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Vishnudas, Vivek Kannoth

Biology

2006

PhD

The high power output necessary for insect flight has resulted in the evolution of muscles with large and abundant myofibrils, the so called 'myofibrillar' muscles. In principle, this modification should come with a trade-off as the broader diameter of the myofibril would slow ATPIADP flux and potentially constrain muscle speed (myosin ATPase). However asynchronous flight muscle exhibits no such trade-off as it simultaneously displays speed, power, and endurance. The asynchronous Drosophila indirect flight muscle (IFM) is an excellent choice for examining functional and molecular adaptations that enable high power out put for flight. Drosophila IFM kinetics appears to be dramatically different from slower muscles. Using fiber mechanics we demonstrate that the Pi release step is the rate limiting step in the IFM myosin and the myosin's affinity for ATP (Km= 5.9 mM) is the lowest ever recorded. This is suggestive of competition between ATP and Pi for myosin, imposing a high ATP concentration requirement in the IFM. The IFM also has a fast actin- myosin detachment rate (3698 s⁻¹), and a fast ADP release rate. Both these requirements (high ATP concentration, fast ADP release rate) coupled with a fast ATPase rate, known from previous experiments and the absence of a phosphoarginine shuttle, necessitates molecular adaptations in the myofibril that would ensure low ADP/ATP ratios in the myofibril. Proteomic analysis of detergent enriched myofibrillar fractions identified predominantly two mitochondrial proteins, stress sensitive B which is an adenine nucleotide transporter and a phosphate transporter along with 19 myofibrillar proteins.
The discovery of cytoskeletal LIM proteins is suggestive of functional and structural interactions between the mitochondria myofibrils and cytoskeleton involving the exchange of ATP, ADP and Pi. The characterization of ANT distribution in the IFM using immunogold transmission electron microscopy with an ANT antibody confirmed that ANT is distributed in equal proportions to the mitochondria and myofibrils. The synchronous TDT (jump muscle) however lacks the myofibrillar ANT. Further studies on a transgenic line that expresses an amino terminal GFP fused to the sesB coding sequence revealed that GFP-sesB protein is present in Drosophila IFM myofibril. Biochemical fractionation of the IFM under iso osmolar conditions, involving extraction of i) soluble glycolytic enzymes by saponin, ii) myosin by high salt extraction, iii) actin by 2M urea and iv) a residual organellar fraction, followed by western blot analysis of fractions revealed that ANT protein is enriched in myosin fraction ii) and organellar fraction iv). This observation raises the possibility that direct channeling of nucleotide between mitochondria and myofibrils is assisted by an ANT protein thereby circumventing the need for a phosphagen shuttle in the IFM. The myofibrillar ANT represents a unique adaptation in the muscles that require efficient exchange of nucleotides between mitochondria and myofibrils.