mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation

The regulatory subunit 1-alpha (RIalpha) of protein kinase A (PKA) and the mTOR kinase are involved in a common pathway regulating mammalian autophagy. RIalpha was found to localize on Rab7-positive late endosomes and on LC3-positive autophagosomal membranes in cultured cells. RIalpha was also shown to physically interact with mTOR kinase and affect its phosphorylation and activity. In this addendum, we further explore the subcellular distribution of mTOR related to RIalpha and LC3. We present experiments showing that mTOR colocalizes with RIalpha-, Rab7- and LC3-positive membranes in cultured cells. Because RIalpha regulates the phosphorylation and activity of mTOR kinase, which we now show localizes on autophagosomal membranes, the possibility emerges that the RIalpha-mTOR complex acts at the level of autophagosome maturation.     

Effect of ankle joint position and electrode placement on the estimation of the antagonistic moment during maximal plantarflexion.

During maximal efforts, antagonistic activity can significantly influence the joint moment. During maximal voluntary "isometric" contractions, certain joint rotation can not be avoided. This can influence the estimation of the antagonistic moment from the EMG activity. Our study aimed to quantify the influence on the calculated agonistic moment produced during maximal voluntary isometric plantarflexions (a) when estimating antagonistic moments at different ankle angles and (b) when placing the EMG electrodes at different portions over the m. tibialis anterior. Ten subjects performed maximal voluntary isometric plantarflexions at 90 degrees ankle angle. In order to estimate the antagonistic moment, submaximal isometric dorsiflexions were performed at various ankle angles. Moment and EMG signals from mm. triceps surae and tibialis anterior were measured. The RMS differences between plantarflexors moment calculated considering the antagonistic cocontraction estimated at the same ankle angle at which the maximal plantarflexion moment was achieved and at different ankle angles ranged from 0.10 to 2.94 Nm. The location of the electrodes led to greater RMS differences (2.35-5.18 Nm). In conclusion, an angle 10 degrees greater than the initial plantarflexion angle is enough to minimize the effect of the change in length of the m. tibialis anterior during the plantarflexion on the estimation of the plantarflexors moment. The localisation of the electrodes over the m. tibialis anterior can influence the estimation of its cocontraction during maximal plantarflexion efforts.     

Orthotic effect of a stabilising mechanism in the surface of gymnastic mats on foot motion during landings.

The purpose of this study was to examine two hypotheses: (a) mat hardness affects foot motion during landing; (b) the influence of a surface stabilising interface integrated in a mat on foot motion is detectable. Two studies were carried out: In the first one, six female gymnasts performed barefoot landings from different falling heights onto three mats having different hardness. In the second study, a stabilising mechanism was integrated in the surface of three new mats with different hardness. Three high speed video cameras (250Hz) captured the motion of the left leg and foot. These were modelled by means of a four rigid body system. The maximal eversion at the ankle joint was not influenced by the different mats (hard: 4.6 degrees +/-1.9 to 9.3 degrees +/-3.4, medium: 3.1 degrees +/-2.7 to 7.4 degrees +/-3.5, soft: 4.8 degrees +/-2.1 to 8.4 degrees +/-3.5). The soft mat without the stabilised surface showed higher eversion values (p<0.05) between forefoot and rearfoot (medial joint: hard: 5.1 degrees +/-3.2 to 7.3 degrees +/-3.3, medium: 6.9 degrees +/-3.1 to 7.5 degrees +/-2.9, soft: 12.7 degrees +/-4.1 to 13.4 degrees +/-3.3; lateral joint: hard: 8.5 degrees +/-3.1 to 9.7 degrees +/-1.1, medium: 9.5 degrees +/-2.6 to 11.2 degrees +/-3.3, soft: 12.1 degrees +/-2.3 to 15.7 degrees +/-3.3). For the mats with the surface stabilising interface, the different hardness did not cause any significant differences in maximal eversion values at the medial (hard: 1.5 degrees +/-3.3 to 5.5 degrees +/-4.5, medium: 1.3 degrees +/-3.5 to 5.1 degrees +/-3.6, soft: 0.7 degrees +/-4.9 to 5.4 degrees +/-4.2) nor at the lateral (hard: 11.3 degrees +/-4.2 to 17.3 degrees +/-4.2, medium: 12.3 degrees +/-4.8 to 17.1 degrees +/-3.7, soft: 11.5 degrees +/-4.6 to 17.1 degrees +/-4.3) forefoot joints. The structure of the mat and the consequent deformation hollow did not influence the kinematics of the ankle joint during landings, but it influenced the motion at the medial and the lateral forefoot joints. By means of a stabilised surface, it is possible to reduce the influence of mat deformation on the maximal eversion between forefoot and rearfoot.     

Effect of contraction form and contraction velocity on the differences between resultant and measured ankle joint moments

During a maximal isometric plantar flexion effort the moment measured at the dynamometer differs from the resultant ankle joint moment. The present study investigated the effects of contraction form and contraction velocity during isokinetic plantar/dorsal flexion efforts on the differences between resultant and measured moments due to the misalignment between ankle and dynamometer axes. Eleven male subjects (age: 31+/-6 years, mass: 80.6+/-9.6 kg, height: 178.4+/-7.4 cm) participated in this study. All subjects performed isometric-shortening-stretch-isometric contractions induced by electrical stimulation at three different angular velocities (25 degrees /s, 50 degrees /s and 100 degrees /s) on a customised dynamometer. The kinematics of the leg were recorded using the vicon 624 system with eight cameras operating at 250 Hz. The resultant moments at the ankle joint were calculated through inverse dynamics. The relative differences between resultant and measured ankle joint moments due to axis misalignment were fairly similar in all phases of the isometric-shortening-stretch-isometric contraction (in average 5-9% of the measured moment). Furthermore these findings were independent of the contraction velocity. During dynamic plantar/dorsal flexion contractions the differences between measured and resultant joint moment are high enough to influence conclusions regarding the mechanical response of ankle extensor muscles. However the relative differences were not increased during dynamic contractions as compared to isometric contractions.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

Effect of the pole--human body interaction on pole vaulting performance

The purposes of this study were: (a) to examine the interactions between the athlete and the pole and the possibility for the athlete to take advantage of the pole's elasticity by means of muscular work and (b) to develop performance criteria during the interaction between the athlete and the pole in pole vaulting. Six athletes performed 4-11 trials each, at 90% of their respective personal best performance. All trials were recorded using four synchronized, genlocked video cameras operating at 50 Hz. The ground reaction forces exerted on the bottom of the pole were measured using a planting box fixed on a force plate (1000 Hz). The interaction between athlete and pole may be split into two parts. During the first part, energy is transferred into the pole and the total energy of the athlete decreases. The difference between the energy decrease of the athlete and the pole energy is an indicator of the energy produced by the athletes by means of muscular work (criterion 1). During the second part of the interaction, energy is transferred back to the athlete and the total energy of the athlete increases. The difference between the returned pole energy and the amount of energy increase of the athlete defines criterion 2. In general, the function of the pole during the interaction is: (a) store part of the kinetic energy that the athlete achieved during the run up as strain energy and convert this strain energy into potential energy of the athlete, (b) allow the active system (athlete) to produce muscular work to increase the total energy potential.     

Footwear affects the gearing at the ankle and knee joints during running

The objective of the study was to investigate the adjustment of running mechanics by wearing five different types of running shoes on tartan compared to barefoot running on grass focusing on the gearing at the ankle and knee joints. The gear ratio, defined as the ratio of the moment arm of the ground reaction force (GRF) to the moment arm of the counteracting muscle tendon unit, is considered to be an indicator of joint loading and mechanical efficiency. Lower extremity kinematics and kinetics of 14 healthy volunteers were quantified three dimensionally and compared between running in shoes on tartan and barefoot on grass. Results showed no differences for the gear ratios and resultant joint moments for the ankle and knee joints across the five different shoes, but showed that wearing running shoes affects the gearing at the ankle and knee joints due to changes in the moment arm of the GRF. During barefoot running the ankle joint showed a higher gear ratio in early stance and a lower ratio in the late stance, while the gear ratio at the knee joint was lower during midstance compared to shod running. Because the moment arms of the counteracting muscle tendon units did not change, the determinants of the gear ratios were the moment arms of the GRF's. The results imply higher mechanical stress in shod running for the knee joint structures during midstance but also indicate an improved mechanical advantage in force generation for the ankle extensors during the push-off phase.     

Effect of different ankle- and knee-joint positions on gastrocnemius medialis fascicle length and EMG activity during isometric plantar flexion

The purpose of this study was to provide evidence on the fact that the observed decrease in EMG activity of the gastrocnemius medialis (GM) at pronounced knee flexed positions is not only due to GM insufficiency, by examining muscle fascicle lengths during maximal voluntary contractions at different positions. Twenty-two male long distance runners (body mass: 78.5+/-6.7 kg, height: 183+/-6 cm) participated in the study. The subjects performed isometric maximal voluntary plantar flexion contractions (MVC) of their left leg at six ankle-knee angle combinations. To examine the resultant ankle joint moments the kinematics of the left leg were recorded using a Vicon 624 system with 8 cameras operating at 120 Hz. The EMG activity of GM, gastrocnemius lateralis (GL), soleus (SOL) and tibialis anterior (TA) were measured using surface electromyography. Synchronously, fascicle length and pennation angle values of the GM were obtained at rest and at the plateau of the maximal plantar flexion using ultrasonography. The main findings were: (a) identifiable differences in fascicle length of the GM at rest do not necessarily imply that these differences would also exist during a maximal isometric plantar flexion contraction and (b) the EMG activity of the biarticular GM during the MVC decreased at a pronounced flexed knee-joint position (up to 110 degrees ) despite of no differences in GM fascicle length. It is suggested that the decrease in EMG activity of the GM at pronounced knee flexed positions is due to a critical force-length potential of all three muscles of the triceps surae.     

Influence of the Mechanical Properties of the Muscle–tendon Unit on Force Generation in Runners with Different Running Economy

In earlier studies, we found more economical runners having a more compliant quadriceps femoris (QF) tendon at low force levels, and a higher contractile strength and stiffness at the triceps surae (TS). To better understand how these differences influence force generation economy and energy recovery, we simulated contractions using a Hill-type muscle model and the previously determined muscle properties as input parameters. For eight different activation levels, we simulated isovelocity concentric contractions preceded by an isovelocity stretch. The length changes and contraction velocities imposed to the muscle–tendon units (MTU) corresponded to those happening whilst running. The main results of the simulations were: (a) a more compliant tendon at low force levels (QF) led to an advantage in force-generation due to a decrease in shortening velocity of the CE, (b) a higher contractile strength and higher stiffness at the TS led to a disadvantage in force-generation at high activation levels and to an advantage at low activation levels. In addition at the high economy runners both MTUs showed an advantageous energy release during shortening, which at the QF was mainly due to a higher elongation of the SEE and at the TS mainly to the higher contractile strength. Especially at low activation levels both MTUs showed an advantageous force generation per activation and a higher energy release as compared to the low economy runners.