Academic literature on the topic 'Drosophila IFM'

A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA’s complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. Our results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.

Drosophila indirect flight muscle (IFM) contains two different types of tropomyosin: a standard 284-amino acid muscle tropomyosin, Ifm-TmI, encoded by the TmI gene, and two > 400 amino acid tropomyosins, TnH-33 and TnH-34, encoded by TmII. The two IFM-specific TnH isoforms are unique tropomyosins with a COOH-terminal extension of approximately 200 residues which is hydrophobic and rich in prolines. Previous analysis of a hypomorphic TmI mutant, Ifm(3)3, demonstrated that Ifm-TmI is necessary for proper myofibrillar assembly, but no null TmI mutant or TmII mutant which affects the TnH isoforms have been reported. In the current report, we show that four flightless mutants (Warmke et al., 1989) are alleles of TmI, and characterize a deficiency which deletes both TmI and TmII. We find that haploidy of TmI causes myofibrillar disruptions and flightless behavior, but that haploidy of TmII causes neither. Single fiber mechanics demonstrates that power output is much lower in the TmI haploid line (32% of wild-type) than in the TmII haploid line (73% of wild-type). In myofibers nearly depleted of Ifm-TmI, net power output is virtually abolished (< 1% of wild-type) despite the presence of an organized fibrillar core (approximately 20% of wild-type). The results suggest Ifm-TmI (the standard tropomyosin) plays a key role in fiber structure, power production, and flight, with reduced Ifm-TmI expression producing corresponding changes of IFM structure and function. In contrast, reduced expression of the TnH isoforms has an unexpectedly mild effect on IFM structure and function.

Although studies have shown that myomesin 2 (MYOM2) mutations can lead to hypertrophic cardiomyopathy (HCM), a common cardiovascular disease that has a serious impact on human life, the effect of MYOM2 on cardiac function and lifespan in humans is unknown. In this study, dMnM (MYOM2 homologs) knockdown in cardiomyocytes resulted in diastolic cardiac defects (diastolic dysfunction and arrhythmias) and increased cardiac oxidative stress. Furthermore, the knockdown of dMnM in indirect flight muscle (IFM) reduced climbing ability and shortened lifespan. However, regular exercise significantly ameliorated diastolic cardiac dysfunction, arrhythmias, and oxidative stress triggered by dMnM knockdown in cardiac myocytes and also reversed the reduction in climbing ability and shortening of lifespan caused by dMnM knockdown in Drosophila IFM. In conclusion, these results suggest that Drosophila cardiomyocyte dMnM knockdown leads to cardiac functional defects, while dMnM knockdown in IFM affects climbing ability and lifespan. Furthermore, regular exercise effectively upregulates cardiomyocyte dMnM expression levels and ameliorates cardiac functional defects caused by Drosophila cardiomyocyte dMnM knockdown by increasing cardiac antioxidant capacity. Importantly, regular exercise ameliorates the shortened lifespan caused by dMnM knockdown in IFM.

Muscle stretch activation (SA) is critical for optimal cardiac and insect indirect flight muscle (IFM) power generation. The SA mechanism has been investigated for decades with many theories proposed, but none proven. One reason for the slow progress could be that multiple SA mechanisms may have evolved in multiple species or muscle types. Laboratories studying IFM SA in the same or different species have reported differing SA functional properties which would, if true, suggest divergent mechanisms. However, these conflicting results might be due to different experimental methodologies. Thus, we directly compared SA characteristics of IFMs from two SA model systems, Drosophila and Lethocerus, using two different fiber bathing solutions. Compared with Drosophila IFM, Lethocerus IFM isometric tension is 10- or 17-fold higher and SA tension was 5- or 10-fold higher, depending on the bathing solution. However, the rate of SA tension generation was 9-fold faster for Drosophila IFM. The inverse differences between rate and tension in the two species causes maximum power output to be similar, where Drosophila power is optimized in the bathing solution that favors faster muscle kinetics and Lethocerus in the solution that favors greater tension generation. We found that isometric tension and SA tension increased with calcium concentration for both species in both solutions, reaching a maximum plateau around pCa 5.0. Our results favor a similar mechanism for both species, perhaps involving a troponin complex that does not fully calcium activate the thin filament thus leaving room for further tension generation by SA.

Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and α-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM–I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with μ-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

Stretch activation (SA) is a delayed increase in force that enables high power and efficiency from a cyclically contracting muscle. SA exists in various degrees in almost all muscle types. In Drosophila, the indirect flight muscle (IFM) displays exceptionally high SA force production ( FSA), whereas the jump muscle produces only minimal FSA. We previously found that expressing an embryonic (EMB) myosin heavy chain (MHC) isoform in the jump muscle transforms it into a moderately SA muscle type and enables positive cyclical power generation. To investigate whether variation in MHC isoforms is sufficient to produce even higher FSA, we substituted the IFM MHC isoform (IFI) into the jump muscle. Surprisingly, we found that IFI only caused a 1.7-fold increase in FSA, less than half the increase previously observed with EMB, and only at a high Pi concentration, 16 mM. This IFI-induced FSA is much less than what occurs in IFM, relative to isometric tension, and did not enable positive cyclical power generation by the jump muscle. Both isometric tension and FSA of control fibers decreased with increasing Pi concentration. However, for IFI-expressing fibers, only isometric tension decreased. The rate of FSA generation was ~1.5-fold faster for IFI fibers than control fibers, and both rates were Pi dependent. We conclude that MHC isoforms can alter FSA and hence cyclical power generation but that isoforms can only endow a muscle type with moderate FSA. Highly SA muscle types, such as IFM, likely use a different or additional mechanism.

The indirect flight muscles (IFMs) of Lethocerus (giant water bug) and Drosophila (fruitfly) are asynchronous: oscillatory contractions are produced by periodic stretches in the presence of a Ca2+ concentration that does not fully activate the muscle. The troponin complex on thin filaments regulates contraction in striated muscle. The complex in IFM has subunits that are specific to this muscle type, and stretch activation may act through troponin. Lethocerus and Drosophila have an unusual isoform of the Ca2+-binding subunit of troponin, troponin C (TnC), with a single Ca2+-binding site near the C-terminus (domain IV); this isoform is only in IFMs, together with a minor isoform with an additional Ca2+-binding site in the N-terminal region (domain II). Lethocerus has another TnC isoform in leg muscle which also has two Ca2+-binding sites. Ca2+ binds more strongly to domain IV than to domain II in two-site isoforms. There are four isoforms in Drosophila and Anopheles (malarial mosquito), three of which are also in adult Lethocerus. A larval isoform has not been identified in Lethocerus. Different TnC isoforms are expressed in the embryonic, larval, pupal and adult stages of Drosophila; the expression of the two IFM isoforms is increased in the pupal stage. Immunoelectron microscopy shows the distribution of the major IFM isoform with one Ca2+-binding site is uniform along Lethocerus thin filaments. We suggest that initial activation of IFM is by Ca2+ binding to troponin with the two-site TnC, and full activation is through the action of stretch on the complex with the one-site isoform.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5′ promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5′ and 3′ untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

An extensive ethylmethanesulfonate mutagenesis of Drosophila melanogaster was undertaken to isolate the stronger alleles of 3 indirect flight-muscle mutations. We isolated 17 strong mutant lines, with nearly complete penetrance and expressivity, using direct screening under polarized light, from more than 1700 mutagenized chromosomes. On complementation, we found 11 of these 17 mutant lines to be alleles of 3 indirect flight-muscle mutations (Ifm(2)RU1, 3 noncomplementing lines; ifm(2)RU2, 6 alleles; ifm(2)RU3, 2 alleles) of the previously isolated 8 complementation groups (Ifm(2)RU1to ifm(2)RU8). In addition, we found 6 new complementation groups with strong defects in adult-muscle morphology; we named these ifm(2)RS1 to ifm(2)RS6. All mutant lines were mapped by meiotic recombination, and 5 of the 6 new complementation lines were mapped using chromosome deficiencies. ifm(2)RS1 maps to a region that harbors ifm(2)RU4 (a mutation that was isolated previously); however, theses are not alleles because each complements the other mutation, and the mutant-muscle phenotype is very different. We used direct screening under polarized light to find recessive mutations; although this method was labor intensive, it can be used to identify recessive genes involved in myogenesis, unlike screens for flightlessness or wing-position defects. This screen identifies regions on the second chromosome that harbor probable genes that are likely expressed in the mesoderm and are thought to be involved in myogenesis. This screen has generated valuable resources that will help us to understand the role of many molecular players involved in myogenesis.

Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a null mutation for flightin, fln0, that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln0 IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25–30% longer than in wild type. fln0 fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln0 sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

Muscle development and function are highly synchronized processes that involve simultaneous action of several transcription factors, signalling cascades, kinases and phosphatases, ion channels, structural proteins and secondary messengers. Several aspects of muscle biology including contraction, development, growth, regeneration etc. have been extensively studied using model organisms like Mus musculus, C. elegans, Drosophila melanogaster, Danio rerio and Xenopus. Chapter 1 provides an in-detail account of work done previously in the field of muscle biology that includes literature about different types of muscles in vertebrates, their localization and function in the body, arrangement of thick and thin filaments in different types of muscles and their development. Of the several model organisms available, Drosophila has long been a favored model organism for muscle biologists. The indirect flight muscles (IFMs) of Drosophila are structurally similar to vertebrate skeletal muscles and physiologically to cardiac muscles and have been extensively used as a model to study vertebrate muscle development and function. Chapter 1 provides an overview of literature on development and function of IFMs in Drosophila. An essential and probably most important second messenger utilized by muscles for their function is Calcium. Muscles harbor an intricate and elaborate machinery of proteins that can sense calcium, transport calcium in and out of the cell, bind calcium and regulate gene expression and so on. Chapter 1 briefly explains the studies done previously with respect to calcium signalling in muscles. Background, which led to hypotheses for present study, has been described in the chapter. Chapter 2 includes details about all the techniques that have been used to conduct the experiments. Calcium signalling plays an important role not just in functioning of muscles but also in development, growth, injury and regeneration of the tissue. Cells have an elaborate machinery of calcium handling proteins that work in synchrony to achieve a desired state of function. Details of this have been included in Chapter 4. Calcium handling machinery is comprised of ion channels like voltage gated calcium channels, calcium activated potassium channels, calcium binding proteins, structural proteins etc. Chapter 3 talks about one of the calcium binding protein, Calcineurin, whose function has been implicated in fibre type switching in vertebrate muscles. Its expression in the muscles increases during exercise or weightlifting suggesting its role in conversion of fast glycolytic fibres to slow oxidative fibres that are required for maintaining sustained force in the muscles. Calcineurin works in concert with several transcription factors to bring about changes in transcription under conditions of stress. It is known to interact with NFAT transcription factor to regulate fibre type switching. It is also known to work along with Mef2 transcription factor to regulate expression of genes. Current study shows that the reduction in levels of Calcineurin-A subunit in muscles does not affect function or structure of muscles, but over-expression of the protein causes premature death of majority of the organisms and flight lessness in the escaper flies. On the contrary, loss of regulatory subunit of the protein, Calcineurin-B2, causes muscle hypercontraction in IFMs of Drosophila, suggesting crucial role of the protein in IFM development. The slow, progressive degeneration of IFMs in calcineurin-B2 (canB2) mutants is reminiscent of the muscle hypercontracted phenotype observed in mutants of Myosin heavy chain and the Troponin T and Troponin I proteins. Genetic studies of calcineurin with mutants of Troponin T and I show a synergistic interaction between Troponin T mutant up101 and calcineurin-B2. The Drosophila Troponin T mutation, up101, is equivalent to human cardiomyopathy Troponin T mutations, R92Q and I79N. The contractile machinery of the Troponin T mutants shows increased sensitivity towards calcium and can contract at the calcium concentrations below the threshold level. Current study (Chapter 3) highlights the importance of calcineurin in maintaining calcium homeostasis in muscles. Loss of calcineurin leads to arrhythmic spontaneous calcium oscillations in IFMs, which means that the average time for which the contraction machinery remains in contact with calcium is higher in canB2 mutant than control (frequency of oscillations is higher in canB2 mutants than control) and this probably contributes to the enhanced hypercontraction phenotype in a calcium sensitive mutant of Troponin T. Arrhythmicity in the calcium oscillations is observed as early as 50hrs after puparium formation (APF), well before the muscle degeneration phenotype is manifested in canB2 mutant flies. This reflects towards the importance of calcineurin in maintenance of calcium homeostasis in muscles. Chapter 4 describes study of spontaneous calcium oscillations during IFM development. Calcium is a highly versatile signal that works at different time points to regulate several cellular processes. Spontaneous calcium oscillations have been extensively studied in striated muscles, both the cardiac and skeletal muscles. There are different types of oscillations to which muscles respond. Long duration calcium transients (LDTs) have been identified in Xenopus myocytes and they predominantly occur prior to myofibrillogenesis, whereas SDTs (Short duration calcium transients) are spontaneous calcium oscillations of short duration (2- 3sec) that originate in subsarcolemmal space and are ryanodine sensitive, insensitive to changes in membrane potential and are independent of extracellular calcium. Similar to vertebrate muscles spontaneous calcium oscillations are also observed in IFMs of Drosophila throughout development. These oscillations were not reported previously in this system. We observe that the calcium oscillations in IFMs start as early as 34hrs APF, coinciding with the initiation of myofibrillogenesis process. There were no evident oscillations before 34hrs APF (i.e. from 0- 34hrs; the time point that involves processes of muscle splitting and myoblast fusion). The nature of these oscillations is still obscure. These oscillations vary in frequency, peak width and peak area across development. Previous reports have shown that cells often respond to changes in stimulus by varying frequency of calcium waves. These frequency changes are decoded by sophisticated molecular machines that include calcium sensitive proteins like calcium/calmodulin dependent protein kinase II and protein kinase C. The difference in peak frequency observed in the developing IFM could be due to differential expression of ion channels and structural proteins at these stages. Indeed, our results show that channels like Ryanodine, STIM and cacophony are transcriptionally regulated, and their transcripts are expressed only in adults whereas transcripts of channels like SERCA and slowpoke (Calcium gated potassium channels) are detected strongly throughout the development. Current study shows that spontaneous calcium oscillations in IFMs are sensitive to the levels of SERCA channels. These channels localize to the endoplasmic reticulum and are required for transportation of calcium from cytosol to endoplasmic reticulum. SERCA calcium pump and its function of calcium sequestration is essential for both development and functioning of the muscles because majority of the animals with reduced expression of SERCA do not survive till adult stage, rather they die in early larval or early pupal stages. Knockdown of SERCA in IFMs leads to increase in peak area and peak width of the calcium oscillations, which suggests the defect in calcium sequestration ability of the muscles. This abnormality in the calcium quenching could result in irregular muscle contraction in adult flies, which is shown by the contracted state of the adult muscles in escaper flies. Spontaneous oscillations are also sensitive to the changes in intracellular calcium levels. Reduction in the levels of intracellular calcium by over-expression of calcium binding protein, Parvalbumin, reduces the frequency of oscillations in developing IFM. These flies show defects in their flight ability suggesting that calcium is utmost important for the functioning of the muscles. Taken together, these studies reflect upon importance of calcium signalling in muscle development and function.

Muscle development is a complex and multifactorial process involving assembly of thousands of proteins in a precise and synchronized manner over the course of development of an organism. Multiple genes and signalling pathways have been identified to have pivotal roles in the development and function of a healthy and functional muscle. However, the role of many genes in muscle development remains unknown. One such gene are the BCL11A/B genes. BCL11A and BCL11B are paralogous genes which belong to the kruppel-like C2H2 type zinc finger transcription factors. The BCL11A and BCL11B protein sequences are 58% identical and 68% similar to each other. Both, BCL11A and BCL11B mainly have non-overlapping functions in neurogenesis and immune cell development. Recent studies have reported mutations in BCL11A to be associated with muscle-related defects like hypotonia, speech disorder and gross motor impairments, while mutations in BCL11B have been shown to be associated with muscle-related defects like hypertrophic cardiomyopathy and aortic stiffness. However, there are no studies which have addressed the molecular function of BCL11A and BCL11B in muscle development and function. CG9650 is the ortholog of BCL11A and BCL11B in Drosophila melanogaster. Overall, CG9650 bears 84% similarity to the vertebrate BCL11A and BCL11B proteins. The Indirect Flight Muscles (IFMs) of Drosophila melanogaster occupy a majority of the thorax of the adult fly and are responsible for powering the wing stroke during flight. These muscles consist of two opposing sets of muscles, namely the dorso-longitudinal muscles (DLMs; six in number) oriented anterior to posterior, and the dorso-ventral muscles (DVMs; three in number) oriented in a dorsal -to-ventral manner. The DLMs are formed by fusion of myoblasts with 3 pre-existing templates called Larval Oblique Muscles (LOMs), and subsequent splitting to form the 6 DLMs. The DVMs are formed by de novo fusion of myoblasts. Due to various advantages like the spatially and temporally distinct time-course of the development, dispensability to survival, similarity in development of the IFMs and vertebrate myogenesis, etc, the Indirect Flight Muscles (IFMs) of Drosophila melanogaster are an excellent model system to study the function of genes in muscle development. In this study, we have attempted to determine the role of CG9650 in development of the IFMs of Drosophila melanogaster. Our experiments show CG9650 is expressed during the specification (embryonic), proliferation (larval), and migration & fusion (pupal) stages of IFM development and depletion of CG9650 leads to a defect in the pattern of the DLMs (i.e. reduced number of DLMs). The expression of CG9650 during the proliferation, migration and fusion stages is crucial for patterning of the DLMs. This patterning defect could be rescued by transgenic expression of CG9650 during IFM development. The CG9650-depleted flies were compromised in their flight ability; walking ability of these flies remained unaffected. At the fascicular level, these DLMs have a larger cross-sectional area, more fibers per fascicle, but a decreased packing density of myofibrils compared to control flies. The sarcomeres of CG9650-depleted flies are thinner and longer, and expressed of the Z-disc protein Actinin was reduced compared to that control flies. These fascicular and sarcomeric defects are believed to cause reduced flight ability of the CG9650-depleted flies. CG9650 is also shown to affect Notch signalling in a context-dependant manner. Loss of CG9650 leads to upregulation of Notch signalling in myoblasts at the proliferation (larval) stage of IFM development. This leads to increased proliferation of the myoblasts. Additionally, loss of CG9650 leads to reduced Armadillo levels, which in turn leads to reduced wingless signalling, thus the stratification of myoblasts on the notum region of the wing disc. During the migration & fusion stages of IFM development, SnS, a (Ig-domain containing protein) is required for fusion of the migrating myoblast with the developing fiber. Migrating myobalsts show a diffuse expression of SnS. Notch signalling induces the formation of SnS puncta required for fusion of the myoblast with developing fiber. Perturbation of CG9650 during IFM development leads to decreased Notch signalling and a decrease in the formation of SnS puncta. This leads to decreased myoblast fusion and hence, arrested splitting of the developing LOMs. A transcriptomics approach was used to determine the genes/pathways regulated by CG9650. An RNA-seq using DLMs depleted for CG9650 revealed misregulation of genes mainly involved in myogenesis and neurogenesis. Aret, Act88F, thor, CanA, myofilin, Mlp60A, Zasp52 were among the differentially regulated genes with known functions in myogenesis. These genes have functions during the myofibrillogenesis stage of IFM development and their differential expression could account for the fascicular and sarcomeric defects seen in CG9650-depleted DLMs. Notably, expression of Hibris (a gene known to function in conjunction with SnS to regulate myoblast fusion) was also differentially regulated. Multiple genes known to be targets of Notch signalling were also found to be differentially regulated, thus confirming CG9650 as a regulator of the Notch pathway. Differential regulation of expression of genes regulating in cell division, channel proteins, immunity, cuticle formation and gene expression was also observed. In summary, our study highlights CG9650 as a novel modulator of Notch signalling output and novel regulator of DLM patterning during IFM development.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

The Notch sequence from Drosophila is used as the sample data. Notch is thought to control cell fate decisions in development It encodes a large, transmembrane protein which may function through cell adhesion, and it was cloned and sequenced in 1985(WhartonetaI., 1985a&b; Kidd et al., 1986). Notch is an ideal sequence to analyze because it contains many features that computers are good at finding. Figure 1 shows a schematic of the Notch protein and its major features. The Notch sequence is available in the Genbank and EMBL sequence database under accession numbers M16153, M16149, M16150, M16151 and M16152 (see Appendix VIS). The most successful way to approach this chapter is to reproduce the analyses. This will familiarize one with a specific software package, and offers a more accurate picture of the volume of output data produced by many programs than could be allowed in the figures in this chapter. Programs for most of the analyses used in this chapter are widely available on IBM PCs, Macintoshes and mainframe computers. The examples have intentionally been kept generic, but programs from the following sources were used: Genetics Computer Group Sequence Analysis Software (Devereux et al., 1984; Genetics Computer Group Inc., Madison, WI),Genbank Online Services (Benton,1990),NationalLibrary of Medicine Services (Benson et al., 1990), and PC Gene (A. Bairoch, University of Geneva; ™ Intelligenetics Inc., Mountain View, CA and Genofit SA). Unless a researcher is studying nontranslatable segments of DNA, the immediate goal upon the isolation of a new gene is usually to deduce the amino acid sequence of its product. The laboratory approach might go from isolating a cDNA clone, determining its nucleotide sequence, locating alarge open reading frame, and translating the sequence into a putative protein. In this case priority is usually given to analyzing the putative protein, with promoter regions introns being sequenced later to elucidate gene regulation. The organization of the following example analysis of Notch reflects these laboratory priorities by beginning with cDNA analysis, moving to protein analysis, and then returning to DNA analysis for the genomic sequence.

The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.