Kinetic equilibrium of forces and molecular events in muscle contraction

In biomolecular systems, the mechanical transfer of free energy occurs with both high efficiency and high speed. It is shown here that such a transfer can be achieved only if the participating free-energy-storing elements exhibit opposing relationships between their content of free energy and the force they exert in the transfer direction. A kinetic equilibrium of forces (KEF) results, in which the transfer of free energy is mediated essentially by thermal molecular motion. On the basis of present evidence, KEF is used as a guiding principle in developing a mechanical model of the crossbridge cycle in muscle contraction. The model allows the basic features of molecular events to be visualized in terms of plausible structures. Real understanding of the process will require identification of the elements that perform the functions described here. Besides chemomechanical energy transduction, KEF may have a role in other biomolecular processes in which free energy is transferred mechanically over large distances.

Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

β-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the β-actin mRNA near the leading edge is facilitated by a short sequence in the 3′ untranslated region, the “zip code.” Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3′ untranslated region leads to delocalization of β-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after β-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of β-actin protein. These results indicate that delocalization of β-actin mRNA results in delocalization of nucleation sites and β-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.