high shear rate with significant force acting to accelerate bond dissociation, the predictions for break-up of doublets were obtained numerically and compared well with data in both individual and population studies. These comparisons enabled bond kinetic parameters for three types of particles cross-linked by two receptor-ligand systems to be calculated, which agreed well with those computed from Monte Carlo simulations. This work can be extended to analyze kinetics of receptor-ligand binding in cell aggregates, such as those of neutrophils and platelets in the circulation.

Differential regulation of morphology and stemness of mouse embryonic stem cells by substrate stiffness and topography

The maintenance of stem cell pluripotency or stemness is crucial to embryonic development and differentiation. The mechanical or physical microenvironment of stem cells, which includes extracellular matrix stiffness and topography, regulates cell morphology and stemness. Although a growing body of evidence has shown the importance of these factors in stem cell differentiation, the impact of these biophysical or biomechanical regulators remains insufficiently characterized. In the present study, we applied a micro-fabricated polyacrylamide hydrogel substrate with two elasticities and three topographies to systematically test the morphology, proliferation, and stemness of mESCs. The independent or combined impact of the two factors on specific cell functions was analyzed. Cells are able to grow effectively on both polystyrene and polyacrylamide substrates in the absence of feeder cells. Substrate stiffness is predominant in preserving stemness by enhancing Oct-4 and Nanog expression on a soft polyacrylamide substrate. Topography is also a critical factor for manipulating stemness via the formation of a relatively flattened colony on a groove or pillar substrate and a spheroid colony on a hexagonal substrate. Although topography is less effective on soft substrates, it plays a role in retaining cell stemness on stiff, hexagonal or pillar-shaped substrates. mESCs also form, in a timely manner, a 3D structure on groove or hexagonal substrates. These results further the understanding of stem cell morphology and stemness in a microenvironment that mimics physiological conditions.

Impact of Carrier Stiffness and Microtopology on Two-dimensional Kinetics of P-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1) Interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.

Two-dimensional kinetics of β2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.

Mechanomics: an emerging field between biology and biomechanics

Cells sense various in vivo mechanical stimuli, which initiate downstream signaling to mechanical forces. While a body of evidences is presented on the impact of limited mechanical regulators in past decades, the mechanisms how biomechanical responses globally affect cell function need to be addressed. Complexity and diversity of in vivo mechanical clues present distinct patterns of shear flow, tensile stretch, or mechanical compression with various parametric combination of its magnitude, duration, or frequency. Thus, it is required to understand, from the viewpoint of mechanobiology, what mechanical features of cells are, why mechanical properties are different among distinct cell types, and how forces are transduced to downstream biochemical signals. Meanwhile, those in vitro isolated mechanical stimuli are usually coupled together in vivo, suggesting that the different factors that are in effect individually could be canceled out or orchestrated with each other. Evidently, omics analysis, a powerful tool in the field of system biology, is advantageous to combine with mechanobiology and then to map the full-set of mechanically sensitive proteins and transcripts encoded by its genome. This new emerging field, namely mechanomics, makes it possible to elucidate the global responses under systematically-varied mechanical stimuli. This review discusses the current advances in the related fields of mechanomics and elaborates how cells sense external forces and activate the biological responses.

Determining β2-Integrin and Intercellular Adhesion Molecule 1 Binding Kinetics in Tumor Cell Adhesion to Leukocytes and Endothelial Cells by a Gas-driven Micropipette Assay

Interactions between polymorphonuclear neutrophils (PMNs) and tumor cells have been reported to facilitate the adhesion and subsequent extravasation of tumor cells through the endothelium under blood flow, both of which are mediated by binding β2-integrin to intercellular adhesion molecule 1 (ICAM-1). Here the adhesions between human WM9 metastatic melanoma cells, PMNs, and human pulmonary microvascular endothelial cells (HPMECs) were quantified by a gas-driven micropipette aspiration technique (GDMAT). Our data indicated that the cellular binding affinity of PMN-WM9 pair was 3.9-fold higher than that of the PMN-HPMEC pair. However, the effective binding affinities per molecular pair were comparable between the two cell pairs no matter whether WM9 cells or HPMECs were quiescent or cytokine-activated, indicating that the stronger adhesion between PMN-WM9 pair is mainly attributed to the high expression of ICAM-1 on WM9 cells. These results proposed an alternative mechanism, where WM9 melanoma cells adhere first with PMNs near vessel-wall regions and then bind to endothelial cells via PMNs under blood flow. In contrast, the adhesions between human MDA-MB-231 metastatic breast carcinoma cells and PMNs showed a comparable cellular binding affinity to PMN-HPMEC pair because the ICAM-1 expressions on MDA-MB-231 cells and HPMECs are similar. Furthermore, differences were observed in the intrinsic forward and reverse rates of the β2-integrin-ICAM-1 bond between PMN-TC and PMN-EC pairs. This GDMAT assay enables us to quantify the binding kinetics of cell adhesion molecules physiologically expressed on nucleated cells. The findings also further the understanding of leukocyte-facilitated tumor cell adhesion from the viewpoint of molecular binding kinetics.

Low Spring Constant Regulates P-Selectin-PSGL-1 Bond Rupture

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 r(f) (>or=10(2) pN/s), defined as the product of spring constant k and retract velocity v, how the low r(f) (<10(2) pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r(f) <or= 20 pN/s with low k ( approximately 10(-3)-10(-2) pN/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 r(f) increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at the same r(f). The most probable force, f*, was enhanced with the spring constant when k < 47.0 x 10(-3) pN/nm, indicating that the bond dissociation at low r(f) 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 r(f) or k.