Force distribution on multiple bonds controls the kinetics of adhesion in stretched cells
Introduction
It is widely accepted that mechanical forces control normal biological processes and their pathological alterations through cell–environment interactions (Discher et al., 2005). These interactions occur under dynamic conditions, e.g., the flowing blood; the internal tension, through adhesion sites which are protein assemblies through clusters which link the cytoskeleton to the extracellular environment (Geiger and Bershadsky, 2001). In particular, receptor–ligand bond anchoring cells in the presence of flowing liquids are stretched by tensile mechanical forces that balance the drag force on the bound cell. Therefore, stabilization under force and resisting removal to shear stresses – and to intra/extracellular stretching forces–are fundamental properties of cellular adhesion. Moreover, weakening or strengthening adhesion by mechanical force remains an opened question if one wants to understand flow-controlled key cellular interactions such as: macrophage–epithelium , leukocyte–endothelium or bacteria–host cell (Thomas, 2008, Thomas et al., 2008).
A fundamental aspect to consider is the stochastic nature of molecular adhesion and the subsequent bond detachment under force which are extensively described in the literature (Bongrand and Golstein, 1983, Evans, 1998). A less considered aspect deals with the complexity of adhesion structures since cellular adhesion involves not only one but several bonds, which stabilize and resist mechanical forces through different strategies of association and reinforcement (Evans, 2001, Evans and Ritchie, 1994, Leckband et al., 1992). These strategies are most likely influenced by forces generated through the intracellular structure or transiting through the extracellular environment (Féréol et al., 2009) and a number of biochemical and biomechanical processes implicating many signaling pathways (Vogel and Sheetz, 2009). From initial adhesion to focal complex and beyond the mature focal adhesion, the adhesion site configuration changes and the maximal force supported by adhesion sites varies by six orders of magnitude (pN-μN) (Bruinsma, 2005). Although these adhesion sites are made of similar weak bond units – i .e., a non covalent molecular link given to support forces up to the pN – their collective organization allows them supporting a huge range of stress (Geiger and Bershadsky, 2001). The structural changes associated to stabilization and strengthening include: (i) increase in cell–substrate contact area (spreading) (Capo et al., 1981), (ii) recruitment of receptors to anchoring sites including their lateral association (clustering) (Andre et al., 1990, Bell et al., 1984, Cluzel et al., 2005), (iii) interaction with cytoskeleton elements that lead to enhanced force distribution among bound receptors via local cytoskeleton stiffening (focal adhesion assembly) (Pasternak and Elson, 1985). These mechanisms of adhesion strengthening are supported by numerous observations from various cellular systems (Balaban et al., 2001, Choquet et al., 1997, Galbraith et al., 2002, Giannone et al., 2003, Zhu et al., 2008). Trying to understand these mechanisms of adhesion stabilization or reinforcement, and how they are regulated by force, remains challenging questions which require new theoretical knowledge and pertinent analysis of data.
Recognizing the need to expose actions at the submicroscopic level, many research groups have employed ultrasensitive force techniques to probe extremely small regions of adhesive contact between surfaces functionalized by biological molecules (Evans and Ritchie, 1994, Leckband et al., 1992). Most direct measurements of single bond strength have been performed with three types of ultrasensitive probes: the Atomic Force Microscope (AFM) where force is sensed by deflection of a thin silicon nitride cantilever (Binnig et al., 1986), the Biomembrane Force Probe (BFP) where force is sensed by a glass microsphere glued to the pole of a micropipette-pressurized membrane capsule (Evans et al., 1995, Simson et al., 1998); and the laser optical tweezers where force is sensed by displacement of a microsphere trapped in a narrowly focused beam of laser light (Ashkin, 1992, Ashkin et al., 1990). Common limitations associated with these techniques deal with thermal fluctuations, hydrodynamic interactions, and noteworthy the difficulty of controlling the number and the configuration of bonds implicated.
Since most of adhesive interactions are initiated and then sustained under flow conditions (in priority blood but not only), cell resistance to removing by fluid shear stress using a “laminar flow chamber” has become a relevant method to measure the kinetics of receptor-ligand adhesion (Bongrand and Golstein, 1983, Pierres et al., 1996a, Pierres et al., 1996b). Basically, particles or circulating cells covered by receptors are flowing near a surface coated by a ligand and arrested by the formation of limited number of adhesion bonds, e.g., one per cell in many experiments, as far as hydrodynamic forces do not exceed a few pNs (Kaplanski et al., 1993, Pierres et al., 1996a, Smith et al., 1999). The relationship between the number of arrested particles and the duration of their arrest allows (i) determining detachment curves whose initial slope provides the dissociation rate (Chang and Hammer, 1999, Smith et al., 1999, Tissot et al., 1992) and moreover (ii) studying the mechanisms of flow-enhanced cell adhesion (Zhu et al., 2008).
The present paper concerns some under considered aspects of the regulation by mechanical force of collective bond organization implicated in the structuration of different adhesion sites. We purposely apply theories of bond dissociation – issued from stochastic approaches – to different types of bond configurations, e.g., parallel or zipper bonds, on which dissociation forces are exerted. The experimental data purposely used are issued from two different methods: the atomic force microscopy (AFM) and the laminar (viscous) shear flow. It appears that force-control of multiple bonds strongly depends on whether the force distribution on multiple bonds is homogeneous, such as during certain AFM experiments, or heterogeneous, such as during shear flow experiments. Thus, to understand the cell adhesion response to stretch, the knowledge of force distribution is needed down to the smallest scale possible. This reinforces the need of calculating the stress/strain fields exerted on living tissues or cells at various scales and certainly down to the molecular scale.
Section snippets
Single bond
Weak noncovalent interactions between large molecules mediate many of life's functions in cells (Evans, 2001). The strength of interaction is the level of force to disrupt a bond on a particular time scale, knowing that these molecular interactions have limited lifetimes and thus fail under any level of force if pulled on for modest period of time (Evans, 1998). For instance, an isolated bond has no strength on times scales longer than its natural lifetime for spontaneous dissociation
Theoretical aspects
Fig. 2 summarizes the theoretical predictions for lifetimes of multiple uncooperative bonds dissociating randomly (Eqs. (10), (11), (12)). The lifetime of multiple bond (n=N) normalized by the bond lifetime of a single and isolated bond (n=1) is plotted versus the number N of identical bonds constituting the parallel and “zipper” configurations. The main message is that although the lifetime of the collective bonds still decreases with increasing force (as shown by Eqs. (11), (12)), bonds
Conflict of interest
All authors report no actual or potential conflict of interest for the work reported in this paper.
Acknowledgment
For this work, we acknowledge receipt of a grant from Agence Nationale de la Recherche (ANR-09-PIRI-002-03).
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