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Belinda Bullard - One of the best experts on this subject based on the ideXlab platform.
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Through thick and thin: dual regulation of insect Flight Muscle and cardiac Muscle compared
Journal of Muscle Research and Cell Motility, 2019Co-Authors: Belinda Bullard, Annalisa PastoreAbstract:Both insect Flight Muscle and cardiac Muscle contract rhythmically, but the way in which repetitive contractions are controlled is different in the two types of Muscle. We have compared the Flight Muscle of the water bug, Lethocerus , with cardiac Muscle. Both have relatively high resting elasticity and are activated by an increase in sarcomere length or a quick stretch. The larger response of Flight Muscle is attributed to the highly ordered lattice of thick and thin filaments and to an isoform of troponin C that has no exchangeable Ca^2+-binding site. The Ca^2+ sensitivity of cardiac Muscle and Flight Muscle can be manipulated so that cardiac Muscle responds to Ca^2+ like Flight Muscle, and Flight Muscle responds like cardiac Muscle, showing the malleability of regulation. The interactions of the subunits in Flight Muscle troponin are described; a model of the complex, using the structure of cardiac troponin as a template, shows an overall similarity of cardiac and Flight Muscle troponin complexes. The dual regulation by thick and thin filaments in skeletal and cardiac Muscle is thought to operate in Flight Muscle. The structure of inhibited myosin heads folded back on the thick filament in relaxed Lethocerus fibres has not been seen in other species and may be an adaptation to the rapid contractions of Flight Muscle. A scheme for regulation by thick and thin filaments during oscillatory contraction is described. Cardiac and Flight Muscle have much in common, but the differing mechanical requirements mean that regulation by both thick and thin filaments is adapted to the particular Muscle.
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binding partners of the kinase domains in drosophila obscurin and their effect on the structure of the Flight Muscle
Journal of Cell Science, 2015Co-Authors: Anja Katzemich, Mathias Gautel, Atsushi Fukuzawa, John C Sparrow, Sean T Sweeney, Ryan J H West, Belinda BullardAbstract:Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the Muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect Flight Muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal Muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila Muscle.
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the m line protein obscurin in the development of insect Flight Muscle
Biophysical Journal, 2015Co-Authors: Anja Katzemich, John C Sparrow, Kevin Leonard, Sean T Sweeney, Belinda BullardAbstract:The Drosophila M-line protein, obscurin (475 kD) has 21 tandem Ig domains, 3 Fn3 domains, a Rho-GEF signalling domain near the N-terminus and two kinase domains near the C-terminus. Obscurin is needed for the formation of a symmetrical sarcomere in the indirect Flight Muscle (IFM). Obscurin forms periodic striations in the IFM sarcomere 30 h after puparium formation, when kettin and myosin are still in unstructured strands and there are no Z-discs. Early expression of obscurin acts as a template for the formation of symmetrical thick filaments. Reducing expression by RNAi had no effect on sarcomere length in IFM, but the M-line was missing and the H-zone was shifted from the midline of the sarcomere. The length and polarity of thin filaments was determined by the position of the bare zone in adjacent thick filaments. Therefore obscurin is essential for the assembly of correctly overlapped thick and thin filaments in a symmetrical sarcomere. We have identified ligands of the two kinase domains in vivo. Kinase constructs with tags were injected into embryos and individual kinases with bound proteins were isolated from IFM. Kinase 1 bound Ball (an active kinase) and kinase 2 bound MASK (a 400 kD protein with ankyrin repeats). Reducing expression of these proteins by RNAi produced a phenotype in IFM that was similar to that produced by reducing obscurin, although obscurin itself was present at wild-type levels. Therefore Ball and MASK probably act downstream of obscurin. These proteins are linked via signalling pathways involved in the development of Drosophila Muscle.
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the m line protein obscurin in the early development of drosophila Flight Muscle
Biophysical Journal, 2013Co-Authors: Anja Katzemich, John C Sparrow, Christopher J H Elliott, Kevin Leonard, Sean T Sweeney, Belinda BullardAbstract:Obscurin is a 475 kDa protein in the M-line of Drosophila Muscles. The protein is made up of Ig and Fn3 domains, a Rho-GEF domain near the N-terminus and two kinase domains near the C-terminus. The expression of obscurin was reduced by a P-element insertion, or by RNAi. Knockdown of obscurin by RNAi was targeted to all Muscles, or specifically to the Flight Muscle (IFM). In P-element mutants and RNAi lines, embryo, larva and pupa developed normally; adults could walk and jump, but were Flightless. In the wild-type pupa, obscurin in the IFM appeared in striations at 30 hours after puparium formation, when kettin (a Z-disc protein) and myosin were in amorphous strands. Thus, obscurin in the M-line precedes the regular assembly of the Z-disc and A-band. In the IFM of flies with reduced obscurin, the sarcomere length was normal but the M-line was missing and H-zone irregular. Isolated thick filaments were asymmetrical with the bare zone shifted from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depended on the position of the bare zone in adjacent thick filaments. Thus, the early expression of obscurin nucleates the assembly of a symmetrical thick filament, which leads to thin filaments of uniform length. Ligands of the kinase domains are: ball (another kinase) binding to kinase 1 and MASK (an ankyrin-repeat protein) binding to kinase 2. Confirmation of these interactions by injecting embryos with kinase constructs will be described. The abnormalities in the IFM sarcomere in RNAi lines with reduced ball or MASK were similar to those in obscurin knockdown flies. The expression of obscurin was normal in RNAi lines of both ligands, demonstrating the importance of obscurin, ball and MASK to sarcomere assembly.
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Regulating the contraction of insect Flight Muscle
Journal of Muscle Research and Cell Motility, 2011Co-Authors: Belinda Bullard, Annalisa PastoreAbstract:The rapid movement of the wings in small insects is powered by the indirect Flight Muscles. These Muscles are capable of contracting at up to 1,000 Hz because they are activated mechanically by stretching. The mechanism is so efficient that it is also used in larger insects like the waterbug, Lethocerus . The oscillatory activity of the Muscles occurs a low concentration of Ca^2+, which stays constant as the Muscles contract and relax. Activation by stretch requires particular isoforms of tropomyosin and the troponin complex on the thin filament. We compare the tropomyosin and troponin of Lethocerus and Drosophila with that of vertebrates. The characteristics of the Flight Muscle regulatory proteins suggest ways in which stretch-activation works. There is evidence for bridges between troponin on thin filaments and myosin crossbridges on the thick filaments. Recent X-ray fibre diffraction results suggest that a pull on the bridges activates the thin filament by shifting tropomyosin from a blocking position on actin. The troponin bridges are likely to contain extended sequences of tropomyosin or troponin I (TnI). Flight Muscle has two isoforms of TnC with different Ca^2+-binding properties: F1 TnC is needed for stretch-activation and F2 TnC for isometric contractions. In this review, we describe the structural changes in both isoforms on binding Ca^2+ and TnI, and discuss how the steric model of Muscle regulation can apply to insect Flight Muscle.
Marcus F Oliveira - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial glycerol phosphate oxidation is modulated by adenylates through allosteric regulation of cytochrome c oxidase activity in mosquito Flight Muscle
Insect Biochemistry and Molecular Biology, 2019Co-Authors: A Gaviraghi, Juliana Correa B R Soares, Julio A Mignaco, Carlos Frederico Leite Fontes, Marcus F OliveiraAbstract:Abstract The huge energy demand posed by insect Flight activity is met by an efficient oxidative phosphorylation process that takes place within Flight Muscle mitochondria. In the major arbovirus vector Aedes aegypti , mitochondrial oxidation of pyruvate, proline and glycerol 3-phosphate (G3P) represent the major energy sources of ATP to sustain Flight Muscle energy demand. Although adenylates exert critical regulatory effects on several mitochondrial enzyme activities , the potential consequences of altered adenylate levels to G3P oxidation remains to be determined. Here, we report that mitochondrial G3P oxidation is controlled by adenylates through allosteric regulation of cytochrome c oxidase (COX) activity in A. aegypti Flight Muscle. We observed that ADP significantly activated respiratory rates linked to G3P oxidation, in a protonmotive force-independent manner. Kinetic analyses revealed that ADP activates respiration through a slightly cooperative mechanism. Despite adenylates caused no effects on G3P-cytochrome c oxidoreductase activity, COX activity was allosterically activated by ADP. Conversely, ATP exerted powerful inhibitory effects on respiratory rates linked to G3P oxidation and on COX activity. We also observed that high energy phosphate recycling mechanisms did not contribute to the regulatory effects of adenylates on COX activity or G3P oxidation. We conclude that mitochondrial G3P oxidation in A. aegypti Flight Muscle is regulated by adenylates through the allosteric modulation of COX activity, underscoring the bioenergetic relevance of this novel mechanism and the potential consequences for mosquito dispersal.
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mitochondrial glycerol phosphate oxidation is modulated by adenylates through allosteric regulation of cytochrome c oxidase activity in mosquito Flight Muscle
bioRxiv, 2019Co-Authors: A Gaviraghi, Juliana Correa B R Soares, Julio A Mignaco, Carlos Frederico Leite Fontes, Marcus F OliveiraAbstract:Abstract The huge energy demand posed by insect Flight activity is met by an efficient oxidative phosphorylation process that takes place within Flight Muscle mitochondria. In the major arbovirus vector Aedes aegypti, mitochondrial oxidation of pyruvate, proline and glycerol 3 phosphate (G3P) represent the major energy sources of ATP to sustain Flight Muscle energy demand. Although adenylates exert critical regulatory effects on several mitochondrial enzyme activities, the potential consequences of altered adenylate levels to G3P oxidation remains to be determined. Here, we report that mitochondrial G3P oxidation is controlled by adenylates through allosteric regulation of cytochrome c oxidase (COX) activity in A. aegypti Flight Muscle. We observed that ADP significantly activated respiratory rates linked to G3P oxidation, in a protonmotive force-independent manner. Kinetic analyses revealed that ADP activates respiration through a slightly cooperative mechanism. Despite adenylates caused no effects on G3P-cytochrome c oxidoreductase activity, COX activity was allosterically activated by ADP. Conversely, ATP exerted powerful inhibitory effects on respiratory rates linked to G3P oxidation and on COX activity. We also observed that high energy phosphate recycling mechanisms did not contribute to the regulatory effects of adenylates on COX activity or G3P oxidation. We conclude that mitochondrial G3P oxidation by A. aegypti Flight Muscle is regulated by adenylates essentially through the allosteric modulation of COX activity, underscoring the bioenergetic relevance of this novel mechanism and the potential consequences for mosquito dispersal.
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a method for assessing mitochondrial physiology using mechanically permeabilized Flight Muscle of aedes aegypti mosquitoes
Analytical Biochemistry, 2019Co-Authors: Alessandro Gaviraghi, Marcus F OliveiraAbstract:Abstract Aedes aegypti is the most important and widespread vector of arboviruses, including dengue and zika. Insect dispersal through the Flight activity is a key parameter that determines vector competence, and is energetically driven by oxidative phosphorylation in Flight Muscle mitochondria. Analysis of mitochondrial function is central for a better understanding of cellular metabolism, and is mostly studied using isolated organelles. However, this approach has several challenges and methods for assessment of mitochondrial function in chemically-permeabilized tissues were designed. Here, we described a reliable protocol to assess mitochondrial physiology using mechanically permeabilized Flight Muscle of single A. aegypti mosquitoes in combination with high-resolution respirometry. By avoiding the use of detergents, high respiratory rates were obtained indicating that substrate access to mitochondria was not limited. This was confirmed by using selective inhibitors for specific mitochondrial substrates. Additionally, mitochondria revealed highly coupled, as ATP synthase or adenine nucleotide translocator inhibition strongly impacted respiration. Finally, we determined that pyruvate and proline induced the highest respiratory rates compared to other substrates tested. This method allows the assessment of mitochondrial physiology in mosquito Flight Muscle at individual level, and can be used for the identification of novel targets aiming rational insect vector control.
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Blood-feeding induces reversible functional changes in Flight Muscle mitochondria of Aedes aegypti mosquito.
PloS one, 2009Co-Authors: Renata L.s. Goncalves, Gabriela O. Paiva-silva, Pedro L. Oliveira, Marisa M. Momoli, Marcos A. Vannier-santos, Ana Carolina Loyola Machado, Marcos Henrique Ferreira Sorgine, Jose Henrique M. Oliveira, Antonio Galina, Marcus F OliveiraAbstract:Background: Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energytransducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on Flight Muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever. Methodology/Principal Findings: Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a3 levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H2O2 formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed. Conclusion: Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feeding
Shigeo Hayashi - One of the best experts on this subject based on the ideXlab platform.
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mechano chemical enforcement of tendon apical ecm into nano filaments during drosophila Flight Muscle development
Current Biology, 2021Co-Authors: Weichen Chu, Shigeo HayashiAbstract:Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the muscular force is transmitted to the exoskeleton through the tendon cells and tendon apical extracellular matrix (ECM). In Drosophila, we found tendon cells secrete Dumpy (Dpy), a zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. We showed that Dpy undergoes filamentous conversion in response to the tension increment during indirect Flight Muscle development. We also found another ZPD protein Quasimodo (Qsm) protects the notum epidermis from collapsing under the Muscle tension by enhancing the tensile strength of Dpy filaments. Qsm is co-transported with Dpy in the intracellular vesicles and diffuses into the exuvial space after secretion. Tissue-specific qsm expression rescued the qsm mutant phenotypes in distant tissues, suggesting Qsm can function in a long-range, non-cell-autonomous manner. In the cell culture assay, Qsm interacts with Dpy-ZPD and promotes secretion and polymerization of Dpy-ZPD. The roles of Qsm underlies the positive feedback mechanism of force-dependent organization of Dpy filaments, providing new insights into apical ECM remodeling through the unconventional interaction of ZPD proteins.
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mechano chemical enforcement of tendon apical ecm into nano filaments during drosophila Flight Muscle development
Social Science Research Network, 2020Co-Authors: Weichen Chu, Shigeo HayashiAbstract:Contractile tension is critical for musculoskeletal system development and maintenance. In insects, the force transmission from the Muscle to the exoskeleton depends on the tendon cells and tendon apical extracellular matrix (aECM). In Drosophila, tendon cells secrete Dumpy (Dpy), a Zona pellucida domain (ZPD) protein, to form the force-resistant filaments in the exuvial space, anchoring the tendon cells to the pupal cuticle. By live imaging, we showed that Dpy filaments undergo remodeling in response to the tension increase during indirect Flight Muscle development. Additionally, we found another ZPD protein, Quasimodo (Qsm), which plays a long-range, non-cell-autonomous role in Dpy filaments enforcement. Inside the cells, Qsm promotes secretion and polymerization of Dpy-ZPD. The dual role of Qsm underlies the positive feedback mechanism of force-dependent remodeling of Dpy filaments, providing new insights into aECM remodeling through the unconventional interaction of ZPD proteins.
Hiroyuki Iwamoto - One of the best experts on this subject based on the ideXlab platform.
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The tymbal Muscle of cicada has Flight Muscle-type sarcomeric architecture and protein expression
Zoological Letters, 2017Co-Authors: Hiroyuki IwamotoAbstract:Background The structural and biochemical features of the tymbal (sound-producing) Muscle of cicadas were studied by X-ray diffraction and immunochemistry, and compared with those of Flight Muscles from the same species. Results The X-ray diffraction pattern of the tymbal Muscle was very similar to that of the dorsal longitudinal Flight Muscle: In both Muscles, the 2,0 equatorial reflection is much more intense than the 1,1, indicating that both Muscles have a Flight Muscle-type myofilament lattice. In rigor, the first myosin/actin layer line reflection was finely lattice-sampled, indicating that the contractile proteins are arranged with a crystalline regularity as in asynchronous Flight Muscles. In contrast, the diffraction pattern from the tensor Muscle, which modulates the sound by stressing the tymbal, did not show signs of such high regularity or Flight Muscle-type filament lattice. Electrophoretic patterns of myofibrillar proteins were also very similar in the tymbal Muscle and Flight Muscles, but distinct from those from the tensor or leg Muscles. The antibody raised against the Flight Muscle-specific troponin-I isoform reacted with an 80-kDa band from both tymbal and Flight Muscles, but with none of the bands from the tensor or leg Muscles. Conclusion The close similarities of the structural and biochemical profiles between the tymbal and the Flight Muscles suggest the possibility that a set of Flight Muscle-specific proteins is diverted to the tymbal Muscle to meet its demand for fast, repetitive contractions.
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Additional file 4: Figure S4. of The tymbal Muscle of cicada has Flight Muscle-type sarcomeric architecture and protein expression
2017Co-Authors: Hiroyuki IwamotoAbstract:SDS gel electrophoretic and immunoblot patterns of the Muscle fibers from Graptopsaltria nigrofuscata. (A), Coomassie brilliant blue-stained SDS gel electrophoretic pattern; (B), Western blot pattern obtained by using an antibody against Flight Muscle-specific troponin-I (troponin-H). Lanes: a, DLM; b, DVM; c, forewing basalar; d, forewing subalar; e, hindwing basalar; f, hindwing subalar; g, forewing 3Ax, h, hindwing 3Ax; i, tymbal; j, tensor; k; leg. (TIFF 980 kb
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a beetle Flight Muscle displays leg Muscle microstructure
Biophysical Journal, 2016Co-Authors: Toshiki Shimomura, Hiroyuki Iwamoto, Tat Thang Vo Doan, Shinichi Ishiwata, Hirotaka Sato, Madoka SuzukiAbstract:In contrast to major Flight Muscles in the Mecynorrhina torquata beetle, the third axillary (3Ax) Muscle is a minor Flight Muscle that uniquely displays a powerful mechanical function despite its considerably small volume, ∼1/50 that of a major Flight Muscle. The 3Ax Muscle contracts relatively slowly, and in Flight strongly pulls the beating wing to attenuate the stroke amplitude. This attenuation leads to left-right turning in Flight or wing folding to cease flying. What enables this small Muscle to be so powerful? To explore this question, we examined the microstructure of the 3Ax Muscle using synchrotron x-ray diffraction, optical microscopy, and immunoblotting analysis. We found that the 3Ax Muscle has long (∼5 μm) myofilaments and that the ratio of thick (myosin) filaments to thin (actin) filaments is 1:5 or 1:6. These characteristics are not observed in the major Flight Muscles, which have shorter myofilaments (∼3.5 μm) with a smaller ratio (1:3), and instead are more typical of a leg Muscle. Furthermore, the Flight-Muscle-specific troponin isoform, TnH, is not expressed in the 3Ax Muscle. Since such a microstructure is suitable for generating large tension, the 3Ax Muscle is appropriately designed to pull the wing strongly despite its small volume.
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x ray diffraction pattern from the Flight Muscle of toxorhynchites towadensis reveals the specific phylogenic position of mosquito among diptera
Zoological Letters, 2015Co-Authors: Hiroyuki IwamotoAbstract:The Diptera are a group of insects with only a single pair of wings (forewings), and are considered monophyletic (originating from a common ancestor). The Flight Muscle in Diptera has features not observed in other insects, such as the long Pro-Ala-rich peptide associated with tropomyosin, not with troponin-I as in other insects, and the formation of a superlattice by myosin filaments analogous to that in vertebrate skeletal Muscle. Here we describe X-ray diffraction patterns from the Flight Muscle of a mosquito, Toxorhynchites towadensis (Culicidae), belonging to a primitive group of Diptera. The diffraction pattern indicates that myosin filaments in the Flight Muscle of this species do not form a superlattice. X-ray diffraction also shows meridional reflections that are not observed in other dipterans, but are present in the patterns from bumblebee (Hymenoptera) Flight Muscle. These observations suggest that the superlattice structure evolved after the common ancestor of Diptera had diverged from other insects. The Flight Muscle of mosquito may retain primitive structural features that are shared by Hymenoptera.
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a peculiar meridional reflection in the x ray diffraction pattern from dipteran Flight Muscle suggests an alternating arrangement of tropomyosin isoforms
Biophysical Journal, 2009Co-Authors: Hiroyuki IwamotoAbstract:The X-ray diffraction pattern from the Flight Muscle of a cranefly, Ctenacroscelis mikado (Diptera), exhibits a prominent meridional reflection not observed in Lethocerus at a spacing of 25.8 nm. Since this spacing is two thirds of the pseudo-repeat of the long-pitched actin helix (38.7 nm), the reflection is likely to be of thin filament-origin. Its occurrence is fully explained if the scattering objects have a basic axial repeat of 77.4 nm (= 2 x 38.7 nm), and the six thin filaments surrounding a thick filament are arranged with an axial stagger of 25.8 nm (= 77.4/3).A possible mechanism to create the 77.4-nm repeat is the presence of two different tropomyosin isoforms. Dipteran Flight Muscle is known to express usual (∼35 kDa) and heavy (∼80 kDa) tropomyosin isoforms, and the extra mass of the latter is ascribed to the C-terminal extension of a pro- and ala-rich sequence. Tropomyosin is a uniform alpha-helical protein that forms a dimer with a typical coiled-coil structure, but the mass of the C-terminal extension would be localized. Thus, the reflection is most readily explained if the two isoforms produce an alternating array of homodimers. However, a cross-linking study suggests that the Ctenacroscelis isoforms produce heterodimers. In Drosophila, two heavy isoforms are known to exist (TmH-33 and TmH-34), and glutathione S-transferase-2 is stably associated with them (Clayton et al., 1998). Then an alternative explanation is that these isoforms also exist in Ctenacroscelis, and they are alternately arranged and only one of them binds GST-2. The scattering object remains to be identified, but the alternating arrangement of tropomyosin isoforms is the most conceivable mechanism to provide the periodicity needed to create the peculiar meridional reflection.
Anja Katzemich - One of the best experts on this subject based on the ideXlab platform.
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binding partners of the kinase domains in drosophila obscurin and their effect on the structure of the Flight Muscle
Journal of Cell Science, 2015Co-Authors: Anja Katzemich, Mathias Gautel, Atsushi Fukuzawa, John C Sparrow, Sean T Sweeney, Ryan J H West, Belinda BullardAbstract:Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the Muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect Flight Muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal Muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila Muscle.
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the m line protein obscurin in the development of insect Flight Muscle
Biophysical Journal, 2015Co-Authors: Anja Katzemich, John C Sparrow, Kevin Leonard, Sean T Sweeney, Belinda BullardAbstract:The Drosophila M-line protein, obscurin (475 kD) has 21 tandem Ig domains, 3 Fn3 domains, a Rho-GEF signalling domain near the N-terminus and two kinase domains near the C-terminus. Obscurin is needed for the formation of a symmetrical sarcomere in the indirect Flight Muscle (IFM). Obscurin forms periodic striations in the IFM sarcomere 30 h after puparium formation, when kettin and myosin are still in unstructured strands and there are no Z-discs. Early expression of obscurin acts as a template for the formation of symmetrical thick filaments. Reducing expression by RNAi had no effect on sarcomere length in IFM, but the M-line was missing and the H-zone was shifted from the midline of the sarcomere. The length and polarity of thin filaments was determined by the position of the bare zone in adjacent thick filaments. Therefore obscurin is essential for the assembly of correctly overlapped thick and thin filaments in a symmetrical sarcomere. We have identified ligands of the two kinase domains in vivo. Kinase constructs with tags were injected into embryos and individual kinases with bound proteins were isolated from IFM. Kinase 1 bound Ball (an active kinase) and kinase 2 bound MASK (a 400 kD protein with ankyrin repeats). Reducing expression of these proteins by RNAi produced a phenotype in IFM that was similar to that produced by reducing obscurin, although obscurin itself was present at wild-type levels. Therefore Ball and MASK probably act downstream of obscurin. These proteins are linked via signalling pathways involved in the development of Drosophila Muscle.
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the m line protein obscurin in the early development of drosophila Flight Muscle
Biophysical Journal, 2013Co-Authors: Anja Katzemich, John C Sparrow, Christopher J H Elliott, Kevin Leonard, Sean T Sweeney, Belinda BullardAbstract:Obscurin is a 475 kDa protein in the M-line of Drosophila Muscles. The protein is made up of Ig and Fn3 domains, a Rho-GEF domain near the N-terminus and two kinase domains near the C-terminus. The expression of obscurin was reduced by a P-element insertion, or by RNAi. Knockdown of obscurin by RNAi was targeted to all Muscles, or specifically to the Flight Muscle (IFM). In P-element mutants and RNAi lines, embryo, larva and pupa developed normally; adults could walk and jump, but were Flightless. In the wild-type pupa, obscurin in the IFM appeared in striations at 30 hours after puparium formation, when kettin (a Z-disc protein) and myosin were in amorphous strands. Thus, obscurin in the M-line precedes the regular assembly of the Z-disc and A-band. In the IFM of flies with reduced obscurin, the sarcomere length was normal but the M-line was missing and H-zone irregular. Isolated thick filaments were asymmetrical with the bare zone shifted from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depended on the position of the bare zone in adjacent thick filaments. Thus, the early expression of obscurin nucleates the assembly of a symmetrical thick filament, which leads to thin filaments of uniform length. Ligands of the kinase domains are: ball (another kinase) binding to kinase 1 and MASK (an ankyrin-repeat protein) binding to kinase 2. Confirmation of these interactions by injecting embryos with kinase constructs will be described. The abnormalities in the IFM sarcomere in RNAi lines with reduced ball or MASK were similar to those in obscurin knockdown flies. The expression of obscurin was normal in RNAi lines of both ligands, demonstrating the importance of obscurin, ball and MASK to sarcomere assembly.