Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.     

The role of passive structures in force enhancement of skeletal muscles following active stretch

We recently found that force enhancement following active stretch in skeletal muscles is accompanied by an increase in passive force following deactivation (J. Exp. Biol. 205 (2002) 1275). However, it is not known if this increase in passive force contributes to the force enhancement observed in the active muscle, and if it is observed at all muscle lengths. The purposes of this study were to quantify the amount of passive force increase as a function of muscle lengths, and to determine if this passive force contributes to the force enhancement observed in the active muscle. Experiments were performed on cat soleus (n = 24) using techniques published previously (J. Biomech. 30(9) (1997) 865). Conceptually, tests involved comparisons of force enhancement and passive force increase for a variety of stretch tests in soleus. Furthermore, in one test, activation of the soleus was interrupted for 1s in the force-enhanced state, and soleus was then re-activated. We found that total force enhancement and passive force increase were positively correlated for all test conditions, that passive force increase following stretch of the active soleus only occurred at muscle lengths corresponding to the descending limb of the force-length relationship, that increases in passive force for a given stretch magnitude became greater at long muscle lengths, and that upon reactivation, there was a remnant passive force enhancement. We conclude from these results that the passive force enhancement following stretch of an active muscle contributes to the total force enhancement, that this passive contribution increases with increasing muscle length, and that there must be at least one other factor than passive force increase that contributes to the total force enhancement, as the passive force increase was always smaller than the total force enhancement. A by-product of this investigation was that we observed a shift in the passive force-length relationship that was dependent on muscle activation, stretch magnitude and muscle length. Therefore, the passive force-length relationship is not a constant property of skeletal muscle, but depends critically on the muscle's contractile history.     

Frequency and length-dependent effects of Botulinum toxin-induced muscle weakness

While the pathogenesis of Botulinum toxin type A (BTX-A)-induced muscle weakness has been systematically researched, little is known about the effects of motor fibre paralysis on the mechanical properties of skeletal muscle. Here, the long-term effect of BTX-A injection on the force-length and force-frequency properties of rabbit knee extensors is investigated. BTX-A-induced muscle weakness was greater at short compared to long muscle lengths and at low compared to high stimulation frequencies four weeks following intervention. Therefore, we conclude that the paralysing effects of BTX-A are not uniform, and must be considered in biomechanical models of musculoskeletal rehabilitation in which BTX-A is used therapeutically, as for example in patients with cerebral palsy. Although the present results could be explained through a variety of mechanisms, this study does not allow for drawing firm conclusions about the length and frequency-dependent effects of BTX-A.     

Is the force-length relationship a useful indicator of contractile element damage following eccentric exercise?

Eccentric exercise has been shown to have a measurable effect on the force-length relationship (FLR), as peak force is shifted to longer muscle lengths following exercise. Recently, this shift in the FLR has been proposed as a "simple, reliable indicator" for assessing contractile element damage following eccentric exercise. However, eccentric exercise causes fatigue and damage, and there is evidence that fatigue alone may also cause a shift in the FLR. The purpose of this paper was to assess the role of fatigue on the FLR (as measured by a torque-joint angle relationship) following isometric and eccentric exercise in the New Zealand white (NZW) rabbit. Six NZW rabbits were divided into two groups for eccentric or isometric contractions of the hindlimb dorsiflexor muscles. Pre- and post-exercise torque-joint angle relationships were measured, and the shift from the pre- to the post-exercise relationship was measured as the change in joint angle at which peak torque was produced. Eccentric exercise resulted in a rightward shift of seven degrees; isometric exercise, which is thought to not cause damage, resulted in a shift of four degrees. Furthermore, torque production was reduced to a greater extent at short compared to long muscle lengths for the eccentric and isometric exercise, resulting in a post-exercise torque-joint angle relationship that was altered in shape. We conclude from these results, that the shift in peak torque may not be a simple and reliable indicator of muscle damage, but is caused by a combination of damage and post-exercise fatigue.     

Effects of shortening on stretch-induced force enhancement in single skeletal muscle fibers

The main purpose of this study was to evaluate the effects of shortening on the stretch-induced force enhancement in single muscle fibers, and indirectly test the hypothesis that force enhancement may be associated with the engagement of a passive element upon activation. Fibers were placed on the descending limb of the force-length relationship, and stretch and shortening contractions were performed. Fibers underwent two sets of shortening-stretch cycles. First, fibers were shortened by a fixed amplitude and speed (10% fiber length, and at 40% fiber length/s), and then were stretched (10% fiber length, and at 40% fiber length/s) immediately following shortening, or 500 or 1000 ms following the shortening. Second, fibers were shortened by varying amounts (5%, 10% and 15% fiber length) and at a constant speed (40% fiber length/s) immediately preceding a given fiber stretch (10% fiber length, and at 40% fiber length/s). When stretching was immediately preceded by shortening, force enhancement was decreased proportionally with the shortening magnitude. When intervals were introduced between shortening and stretch, the effects of shortening on the stretch-induced force enhancement became less prominent. We concluded that, in contrast to published suggestions, shortening affects the stretch-induced force enhancement in an amplitude-dependent manner in single fibers, as it does in whole muscles, but this effect is diminished by increasing the time period between the shortening and stretch phases.     

Strain-rate dependence of cartilage stiffness in unconfined compression: the role of fibril reinforcement versus tissue volume change in fluid pressurization

The strain and strain-rate-dependent response of articular cartilage in unconfined compression was studied theoretically. The transient stress and stiffness of cartilage were determined for strain rates ranging from zero to infinity. It is shown, for a given compressive strain, that the axial stress initially increases quickly as a function of strain rate, and then increases progressively more slowly towards the stress corresponding to the instantaneous response. The volume change of the tissue does not give its transient stiffness uniquely, because of the strong strain-rate dependence. The variation of tissue stiffness is primarily determined by the transient stiffness of the radial fibrils. Load sharing between the solid matrix and fluid pressurization also depends on the strain rate. At 15% axial compression, the matrix bears more than 80% of the applied load at a strain rate of 0.005%/s, while the fluid pressurization contributes more than 80% of the load at a strain rate of 0.15%/s. These results show the interplay between fibril reinforcement and fluid pressurization in articular cartilage: the fluid drives fibril stiffening which in turn produces high pore pressure at high strain rates.As a secondary objective of the present work, a fibrillar continuum element was formulated to replace the fibrillar spring element used previously in fibril-reinforced modeling, in order to eliminate the deformation incompatibility between the spring system and the nonfibrillar matrix. The results obtained using the two fibrillar elements were compared with the closed-form solutions for the static and instantaneous responses for the case of large deformation. It was found for unconfined compression that using the spring elements did not generally result in greater numerical errors than using the fibrillar continuum elements.     

Considerations on Joint and Articular Cartilage Mechanics

When studying joint degeneration leading to osteoarthritis (OA), it seems imperative that local joint tissue loading is known during normal everyday movement and that the adaptive/degenerative effects of this loading are quantified systematically. Philosophically, we believe the best way to approach this problem is by studying joint degeneration and osteoarthritis in long-term experimental models and by representing diarthrodial joints and the associated tissues with accurate, geometric and structural, theoretical models. Here, we present selected examples of our work representing this approach. Experimentally, we demonstrate that the local loading of joints changes continuously in experimental models of OA, not only because of the changing external and internal loading, but also because of the continuous alterations in joint contact geometry and tissue mechanical properties. Furthermore, we show that single bouts of joint loading affect gene expression, and that gene expression, as well as subsequent joint degeneration is site-specific. In fact, opposing articular surfaces that are exposed to the same loading may degenerate at completely different rates. Finally, we propose a series of theoretical models of articular cartilage and contact mechanics, demonstrating that many of the anisotropic and inhomogeneous properties can be explained by structural elements and their orientation and volumetric concentration across the tissue.     

Y2 and Y4 receptor signaling synergistically act on energy expenditure and physical activity

Neuropeptide Y receptors are critical regulators of energy homeostasis and are well known for their powerful influence on feeding, but their roles in other important aspects of energy homeostasis, such as energy expenditure and their functional interactions in these processes, are largely unknown. Here we show that mice lacking both Y2 and Y4 receptors exhibited a reduction in adiposity, more prominent in intra-abdominal vs. subcutaneous fat, and an increase in lean mass as determined by dual-energy X-ray absorptiometry. These changes were more pronounced than those seen in mice with Y2 or Y4 receptor single deletion, demonstrating the important roles and synergy of Y2 and Y4 signaling in the regulation of body composition. These changes in body composition occurred without significant changes in food intake, but energy expenditure and physical activity were significantly increased in Y4(-/-) and particularly in Y2(-/-)Y4(-/-) but not in Y2(-/-) mice, suggesting a critical role of Y4 signaling and synergistic interactions with Y2 signaling in the regulation of energy expenditure and physical activity. Y2(-/-) and Y4(-/-) mice also exhibited a decrease in respiratory exchange ratio with no further synergistic decrease in Y2(-/-)Y4(-/-) mice, suggesting that Y2 and Y4 signaling each play important and independent roles in the regulation of substrate utilization. The synergy between Y2 and Y4 signaling in regulating fat mass may be related to differences in mitochondrial oxidative capacity, since Y2(-/-)Y4(-/-) but not Y2(-/-) or Y4(-/-) mice showed significant increases in muscle protein levels of peroxisome proliferator-activated receptor (PPAR)γ coactivator (PGC)-1α, and mitochondrial respiratory chain complexes I and III. Taken together, this work demonstrates the critical roles of Y2 and Y4 receptors in the regulation of body composition and energy metabolism, highlighting dual antagonism of Y2 and Y4 receptors as a potentially effective anti-obesity treatment.