Biomedical subjects

J J Meister

Publications and source records attributed to J J Meister.

At least 19 recordsLinked to original sources

PubMed2006

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.

Actins↗

PubMed2005

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.

Action Potentials↗

PubMed2001

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 (0.01Hz 0.05). 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.

Analysis of Variance↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Acetylcholine↗

PubMed2001

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.

Adaptation, Physiological↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Adaptation, Physiological↗

PubMed2001

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.

Acetylcholine↗

PubMed2000

Static compression is associated with decreased diffusivity of dextrans in cartilage explants.

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

3T3 Cells↗

PubMed2000

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.

3T3 Cells↗

PubMed1999

Microplates: a new tool for manipulation and mechanical perturbation of individual cells.

We present a new type of microinstrument allowing manipulation and mechanical perturbation of individual cells under an optical microscope. These instruments, which we call microplates, are pulled from rectangular glass bars. They have flat tips, typically 2 microm thick x 20 microm wide, whose specific shape and stiffness can be adjusted through the pulling protocol. After appropriate chemical treatment, microplates can support cell adhesion and/or spreading. Rigid microplates are used to hold cells, whereas more flexible ones serve as stress sensors, i.e. their deflexion is used to probe forces in the range of 1-1000 nN. The main advantages of microplates are their simple geometry and surface properties, and their ability to provide mechanical measurements. In this methodological paper, we give details about microplate preparation and adhesiveness, manipulation set-up, force calibration, and image analysis. Several manipulations have already been carried out on fibroblasts, including uniaxial deformation, micropipet aspiration of adherent cells, and cell-substrate separation. Our results to date provide new insights into the morphology, mechanical properties, and adhesive resistance of cells. Many future applications can be envisaged.

Animals↗

PubMed1999

Changes in the mechanical properties of fibroblasts during spreading: a micromanipulation study.

Cell morphology is controlled in part by physical forces. If the main mechanical properties of cells have been identified and quantitated, the question remains of how the cell structure specifically contributes to these properties. In this context, we addressed the issue of whether cell rheology was altered during cell spreading, taken as a fundamental morphological change. On the experimental side, we used a novel dual micromanipulation system. Individual chick fibroblasts were allowed to spread for varying amounts of time on glass microplates, then their free extremity was aspirated into a micropipet at given pressure levels. Control experiments were also done on suspended cells. On the theoretical side, the cell was modeled as a fluid drop of viscosity mu, bounded by a contractile cortex whose tension above a resting value was taken to be linearly dependent on surface area expansion. The pipet negative pressure was first adjusted to an equilibrium value, corresponding to formation of a static hemispherical cap into the pipet. This allowed computation, through Laplace's law, of the resting tension (tau 0), on the order of 3 x 10(-4) N/m. No difference in tau 0 was found between the different groups of cells studied (suspended, adherent for 5 min, spread for 0.5 h, and spread for 3 h). However, tau 0 was significantly decreased upon treatment of fibroblasts with inhibitors of actin polymerization or myosin function. Then, the pressure was set at 30 mmH2O above the equilibrium pressure. All cells showed a biphasic behavior: (1) a rapid initial entrance corresponding to an increase in surface area, which was used to extract an area expansion elastic modulus (K), in the range of 10(-2) N/m; this coefficient was found to increase up to 40% with cell spreading; (2) a more progressive penetration into the pipet, linear with time; this phase, attributed to viscous behavior of the cytoplasm, was used to compute the apparent viscosity (mu, in the range of 2-5 x 10(4) Pa s) which was found to increase by as much as twofold with cell spreading. In some experiments the basal force at the cell-microplate interface was quantitated with flexible microplates and found to be around 1 nN, in agreement with values calculated from the model. Taken together, our results indicate a stiffening of fibroblasts upon spreading, possibly correlated with structural organization of the cytoskeleton during this process. This study may help understand better the morphology of fibroblasts and their mechanical role in connective tissue integrity.

Actins↗

PubMed1999

Pulse wave attenuation measurement by linear and nonlinear methods in nonlinearly elastic tubes.

Reasons for the continuing difficulty in making definitive measurements of pulse wave attenuation in elastic tubes and arteries in the presence of reflections are sought. The measurement techniques available were re-examined in elastic tubes mimicking the arterial compliance nonlinearity, under conditions of strong reflection. The pulse was of physiological shape, and two different pulse amplitudes in the physiological range were used. Measurements of pressure, flow-rate and diameter pulsation allowed the deployment of four of the classical linear methods of analysis. In addition, a method of separating the forward- and backward-travelling waves that does not require linearising assumptions was used, and the attenuation in the forward and reverse directions was calculated from the resulting waveforms. Overall, the results obtained here suggest that a fully satisfactory way of measuring arterial attenuation has yet to be devised. The classical linear methods all provided comparable attenuation estimates in terms of average value and degree of scatter across frequency. Increased scatter was generally found at the higher pulse amplitude. When the forward waveforms from the separation were similarly compared in terms of frequency components, the average value at energetic harmonics was similar to both the value indicated by the linear methods and the values predicted from linear theory on the basis of estimated viscous and viscoelastic parameter data. The backward waveforms indicated a physically unreasonable result, attributed as the expression for this technique of the same difficulties that normally manifest in scatter. Data in the literature suggesting that one of the classical methods, the three-point, systematically over-estimates attenuation were not supported, but it was confirmed that this method becomes prone to negative attenuation estimates at low harmonics as pulse amplitude increases. Although the goal of definitive attenuation measurement remains elusive, the task provides a sensitive tool for the examination of the effect of nonlinearities in the arterial system.

Arteries↗

PubMed1998

Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch.

Hemodynamic forces affect endothelial cell morphology and function. In particular, circumferential cyclic stretch of blood vessels, due to pressure changes during the cardiac cycle, is known to affect the endothelial cell shape, mediating the alignment of the cells in the direction perpendicular to stretch. This change in cell shape proceeds a drastic reorganization at the internal level. The cellular scaffolding, mainly composed of actin filaments, reorganize in the direction which later becomes the cell's long axis. How this external mechanical stimulus is 'sensed' and transduced into the cell is still unknown. Here, we develop a mathematical model depicting the dynamics of actin filaments, and the influence of the cyclic stretch of the substratum based on the experimental evidence that external stimuli may be transduced inside the cell via transmembrane proteins which are coupled with actin filaments on the cytoplasmic side. Based on this view, we investigate two approaches describing the formulation of the transduction mechanisms involving the coupling between filaments and the membrane proteins. As a result, we find that the mechanical stimulus could cause the experimentally observed reorganization of the entire cytoskeleton simply by altering the dynamics of the filaments connected with the integral membrane proteins, as described in our model. Comparison of our results with previous studies of cytoskeletal dynamics reveals that the cytoskeleton, which, in the absence of the effect of stretch would maintain its isotropic distribution, slowly aligns with the precise direction set by the external stimulus. It is found that even a feeble stimulus, coupled with a strong internal dynamics, is sufficient to align actin filaments perpendicular to the direction of stretch.

Actins↗

PubMed1998

Simultaneous measurements of actin filament turnover, filament fraction, and monomer diffusion in endothelial cells.

The analogous techniques of photoactivation of fluorescence (PAF) and fluorescence recovery after photobleaching (FRAP) have been applied previously to the study of actin dynamics in living cells. Traditionally, separate experiments estimate the mobility of actin monomer or the lifetime of actin filaments. A mathematical description of the dynamics of the actin cytoskeleton, however, predicts that the evolution of fluorescence in PAF and FRAP experiments depends simultaneously on the diffusion coefficient of actin monomer, D, the fraction of actin in filaments, FF, and the lifetime of actin filaments, tau (, Biophys. J. 69:1674-1682). Here we report the application of this mathematical model to the interpretation of PAF and FRAP experiments in subconfluent bovine aortic endothelial cells (BAECs). The following parameters apply for actin in the bulk cytoskeleton of subconfluent BAECs. PAF: D = 3.1 +/- 0.4 x 10(-8) cm2/s, FF = 0.36 +/- 0.04, tau = 7.5 +/- 2.0 min. FRAP: D = 5.8 +/- 1.2 x 10(-8) cm2/s, FF = 0.5 +/- 0.04, tau = 4.8 +/- 0.97 min. Differences in the parameters are attributed to differences in the actin derivatives employed in the two studies and not to inherent differences in the PAF and FRAP techniques. Control experiments confirm the modeling assumption that the evolution of fluorescence is dominated by the diffusion of actin monomer, and the cyclic turnover of actin filaments, but not by filament diffusion. The work establishes the dynamic state of actin in subconfluent endothelial cells and provides an improved framework for future applications of PAF and FRAP.

Actins↗