Drosophila Flight Research Articles

From genes to tensile forces: genetic dissection of contractile protein assembly and function in Drosophila melanogaster

Myofibrils, the contractile organelles of skeletal muscle, are highly ordered and precisely regulated actomyosin networks. Investigations of myofibril assembly are revealing the cellular mechanisms by which contractile components are arranged and regulated. In order to facilitate this research we have developed formal molecular genetics for myofibrillar proteins of Drosophila flight muscle. Presently, mutations can be used systematically to perturb or eliminate any of the classical myofibrillar proteins within these fibers, and the in vivo consequences can be conveniently evaluated using protein electrophoresis, electron microscopy, or by assaying flight performance. Here we review some recent progress.

Protein engineering and the study of muscle contraction in Drosophila flight muscles

We describe an experimental approach to the use of genetics to study muscle contraction in Drosophila melanogaster. Mutations induced by in vitro mutagenesis are inserted into the genome of flies using P-element mediated transformation, permitting the effects of the mutant genes to be studied in vivo in the indirect flight muscles (IFMs). Details of how mechanical experiments can be performed on skinned IFMs, despite their small size, are provided. The effects of two in vitro actin mutations, G368E and E316K, are described. The problems of performing biochemical and biophysical experiments on the IFMs and their myofibrillar proteins are described, together with indications as to how these may be overcome.

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains—II. Flightless [formula omitted] fly

1. 1. The Ca 2+-activated and Mg 2+ actin-activated myosin ATPase activities of flightless mfd − mutant Drosophila flight muscle myosin were one-half and one-third of those of the wild-type fly muscle myosin, respectively. 2. 2. In the two-dimensional gel electrophoresis, the spots corresponding to phosphorylated myosin light chains, Lf1 and Lt1, were hardly detected in mfd − mutant myosin. 3. 3. These results support not only the conclusion that phosphorylation of myosin light chains regulates Drosophila myosin ATPase activity but also the assumption that the phosphorylation of myosin light chains is directly involved in flight function of the Drosophila fly.

Troponin of asynchronous flight muscle

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 M r), Tn-T (apparent M r 53,000) and a heavy component, Tn-H (apparent M r 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca 2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-I in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.

The roles of potassium currents in Drosophila flight muscles

The roles of different K+ currents in regulating the generation and waveform of action potentials in Drosophila dorsal longitudinal flight muscles (DLMs) were examined in current-clamp experiments. In response to depolarizing current, DLMs displayed an initial transient rectification of the electronic potential lasting for up to hundreds of milliseconds. This delay in excitation was followed by oscillations or graded spikes that finally gave way to sharply rising spikes. Previous voltage-clamp studies of DLMs have revealed an inward Ca2+ current and at least three K+ currents: IA and IK, which are voltage-dependent, and IC, which is Ca2+ dependent. IA and IC are early inactivating currents, while IK is a slow, noninactivating current. In mature adults, selective elimination of IA either with Shaker (Sh) mutations or with 4-aminopyridine (4-AP), had no effect on spike duration or on the delay in excitation. In contrast, when IC was specifically eliminated with the slowpoke (slo) mutation, there was no delay before excitation, the amplitude of the spikes was significantly increased, and the spike duration was increased by 10-fold. Similar results were obtained by reducing IC in normal muscle by intracellular injections of EGTA or by use of low Ca2+ saline. Furthermore, DLM spikes evoked in slo by stimulation of the motorneuron were also broadened, suggesting that IC functions in a similar manner during normal flight as in current-clamped muscles. Elimination of IK along with IA and IC in saline containing tetraethylammonium or Ba2+ resulted in further prolongation of the DLM spike. In Ba2+ saline, there was an additional increase in spike amplitude as well. We conclude that in mature adults, IC, rather than IA, plays the major role in repolarization of DLM spikes and in the delay before excitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Optomotor control of the force of flight in Drosophila and Musca

Optomotor control of the force of flight in Drosophila and Musca

Optomotor control of the force of flight in Drosophila and Musca

Drift of the retinal images of the surroundings elicits optomotor responses of flight control in the fruitfly, Drosophila melanogaster, and in the housefly, Musca domestica. The present investigation deals with the responses of tethered flies in still air. The responses were elicited by continuous movement of striped patterns in front of the eyes, and characterized by the magnitude and elevation of the resulting force of flight which is the average of the forces produced during a wingbeat cycle. The force of flight is resolved into the upward directed lift and the forward directed thrust. In either species, pattern movement acts upon the magnitude, but not upon the elevation of the force of flight. The elevation relative to the longitudinal body axis is almost invariably 24° in Drosophila, and 29° in Musca. The lift/thrust ratio in still air is fixed accordingly, and can be changed only by variation of the body angle. Keeping an angle of minimum body drag does not contribute significantly to the efficiency of insect flight at very low Reynolds numbers (Re). Control of the lift/thrust ratio by variation of the body angle is, therefore, less surprising in Drosophila where Re is in the order of 102, than in Musca, where Re is in the order of 103. Control of this ratio without variation of the body angle is actually established in insects flying at even higher Re. Covariance of lift and thrust in the investigated flies is achieved by control of wingbeat amplitude or wingbeat frequency, but not by control of wing pitch or stroke plane. A change in the latter parameters would have deflected the force of flight and is, therefore, inconsistent with the constant elevation found in the present experiments. The results obtained, so far, do not exclude active deflections of the force vector during occasional bouts of aerobatics, or passive deflections of this vector during flight at non-zero airspeed.

Drosophila mutants reveal two components of fast outward current.

The gating of potassium ion channels has been shown to be dependent on voltage or Ca2+ ions or both 4,5. A fast transient potassium current (sometimes denoted IA) is found in a wide variety of animals. The Ca2+-sensitivity of this early outward current has been a matter of dispute as reports from different systems have indicated complete insensitivity, marked sensitivity or only partial sensitivity. It is possible that there are two distinct early outward current systems, one Ca2+-sensitive and the other not. Thus, the reports of partial Ca2+-sensitivity would indicate the presence of both systems in the membrane. I now report that in the adult Drosophila flight muscles, the transient outward current is also partially Ca2+-sensitive. The hypothesis that two separate currents are present is strengthened by the discovery that in several mutants of the X-linked Shaker locus (ShKS133, Sh102 and ShK0120): the Ca2+-independent component (IAci) is absent with only the Ca2+-dependent component (IAcd) remaining. Hence, the mutations seem to delete one of two separate current systems. An alternative hypothesis is that only one channel type is present that can be modified by mutation to be totally Ca2+-dependent.

Outward Currents in Developing Drosophila Flight Muscle

The development of two different voltage-sensitive potassium channels was studied in Drosophila flight muscle by voltage clamp techniques. Early in development active channels are not present in the membrane. The first channels to appear are the A current channels, which carry a fast, rapidly inactivating potassium current. The channels for delayed rectification appear later. Channels carrying inward current also appear only after the A current channels. During development, the A current may be easily studied in isolation from other currents and thus provides a desirable system for studying the genetic determinants of this current.

Changes in Membrane Properties of the Drosophila Dorsal Longitudinal Flight Muscle Induced by Sodium Pump Inhibitors

ABSTRACT Drosophila dorsal longitudinal flight muscle fibres made anoxic by passing nitrogen through the tracheal system or treated with 10−5m ouabain or strophanthidin show a reversible fall in resting membrane potential of 16·5 mV (s.e. 0·96), 13·7 mV (s.e. 0·87), and 17·0 mV (s.e. 2·8), respectively. The reversible depolarization obtained with these sodium pump blockers occurred within 10–15 min. The depolarization of the muscle fibres was accompanied by a decrease in input resistance of 21·2% (s.e. 3·8) in anoxia, 21·4% in ouabain, and 25·6% (s.e. 6·7) in strophanthidin. The resistance decrease in strophanthidin and ouabain was transient and returned to above the resting level while the muscle fibres were still exposed to these agents. Recovery of membrane potential in cells exposed to anoxia is biphasic. An initial ‘fast’ phase of recovery occurs within 15 s upon return to air followed by a late ‘slow’ phase lasting several minutes. Recovery of input resistance in cells exposed to N2 coincided with the ‘fast’ phase of the recovery of resting membrane potential. Recovery of membrane potential following exposure to strophanthidin is a long, slow process which occurs at conductance values at the resting level or below. The tendency towards spontaneous action potentials was increased by anoxia and the action potentials occurring in anoxia were elongated into plateau potentials of about 18 s duration. These results are consistent with the hypothesis that anoxia and cardioactive steroids inhibit a metabolic process, possibly an electrogenic ion pump, that is essential for maintenance of the resting membrane potential in Drosophila flight muscle. Exposure to these agents also results in changes in input resistance. Both of these effects could contribute to the depolarization and affect the excitable properties of the muscle fibre membrane.

Output pattern generation by Drosophila flight motoneurons

Output pattern generation by Drosophila flight motoneurons

Ultrastructure and Contractile Properties of Isolated Myofibrils and Myofilaments from Drosophila Flight Muscle

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The Respiratory Exchange of the Desert Locust (Schistocerca Gregaria) before, During and After Flight

ABSTRACT During the last two decades the knowledge of the physiology of heavy muscular work has increased considerably, and the high metabolic rate of flying insects has attracted particular attention. Jongbloed & Wiersma (1935), Chadwick & Gilmour (1940), and Davis & Fraenkel (1940) have reviewed the literature concerning the metabolic rate of flying insects. More recent investigations dealt mainly with the nature of the fuels combusted or with other aspects of insect flight. Most of the experiments referred to below dealt with measurements of the oxygen consumption of insects performing flight movements in a relatively small container. In this respect and because generally the animals were suspended and the natural locomotion was prevented the conditions of flight were abnormal. However, these limitations concerning the conditions of flight are probably of minor importance when we are interested mainly in the order of magnitude of the metabolic rate and in the nature of the fuels combusted, and with these limitations in mind, the present state of knowledge might be summarized: (1) During flight the oxygen consumption is considerably increased and has been estimated at about 100 1. O2/kg. body weight/hr. in bees (Jongbloed & Wiersma, 1935), flies (Davis & Fraenkel, 1940), and butterflies (Zeuthen, in Krogh, 1941). This means that the metabolic rate during flight in some insects increases 100 times or even more compared with the metabolism during rest. In an extensive and very interesting series of experiments Chadwick & Gilmour (1940) and Chadwick (1947) have demonstrated that various species of Drosophila consumed only 20 1. O2/kg./hr., a figure which was indirectly verified by Williams, Barness & Sawyer (1943) and Wigglesworth (1949). In the above-mentioned papers by Chadwick, and a paper by Chadwick & Williams (1949), the metabolic rate was correlated with the wing-beat frequency.(2) The respiratory quotient (R.Q.) during flight was measured in bees (Jongbloed & Wiersma, 1935, in Apis) and flies (Chadwick, 1947, in Drosophila). In both cases the R.Q. equalled unity, indicating the combustion of carbohydrates, and this interpretation seems to be correct, glucose and glycogen being utilized as fuels in Apis (Beutler, 1937) and Drosophila (Williams et al. 1943; Wigglesworth, 1949) respectively.(3) After the cessation of flight a small oxygen debt was demonstrated in Drosophila (Chadwick & Gilmour, 1940). The debt was abolished in less than 2 min. August Krogh’s unpublished analyses of the R.Q. of flying Lepidoptera gave values about unity, and so it seems reasonable to assume that the higher insects derive the energy for flight movements from the combustion of carbohydrates. Many insects generally considered primitive from a morphological point of view are, nevertheless, excellent flyers in one or more respects. The dragonflies, for example, are second to none as far as the flying speed and the refined regulation of the flight movements are concerned, and locusts are able to endure sustained flight for several hours every day during a considerable period of time. As the migrations of locusts are of great economic importance, it is thus of interest from both practical and theoretical points of view to study the metabolism during flight in these relatively primitive insects. The purpose of the present paper has been to study some quantitative and qualitative aspects of the combustion of flying locusts.

The utilization of reserve substances in Drosophila during flight.

ABSTRACT The respiratory quotient in Drosophila during flight is about 1·0 (Chadwick, 1947), and the duration of flight to exhaustion varies with the age of the fly in the same manner as does the total glycogen—increasing steeply during the first 5 days of adult life, remaining at a maximum for about 10 days and then falling gradually until the fly is 5 or 6 weeks old (Williams, Barness & Sawyer, 1943). It appears certain that glycogen is the main or sole reserve substance consumed during flight. It is the object of the present work to provide a histological picture of the distribution of the reserve substances and their mobilization during sustained flight. In the course of the work it has been possible to compare directly the efficiency of different substances as sources of energy for flight. D. melanogaster has been used throughout. The insects have been reared and maintained on standard maize-meal medium at 250C.

CENTRAL EFFECTS OF CENTRIPETAL IMPULSES IN AXONS OF SPINAL VENTRAL ROOTS

CENTRAL EFFECTS OF CENTRIPETAL IMPULSES IN AXONS OF SPINAL VENTRAL ROOTS