Intercellular ultrafast Ca(2+) wave in vascular smooth muscle cells: numerical and experimental study.

Vascular smooth muscle cells exhibit intercellular Ca(2+) waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca(2+) wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca(2+) wave and it was suggested to be the result of the interplay between membrane potential and Ca(2+) dynamics which depended on influx of extracellular Ca(2+), cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca(2+) wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca(2+) wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca(2+) wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca(2+) waves in smooth muscle cells.

An actin length threshold regulates adhesion maturation at the lamellipodium/lamellum interface.

The mechanical coupling between adherent cells and their substrates is a major driver of downstream behavior. This coupling relies on the formation of adhesion sites and actin bundles. How cells generate these elements remains only partly understood. A potentially important mechanism, the length threshold maturation (LTM), has previously been proposed to regulate adhesion maturation and actin bundle stabilization tangential to the leading edge. The LTM describes the process by which cells integrate lamellar myosin forces to trigger adhesion maturation. These forces, cumulated over the length of an actin bundle, are balanced at the anchoring focal complexes. When the bundle length exceeds a certain threshold, the distributed lamellar forces become sufficient to trigger the stabilization of the bundle and its adhesions. In this continuing study, we experimentally challenge the LTM for the first time, by seeding cells on micropatterned substrates with various non-adhesive gaps designed to selectively trigger the LTM. While stable actin bundles were observed on all patterns, their lengths were almost exclusively above 3 µm or 4 µm depending on the cell type. Furthermore, the frequency with which gaps were bridged increased nearly as a step function with increasing gap width, indicating a substrate dependent behavioral switch. These combined observations point strongly to LTM with a threshold above 3 µm. We thus experimentally confirm with two cell types our previous theoretical work postulating the existence of a length dependent threshold mechanism that triggers adhesion maturation and actin bundle stabilization.

High-resolution cell outline segmentation and tracking from phase-contrast microscopy images.

Accurate extraction of cell outlines from microscopy images is essential for analysing the dynamics of migrating cells. Phase-contrast microscopy is one of the most common and convenient imaging modalities for observing cell motility because it does not require exogenous labelling and uses only moderate light levels with generally negligible phototoxicity effects. Automatic extraction and tracking of high-resolution cell outlines from phase-contrast images, however, is difficult due to complex and non-uniform edge intensity. We present a novel image-processing method based on refined level-set segmentation for accurate extraction of cell outlines from high-resolution phase-contrast images. The algorithm is validated on synthetic images of defined noise levels and applied to real image sequences of polarizing and persistently migrating keratocyte cells. We demonstrate that the algorithm is able to reliably reveal fine features in the cell edge dynamics.

Stretch-elicited calcium responses in the intact mouse thoracic aorta.

Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry.

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells.

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress.

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.

Myofibroblast development is characterized by specific cell-cell adherens junctions.

Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of alpha-SMA-positive and -negative fibroblasts in suspension. AJs of plated myofibroblasts are reinforced by alpha-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits alpha-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-alpha-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity.

Analysis of actin dynamics at the leading edge of crawling cells: implications for the shape of keratocyte lamellipodia.

Leading edge protrusion is one of the critical events in the cell motility cycle and it is believed to be driven by the assembly of the actin network. The concept of dendritic nucleation of actin filaments provides a basis for understanding the organization and dynamics of the actin network at the molecular level. At a larger scale, the dynamic geometry of the cell edge has been described in terms of the graded radial extension model, but this level of description has not yet been linked to the molecular dynamics. Here, we measure the graded distribution of actin filament density along the leading edge of fish epidermal keratocytes. We develop a mathematical model relating dendritic nucleation to the long-range actin distribution and the shape of the leading edge. In this model, a steady-state graded actin distribution evolves as a result of branching, growth and capping of actin filaments in a finite area of the leading edge. We model the shape of the leading edge as a product of the extension of the actin network, which depends on actin filament density. The feedback between the actin density and edge shape in the model results in a cell shape and an actin distribution similar to those experimentally observed. Thus, we explain the stability of the keratocyte shape in terms of the self-organization of the branching actin network.

Adaptation of conduit artery vascular smooth muscle tone to induced hypertension.

We studied the changes in vascular smooth muscle (VSM) cell tone during the adaptation of rat common carotids to induced hypertension. Hypertension was induced in 8 week old male Wistar rats by total ligation of the aorta between the two kidneys. Mean blood pressure increased abruptly from 92 +/- 2mm Hg (mean +/- SE) to 145 +/- 4 mm Hg and remained constant thereafter. Rats were sacrificed 2, 4, 8, and 56 days after surgery and the left common carotid artery was excised for analysis. Pressure-diameter curves were measured in vitro under normal, maximally contracted, and totally relaxed VSM. The VSM tone was analyzed in terms of its basal tone (active stress at low strains) and its myogenic tone (increase in active stress at high strains). Our results show that the capacity of the VSM to develop maximal active stress is not altered in hypertension. Basal tone, however, increases rapidly in the acute hypertension phase (2-8 days postsurgery) and drops to nearly control values at 56 days postsurgery. Also, the onset of myogenic response decreases to lower strains following the step change in pressure, to be restored back to control levels at 56 days postsurgery. We conclude that VSM adaptation is most significant in the acute hypertension phase and acts as a first, rapid defense mechanism for the arterial wall. The VSM tone returns back to normal levels once the slower geometrical and structural remodeling is developed sufficiently to restore the biomechanical environment and function of the arterial wall to control levels.

Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro.

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.

Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries.

We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.

In vitro diameter response of rat femoral artery to flow in the presence and absence of endothelium.

We have examined the diameter response of rat femoral artery segments in the presence and absence of endothelium to changes in flow rate. The segments were isolated, mounted on microcannulae, maintained at 37 degrees C, and perfused at 90 mmHg with Tyrode's solution. The external arterial diameter was measured using video-microscopy. The mean control diameter was 741+/-22 microm (mean+/-SEM,n=7). The arteries were preconstricted to 75+/-1% of the control diameter with a superfusion of 1 microM norepinephrine (NE). Endothelial function was verified by perfusion of 1 micro;M acetylcholine (ACh). Two different flow protocols were employed: step changes in flow (n=7) and low-frequency sinusoidal flow changes (0.01Hz0.05). Sinusoidal flow oscillations resulted in sinusoidal diameter oscillations, whose amplitude and phase lag were inversely proportional to the frequency of the flow oscillations. A first-order low-pass filter, with a time constant of 28+/-3 and 30+/-5s for arteries with and without functional endothelium, respectively, was used to describe the relation between oscillatory flow and diameter. The response of the rat femoral arteries to changes in flow was not found to be different whether the endothelium was intact or removed.

Assessing the homogeneity of the elastic properties and composition of the pig aortic media.

Most previous studies of arterial wall elasticity and rheology have assumed that the properties of the wall are uniform across the thickness of the media and, therefore, that the relationship between stress and strain may be described by a constitutive equation based on a single strain energy function. The few studies where this assumption has been questioned, focussed on differences between the adventitia and the media rather than on differences within the media itself. Here, we report in vitro elasticity and residual strain measurements performed separately on the inner and outer half of the pig aortic media, together with a histomorphometric assessment of the radial distribution of elastin, collagen and smooth muscle cell numbers. Although we found that the pressure-diameter relationships of the two halves were dissimilar, when allowance was made for their different unloaded dimensions, their material properties agreed closely, a result in keeping with the observed uniform radial distribution of scleroprotein and vascular smooth muscle. We also found a difference in the opening angle (which is often taken as a measure of residual strain) between the inner and outer medial halves. However, strain analysis showed that the opening angle is an extremely sensitive measure of residual strain and that the difference in the actual magnitudes of residual strain between the two halves of the media was small. We conclude that the media of the porcine thoracic aorta has similar elastic properties throughout its thickness and that this uniformity is matched by a uniform distribution of matrix protein and vascular smooth muscle cells. Furthermore, the distribution of strain in the media can adequately be described by a single-layer model with uniform elastic properties throughout its thickness.

Model of geometrical and smooth muscle tone adaptation of carotid artery subject to step change in pressure.

Recent experimental studies have shown significant alterations of the vascular smooth muscle (VSM) tone when an artery is subjected to an elevation in pressure. Therefore, the VSM participates in the adaptation process not only by means of its synthetic activity (fibronectins and collagen) or proliferative activity (hypertrophy and hyperplasia) but also by adjusting its contractile properties and its tone level. In previous theoretical models describing the time evolution of the arterial wall adaptation in response to induced hypertension, the contribution of VSM tone has been neglected. In this study, we propose a new biomechanical model for the wall adaptation to induced hypertension, including changes in VSM tone. On the basis of Hill's model, total circumferential stress is separated into its passive and active components, the active part being the stress developed by the VSM. Adaptation rate equations describe the geometrical adaptation (wall thickening) and the adaptation of active stress (VSM tone). The evolution curves that are derived from the theoretical model fit well the experimental data describing the adaptation of the rat common carotid subjected to a step increase in pressure. This leads to the identification of the model parameters and time constants by characterizing the rapidity of the adaptation processes. The agreement between the results of this simple theoretical model and the experimental data suggests that the theoretical approach used here may appropriately account for the biomechanics underlying the arterial wall adaptation.

Simultaneous arterial calcium dynamics and diameter measurements: application to myoendothelial communication.

The goal of the present study was to analyze the intercellular calcium communication between smooth muscle cells (SMCs) and endothelial cells (ECs) by simultaneously monitoring artery diameter and intracellular calcium concentration in a rat mesenteric arterial segment in vitro under physiological pressure (50 mmHg) and flow (50 microl/min) in a specially developed system. Intracellular calcium was expressed as the fura 2 ratio. The diameter was measured using a digital image acquisition system. Stimulation of SMCs with the alpha(1)-agonist phenylephrine (PE) caused not only an increase in the free intracellular calcium concentration of the SMCs as expected but also in the ECs, suggesting a calcium flux from the SMCs to the ECs. The gap junction uncoupler palmitoleic acid greatly reduced this increase in calcium in the ECs on stimulation of the SMCs with PE. This indicates that the signaling pathway passes through the gap junctions. Similarly, although vasomotion originates in the SMCs, calcium oscillates in both SMCs and ECs during vasomotion, suggesting again a calcium flux from the SMCs to the ECs.

Short-Term biomechanical adaptation of the rat carotid to acute hypertension: contribution of smooth muscle.

The biomechanical adaptation of the arterial wall to hypertension has been studied extensively in recent years; however, the exact biomechanical contribution of vascular smooth muscle cells (VSMCs) during the adaptation process in conduit vessels is not known. We induced hypertension in 8 wk old Wistar rats by total ligation of the aorta between the two kidneys. Mean blood pressure increased from 92 +/- 2 (mean +/- SE) mm Hg to approximately 150 mmHg. Rats were sacrificed 2, 4, and 8 d after surgery and the left common carotid artery was excised for analysis. Wall thickness increased by 18% in 8 d and the opening angle by 32% in 4 d. The elastic properties were measured under normal VSMC tone (i.e., the amount of VSMC tone under normal conditions also called basal VSMC tone or normal resting VSMC tone), under maximally contracted VSMC (NE, 5 x 10(-7) mol/L) and under totally relaxed VSMC conditions (papaverine, 10(-4) mol/L). The most pronounced modifications were the changes in elastic properties related to normal VSMC tone. The functional contraction ratio at 100 mm Hg, defined as the relative contraction under normal conditions (normal VSMC tone), increased by 439% 4 d after the induction of hypertension. The total contraction capacity of the VSMC increased by 38% within 8 d. The changes in normal VSMC tone led to important changes in the mechanical properties of the arterial wall. Under normal VSMC conditions, compliance at mean pressure (148 mm Hg) increased by 159% within 8 d, whereas in the absence of VSMC tone, compliance did not increase significantly. We conclude that in conduit vessels, the VSMC, which is the sensing and effecting element of the adaptation process, is subjected to large-scale changes during the early phase of arterial adaptation to acute hypertension.

Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis.

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1-16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k+ = 0.3-1.4 s(-1) and koff = 0.05-0.25 s(-1), respectively). The bond distribution is binomial, with an average bond number < or = 10 at these time scales. Increasing the fibronectin density (100-3000 molecules/microm2) raised k+ in a diffusion-dependent manner, leaving koff relatively unchanged. Increasing the temperature (23-37 degrees C) raised both k+ and koff, allowing calculation of the activation energy of the chemical reaction (around 20 kBT). Increasing the compressive force on the cell during contact (up to 60 pN) raised k+ in a logarithmic manner, probably through an increase in the contact area, whereas koff was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond.

Dynamics of adhesive rupture between fibroblasts and fibronectin: microplate manipulations and deterministic model.

To characterize the dynamics of cell-substrate adhesive rupture, we used a novel micromanipulation technique, in which individual fibroblasts seized on a rigid microplate were placed into contact with a fibronectin-coated flexible microplate, then pulled away. The fibronectin density (0-3000 molecules/microm2) and the pulling rate (1-10 microm/s) were controlled. The extent of the contact zone decreased to zero at a time threshold corresponding to adhesive rupture. The uniaxial force at the interface, computed from the deflection of the microplate, increased linearly with time and reached a maximum before dropping to zero. A deterministic model, focusing on the mean number of bonds between fibronectin and its membrane receptor on the cell surface, shows rapid rupture when the force reaches a critical value, in agreement with experimental observations. Increasing the ligand density and the rate of load raises the maximal force (30 200 nN), in reasonably good agreement with the model predictions. Minimization of error between experimental and simulated forces allowed identification of two physicochemical properties of the bond, i.e. its association rate constant (k(2D)on = 3 x 10(-4) microm2/s) and structural length (d = 3 nm). These results may help understand better fibroblast locomotion and interaction with the extracellular matrix.

A probabilistic model for ligand-cytoskeleton transmembrane adhesion: predicting the behavior of microspheres on the surface of migrating cells.

A theoretical model describing the attachment and cytoskeletal coupling of microspheres to the dorsal surface of motile cells was developed. Integral membrane receptors beneath a ligand-coated microsphere are allowed to be either free, attached to the microsphere, bound to the rearward moving actin network, or linked to both the bead and the cytoskeleton, and to switch between these four states. The binding transitions being modeled as chemical reactions governed by rate constants taken from literature, the chance for a receptor to be in each binding state over time is obtained by solving mass-balance equations for the probability functions. The population of n such receptors beneath the microsphere is accounted for by a binomial distribution for each state. Adhesion and transmembrane coupling (resulting in microsphere transport) being defined by a minimal number of ligand-receptor and receptor-cytoskeleton bonds, respectively, the probabilities of attachment and transport of the microsphere over time are expressed in terms of state probability distributions. It is found that increasing the ligand density raises the attachment and transport probabilities, in good quantitative agreement with recent experiments using optical tweezers and accurate position tracking. Increasing the bead size does not affect attachment, but raises the transport probability with a marked transition for bead diameter around 100 nm, as for experimental data. Increasing the restraining force decreases the transport probability, probably by inducing a rupture of receptor-cytoskeleton bonds. This study thus provides a framework that helps understand the process of cortical flow associated with cell locomotion.