Biomedical subjects

J L McGrath

Publications and source records attributed to J L McGrath.

9 recordsLinked to original sources

PubMed2004

A mechanistic model of the actin cycle.

We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for enhancing Pi release from the ADP.Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of available actin. This limitation arises because as the cycle becomes more dynamic, it tends toward the unpolymerized state.

Actins↗

PubMed2000

Steps and fluctuations of Listeria monocytogenes during actin-based motility.

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization. Although the biochemical and genetic aspects of Listeria motility have been intensely studied, biophysical data are sparse. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin. We occasionally observed smaller steps (<3 nm), as well as periods of motion with no obvious pauses. Clearly, bacteria do not sense cytoplasmic viscoelasticity because they fluctuate 20 times less than adjacent lipid droplets. Instead, bacteria bind their own actin 'tails, and the anchoring proteins can 'step' along growing filaments within the actin tail. Because positional fluctuations are unusually small, the forces of association and propulsion must be very strong. Our data disprove the brownian ratchet models and limit alternative models, such as the 'elastic' brownian ratchet or the 'molecular' ratchet.

Actins↗

PubMed2000

Regulation of the actin cycle in vivo by actin filament severing.

Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.

Actin Depolymerizing Factors↗

PubMed2000

The mechanics of F-actin microenvironments depend on the chemistry of probing surfaces.

To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (> 10(4) rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (approximately 100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.

Actins↗

PubMed1998

Measuring actin dynamics in endothelial cells.

Cytoplasmic actin distributes between monomeric and filamentous phases in cells. As cells crawl, actin polymerizes near the plasma membrane of expanding peripheral cytoplasm and depolymerizes elsewhere. Thus, the finite actin filament lifetime, the diffusivity of actin monomer, and the distribution of actin between the polymer and monomer phases are key parameters in cell motility. The dynamics of cellular actin can be determined by following the evolution of fluorescence in the techniques of photoactivated fluorescence (PAF) or fluorescence recovery after photobleaching (FRAP) of microinjected actin derivatives. A mathematical model is discussed that measures monomer diffusion coefficients, filament turnover rates, and the fraction of actin polymerized from measurements of the evolution of fluorescence from a photoactivated band [Tardy et al. (1995) Biophys. J., 69:1674-1682; McGrath et al. (1998) Biophys. J., in press]. Applying this model to subconfluent endothelial cells shows that approximately 40% of the actin is polymer and that these filaments turn over on average every 6 minutes. This report discusses how PAF and FRAP can be combined with more traditional biochemistry to probe actin cytoskeleton remodeling in endothelial cells.

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↗

PubMed1998

Effect of endogenous nitric oxide inhibition on airway responsiveness to histamine and adenosine-5'-monophosphate in asthma.

BACKGROUND: Nitric oxide (NO) may be bronchoprotective in asthma, possibly due to a direct action on airway smooth muscle or through mast cell stabilisation. To investigate this the effects of two doses of nebulised NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NO synthase (NOS) inhibitor, on exhaled NO levels and airway responsiveness to histamine, a direct smooth muscle spasmogen, and adenosine-5'-monophosphate (AMP), an indirect spasmogen which activates mast cells, were evaluated in patients with mild asthma. METHODS: The study consisted of two phases each with a double blind, randomised, crossover design. In phase 1, 15 subjects inhaled either L-NAME 54 mg or 0.9% saline 30 minutes before histamine challenge. Nine of these subjects were studied in a similar fashion but were also challenged with AMP. In phase 2, 13 subjects (eight from phase 1) performed the same protocol but inhaled L-NAME in a dose of 170 mg or 0.9% saline before being challenged with histamine and AMP. RESULTS: The mean (95% CI) reduction in exhaled NO levels after L-NAME 54 mg was 78% (66 to 90) but this did not alter airway responsiveness; the geometric mean (SE) concentration provoking a fall of 20% or more in forced expiratory volume in one second (PC20) after L-NAME and saline was 0.59 (1.26) and 0.81 (1.26) mg/ml, respectively, for histamine and 20.2 (1.7) and 17.2 (1.6) mg/ml, respectively, for AMP. In contrast, L-NAME 170 mg reduced NO levels to a similar extent (81% (95% CI 76 to 87)) but increased airway responsiveness by approximately one doubling dose to both spasmogens; the geometric mean (SE) PC20 for histamine after L-NAME 170 mg and saline was 0.82 (1.29) and 1.78 (1.19) mg/ml, respectively (p < 0.001), and for AMP was 11.8 (1.5) and 24.3 (1.4) mg/ml, respectively (p < 0.001). CONCLUSIONS: These results suggest that L-NAME increases airway responsiveness in asthma. This may occur through mechanisms separate from NO inhibition or through pathways independent of those responsible for production of NO measured in exhaled air.

Adenosine Monophosphate↗

PubMed1998

Allergen-induced early and late asthmatic responses are not affected by inhibition of endogenous nitric oxide.

Endogenous exhaled nitric oxide (NO) is increased during the late response to inhaled allergen in patients with asthma and may be bronchoprotective in asthma or have a deleterious effect when generated in excess under inflammatory conditions. To investigate this, we evaluated the effect of inhibiting endogenous NO production with nebulized NG-nitro-L-arginine methyl ester (L-NAME), a nonselective NO synthase (NOS) inhibitor, on early and late asthmatic responses to inhaled allergen in patients with mild allergic asthma. After a screening allergen challenge (AC), 22 male patients attended two visits conducted in a double-blind, randomized, placebo-controlled, crossover manner. Twelve patients demonstrating an early asthmatic response only (single responders) inhaled either L-NAME 170 mg or 0.9% saline 20 min before AC, with exhaled NO and FEV1 measured for 3 h. Ten patients demonstrating both early and late asthmatic responses (dual responders) were studied in a similar fashion but inhaled two further doses of L-NAME or placebo 3.5 and 7 h after the initial dose, with exhaled NO and FEV1 measured for 10 h. L-NAME reduced exhaled NO levels by 77 +/- 5% (p < 0.01) and 71 +/- 7% (p < 0.01) in single and dual responders, respectively, but had no significant effect on early or late asthmatic responses. Following AC in single responders, the mean (+/- SEM) maximum fall in FEV1 after L-NAME and saline was 21.2 +/- 2.9% and 23.8 +/- 3.0%, respectively, and in dual responders, 31.2 +/- 4.5% and 31.8 +/- 5. 8% during the early asthmatic responses, and 27.4 +/- 3.9% and 30.6 +/- 4.5% during the late asthmatic responses, respectively. Area under the curve (AUC) did not significantly differ. AUC0-2 h in single responders after L-NAME and saline was 20.2 +/- 3.9 and 24.9 +/- 4.4 Delta% FEV1/h, and in dual responders, 37.6 +/- 8.4 and 36.7 +/- 8.4 Delta% FEV1/h, respectively, and 106.2 +/- 18.9 and 117.1 +/- 22.4 Delta% FEV1/h, respectively, for the AUC4-10 h. This study suggests that in mild allergic asthma, endogenous NO neither protects against nor contributes to the processes underlying airway responses to inhaled allergen.

Adult↗

PubMed1995

Interpreting photoactivated fluorescence microscopy measurements of steady-state actin dynamics.

A continuum model describing the steady-state actin dynamics of the cytoskeleton of living cells has been developed to aid in the interpretation of photoactivated fluorescence experiments. In a simplified cell geometry, the model assumes uniform concentrations of cytosolic and cytoskeletal actin throughout the cell and no net growth of either pool. The spatiotemporal evolution of the fluorescent actin population is described by a system of two coupled linear partial-differential equations. An analytical solution is found using a Fourier-Laplace transform and important limiting cases relevant to the design of experiments are discussed. The results demonstrate that, despite being a complex function of the parameters, the fluorescence decay in photoactivated fluorescence experiments has a biphasic behavior featuring a short-term decay controlled by monomer diffusion and a long-term decay governed by the monomer exchange rate between the polymerized and unpolymerized actin pools. This biphasic behavior suggests a convenient mechanism for extracting the parameters governing the fluorescence decay from data records. These parameters include the actin monomer diffusion coefficient, filament turnover rate, and ratio of polymerized to unpolymerized actin.

Actins↗