Stabilizing to disruptive transition of focal adhesion response to mechanical forces

Strong mechanical forces can, obviously, disrupt cell-cell and cell-matrix adhesions, e.g., cyclic uniaxial stretch induces instability of cell adhesion, which then causes the reorientation of cells away from the stretching direction. However, recent experiments also demonstrated the existence of force dependent adhesion growth (rather than dissociation). To provide a quantitative explanation for the two seemingly contradictory phenomena, a microscopic model that includes both integrin-integrin interaction and integrin-ligand interaction is developed at molecular level by treating the focal adhesion as an adhesion cluster. The integrin clustering dynamics and integrin-ligand binding dynamics are then simulated within one unified theoretical frame with Monte Carlo simulation. We find that the focal adhesion will grow when the traction force is higher than a relative small threshold value, and the growth is dominated by the reduction of local chemical potential energy by the traction force. In contrast, the focal adhesion will rupture when the traction force exceeds a second threshold value, and the rupture is dominated by the breaking of integrin-ligand bonds. Consistent with the experiments, these results suggest a force map for various responses of cell adhesion to different scales of mechanical force. PMID: 20542514

Stability Of Adhesion Clusters And Cell Reorientation Under Lateral Cyclic Tension

This work is motivated by experimental observations that cells on stretched substrate exhibit different responses to static and dynamic loads. A model of focal adhesion that can consider the mechanics of stress fiber, adhesion bonds, and substrate was developed at the molecular level by treating the focal adhesion as an adhesion cluster. The stability of the cluster under dynamic load was studied by applying cyclic external strain on the substrate. We show that a threshold value of external strain amplitude exists beyond which the adhesion cluster disrupts quickly. In addition, our results show that the adhesion cluster is prone to losing stability under high-frequency loading, because the receptors and ligands cannot get enough contact time to form bonds due to the high-speed deformation of the substrate. At the same time, the viscoelastic stress fiber becomes rigid at high frequency, which leads to significant deformation of the bonds. Furthermore, we find that the stiffness and relaxation time of stress fibers play important roles in the stability of the adhesion cluster. The essence of this work is to connect the dynamics of the adhesion bonds (molecular level) with the cell's behavior during reorientation (cell level) through the mechanics of stress fiber. The predictions of the cluster model are consistent with experimental observations.

Two-Dimensional Kinetics Of Beta(2)-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.

细胞黏附的力学建模及其力敏感性的机理研究

细胞黏附在机体的生理和病理过程中起着重要的作用。作为细胞内、外信息交流和传递的通道，细胞黏附斑具有独特的力敏感性。实验表明，在力的作用下，黏附斑不仅可以生长、成熟和破坏，而且还能感知外部环境的力学性质，如基底硬度、硬度梯度和形貌等等。细胞黏附如何响应不同的力学刺激，物理机理是什么，如何定量描述这些物理机理？这些问题是细胞生物学和细胞力学中的重要问题。本论文通过在分子和亚细胞尺度上的力学建模研究了黏附斑的力敏感性机理，主要包括以下几方面的内容： (1) 发展了一个非线性的撕裂模型，研究了细胞黏附的稳定性和边缘依赖性。通过引入黏附分子键的非线性本构关系，并考虑黏附分子键的多种分布形式，我们发现黏附分子键的非线性效应对维持细胞黏附的稳定性起着至关重要的作用。黏附分子键的非线性力学性质使黏附分子键可以同时承载，降低了细胞对黏附分子键分布的依赖性，大大提高了细胞的黏附强度。本文的预测结果与实验结果一致。 (2) 建立了细胞黏附的细观力学模型，研究了在力作用下黏附斑生长和失稳的分子机理。在细观力学模型中，引入了“整联蛋白的聚集”和“整联蛋白-配体的反应”两个分子作用机理，并用两个化学反应来描述。通过基于Monte Carlo思想的Gillespie算法模拟了细胞黏附在不同载荷下的响应。我们发现黏附斑只能在一定范围的张力下生长，在这个范围内整联蛋白的聚集机制占主导。而当张力大于某个临界值时，黏附斑将失稳并导致破坏，这时整联蛋白-配体分子键的解离机制占主导。因此，黏附斑对作用力的不同响应，是不同分子作用机制在力作用下相互消长的结果。同时我们还建立了一个唯象的热力学模型中，验证了我们的细观力学模型。 (3) 基于细胞黏附的细观力学模型，研究了周期性载荷下细胞的重排和转向机理。在细观力学模型中，通过黏附块(adhesion plaque)，将整联蛋白-配体分子键和细胞骨架联系起来。基于Monte Calro模拟，我们发现存在一个载荷临界值，当外载大于临界值时，细胞将进行重排。细胞重排的原因是在周期性载荷下黏附斑的失稳。通过引入整联蛋白-配体成键的化学反应动力学和应力纤维的粘弹性性质，解释了细胞黏附稳定性的频率依赖性。本文预测的细胞转向临界载荷和重排方向，与实验结果一致。

Interaction between single molecules of Mac-1 and ICAM-1 in living cells: An atomic force microscopy study

The interaction between integrin macrophage differentiation antigen associated with complement three receptor function (Mac-1) and intercellular adhesion molecule-1 (ICAM-1), which is controlled tightly by the ligand-binding activity of Mac-1, is central to the regulation of neutrophil adhesion in host defense. Several "inside-out" signals and extracellular metal ions or antibodies have been found to activate Mac-1, resulting in an increased adhesiveness of Mac-1 to its ligands. However, the molecular basis for Mac-1 activation is not well understood yet. In this work, we have carried out a single-molecule study of Mac-1/ICAM-1 interaction force in living cells by atomic force microscopy (AFM). Our results showed that the binding probability and adhesion force of Mac-1 with ICAM-1 increased upon Mac-1 activation. Moreover, by comparing the dynamic force spectra of different Mac-1 mutants, we expected that Mac-1 activation is governed by the downward movement of its alpha 7 helix. (c) 2007 Elsevier Inc. All rights reserved.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

文昌鱼RACK1基因在胚胎发育中的表达

脊椎动物活化的(蛋白)激酶C受体1(receptor for activated C-kinase 1,RACK1)在胚胎发育过程中起着至关重要的作用.克隆了RACK1基因在青岛文昌鱼(Branchiostoma belcheri)中的同源基因AmphiRACK1,对其进行了系统的进化学分析,并研究了该基因在正常胚胎发育过程中和经LiCl处理胚胎发育中的时空表达图式.系统进化学分析结果表明,文昌鱼RACK1位于脊椎动物进化枝的基部.在正常胚胎发育中,AmphiRACK1基因在脑泡、神经管和体节中都有明显的表达.在经LiCl处理的胚胎中,该基因在体节中的分节型表达变模糊,表达下调;在脑泡和神经管中的表达下调甚至消失.此外,在成体文昌鱼的轮器、鳃血管、肝盲囊和肠上皮以及精巢中都检测到AmphiRACK1基因不同程度的表达.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Nonlinear Mechanical Modeling Of Cell Adhesion

Cell adhesion, which is mediated by the receptor-ligand bonds, plays an essential role in various biological processes. Previous studies often described the force-extension relationship of receptor-ligand bond with linear assumption. However, the force-extension relationship of the bond is intrinsically nonlinear, which should have significant influence on the mechanical behavior of cell adhesion. In this work, a nonlinear mechanical model for cell adhesion is developed, and the adhesive strength was studied at various bond distributions. We find that the nonlinear mechanical behavior of the receptor-ligand bonds is crucial to the adhesive strength and stability. This nonlinear behavior allows more bonds to achieve large bond force simultaneously, and therefore the adhesive strength becomes less sensitive to the change of bond density at the outmost periphery of the adhesive area. In this way, the strength and stability of cell adhesion are soundly enhanced. The nonlinear model describes the cell detachment behavior better than the linear model. (C) 2007 Elsevier Ltd. All rights reserved.

Comparison of Various Adhesion Contact Theories and the Influence of Dimensionless Load Parameter

Three models, JKR (Johnson, Kendall and Roberts), DMT (Derjaguin, Muller, and Toporov) andMD (Maugis-Dugdale),are compared with the Hertz model in dealing with nano-contact problems. It has been shown that both the dimensionless load parameter, P D P=.1/4

Quantifying the Effects of Contact Duration, Loading Rate, and Approach Velocity on P-Selectin-PSGL-1 Interactions Using AFM

Kinetics and its regulation by extrinsic physical factors govern selectin-ligand interactions that mediate tethering and rolling of circulating cells on the vessel wall under hemodynamic forces. While the force regulation of off-rate for dissociation of selectin-ligand bonds has been extensively studied, much less is known about how transport impacts the on-rate for association of these bonds and their stability. We used atomic force microscopy (AFM) to quantify how the contact duration, loading rate, and approach velocity affected kinetic rates and strength of bonds of P-selectin interacting with P-selectin glycoprotein ligand I (PSGL-1). We found a saturable relationship between the contact time and the rupture force, a biphasic relationship between the adhesion probability and the retraction velocity, a piece-wise linear relationship between the rupture force and the logarithm of the loading rate, and a threshold relationship between the approach velocity and the rupture force. These results provide new insights into how physical factors regulate receptor-ligand interactions.

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) (>= 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) <= 20 pN/s with low k (similar to 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

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.

A special method to evaluate the strong adhesion between brittle coating and ductile substrate

The evaluation of the interfacial adhesion of coating system has always been a rough task. In this paper, a special testing method of cross-sectional indentation is applied on a model coating system, i.e. electroplated chromium on a steel substrate which is generally regarded as an example of materials pair with strong adhesion. Based on fractography analysis with SEM and interfacial stress simulation with FEM, it is found that interfacial shear stress may induce coating spalling. More interestingly, spalling location is sensitive to substrate pretreatment process. This shows the feasibility of cross-sectional indentation to distinguish interfacial strength at a high level.

IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into understanding how L-selectin shedding regulates leukocyte rolling and adhesion.