Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.

Cell polarization is essential in a wide range of biological processes such as morphogenesis, asymmetric division, and directed migration. In this study, we show that two tumor suppressor proteins, adenomatous polyposis coli (APC) and Dlg1-SAP97, are required for the polarization of migrating astrocytes. Activation of the Par6–PKCζ complex by Cdc42 at the leading edge of migrating cells promotes both the localized association of APC with microtubule plus ends and the assembly of Dlg-containing puncta in the plasma membrane. Biochemical analysis and total internal reflection fluorescence microscopy reveal that the subsequent physical interaction between APC and Dlg1 is required for polarization of the microtubule cytoskeleton.

Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.

1. The rate of appearance of inorganic phosphate (Pé) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P 3 -1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca¥. Pé appearance was monitored from the fluorescence signal of a Pé-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the phosphate-binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca¥ at 20°C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s¢ in the first turnover based on a myosin subfragment 1 concentration of 150 ìÒ. This was calculated from a linear regression of the fluorescence signal reporting 20-150 ìÒ of Pé release. Tension was at 67% of its isometric level by the time 150 ìÒ Pé was released. ATPase activities were 36 and 31 s¢ for Pé released in the ranges of 150-300 ìÒ and 300-450 ìÒ, respectively. The ATPase activity had a QÔÑ value of 2·9 based on measurements at 5, 12 and 20°C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12·7 s¢ at 20°C.The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s¢. 4. On photolysis of NPE-caged ATP in the absence of Ca¥ the ATPase activity was 0·11 s¢ at 20°C with no discernible rapid transient phase of Pé release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca¥ was also measured on activation from the relaxed state by photolytic release of Ca¥ from a caged Ca¥ compound, nitrophenyl-EGTA. At 5°C the ATPase rate was 5·8 and 4·0 s¢ in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pé on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0·5 mÒ) decreased the initial ATPase rate by 23%. Pé (10 mÒ) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5°C in the presence of Ca¥ the ATPase activity was biphasic being 15·0 s¢ during the first turnover (based on 180 ìÒ myosin subfragment 1). Tension was 74% of its isometric level by the time 180 ìÒ Pé was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometr...