Intersegmental Muscles Research Articles

Post-transcriptional regulation of gene expression during the programmed death of insect skeletal muscle.

While considerable attention has focused on the role of specific proteins in mediating programmed cell death, few studies have examined the possible involvement of post-transcriptional regulation of mRNAs associated with this developmental process. We have examined developmental changes in transcript stability and translatability using protein extracts generated from the intersegmental muscles (ISM) of the moth Manduca sexta as a cell-free model system to examine three genes that are representative of the patterns of expression observed in condemned ISMs: repressed (actin), induced (polyubiquitin) and constitutively expressed (ubiquitin-fusion 80; ubf80). In addition, we have used luciferase mRNA as a generic reporter transcript to determine if there are sequence-specific controls of mRNA function related to programmed cell death. Among the three Manduca transcripts, polyubiquitin displayed the shortest half-life (t1/2) in all ISM extracts tested. The stability and translatability of all mRNAs were most affected in extracts from muscle cells from day 17 animals, just prior to the commitment of the muscles to die. Transfer of the 3' untranslated regions from the Manduca transcripts to luciferase mRNA did not appreciably change the stability or translatability of this test transcript. These data suggest that there may be global removal of cellular transcripts just prior to death to allow newly expressed mRNAs to rapidly accumulate to high levels. Such changes in message abundance, translatability and stability may facilitate the efficient activation of death (and perhaps other differentiation programs) in some developmental systems.

Proteolytic activity of 26s proteasomes isolated from muscles of the tobacco hornworm, Manduca sexta: differences between surviving muscles and those undergoing developmentally programmed cell death.

The intersegmental muscles (ISMs) of tobacco hornworm,Manduca sexta are a well-characterised model system for examining the biochemical changes that accompany programmed cell death during development. When the ISMs become committed to die, there are dramatic increases in both the ubiquitin-expression, and ubiquitin-dependent proteolysis. Since the 26S proteasome is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its enzymatic properties. Specific chymotrypsin-like proteolytic activity of 26S proteasomes isolated from ISM is four times higher than that of surviving flight muscle (FM). However, specific activity does not change between developmental stages within ISM or FM. The difference between proteolytic capacity of the two kinds of muscles is even higher when the ISM become committed to die because 26S proteasome content of ISM increases just before cell death. These observations underline the role of 26S proteasome in programmed cell death.

NOT ALL MUSCLES MEET THE SAME FATE WHEN THEY DIE

Two general patterns of cell death are usually described in animals: necrosis and apoptosis. The former is a passive process that displays cellular swelling and lysis, while the latter involves cellular shrinkage and gene-mediated, ATP-dependent processes. Independent of the proximal cause of cell death, cell corpses are almost always removed by phagocytic cells. This is far from universal for all cells however, since phagocytic cells have not been noted during the programmed death of some skeletal muscles in insects. To further explore this, we used a variety of anatomical methods to examine the death of the intersegmental muscles (ISMs) of the moth Manduca sexta. The ISMs are giant cells that die during the 30h following adult emergence. At no stage examined were hemocytes or other cells associated with the sarcolemma. The failure to detect macrophages was not due to technical limitations since immunohistochemical and functional studies demonstrate their presence in the hemolymph. The absence of phagocytosis to remove ISM corpses suggests that all of the biochemical machinery required for cellular destruction is resident within the ISMs themselves. This is consistent with analysis suggesting that Manduca does not possess sufficient numbers of macrophages to consume the ISMs. Given that insects do not have adaptive immunity, the ability to use a completely cell autonomous process may be a developmental option that cannot be exploited in vertebrates.

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.

Molecular and Cytological Characterization of an Actin Gene from Hessian Fly (Diptera: Cecidomyiidae)

Abstract A clone of the Drosophila melanogaster (Meigen) gene Act5C was used to isolate an actin gene from a Hessian fly, Mayetiola destructor (Say), genomic library in phage lambda. A combined molecular and cytological analysis with the Hessian fly actin gene (designated MdA1) was undertaken. The coding region of this gene was contiguous and encoded a protein that was 99% identical to the intersegmental muscle actins 57A and 87E from D. melanogaster. Additionally, the protein was >97% identical to the flight-muscle-specific actin 88E from D. melanogaster as well as muscle actins 1 and 2 from Bombyx mori (L.). Only the muscle actin 79B from D. melanogaster, a muscle actin in leg and thorax, showed <97% identity with actin 1 from Hessian fly. The actin 1 from Hessian fly shared less amino acid identity (94–95%) with the cytoplasmic actins from D. melanogaster, Anopheles gambiae (Giles), and B. mori, with differences occurring in a conserved region of the cytoplasmic actins proposed to function in interacti...

New experimental evidence for somite resegmentation

According to the concept of resegmentation, the boundaries of vertebrae are shifted one half a segment compared with somite boundaries. This theory has been experimentally confirmed by interspecific transplantations of single somites. Due to the difficulty of exactly orientating individual somites in the host embryo, the outcome and interpretations of these experiments have occasionally been questioned. This is especially true for the formation of neural arches, their processes, and the ribs. We reinvestigated the formation of vertebrae in the avian embryo by grafting one and one half somites from quail to chick embryos. This method eliminates the possibility of a wrong somite orientation in the host embryo. Results show that the vertebral body, the neural arch and its processes are made up of material of two adjacent somites. This is also true for the rib, with the exception of the costal head, which is formed by only one somite. Whereas in the proximal part of the costal body the chick and quail cell regions border on each other in the middle of the rib, in its distal part quail cells gradually begin to mix with chick cells. The intersegmental muscles and their skeletal attachments sites are formed from the same somite. These results support and complete the data of previous studies and confirm the resegmentation concept.

Cloning and analysis of small cytoplasmic leucine-rich repeat protein (SCLP), a novel, phylogenetically-conserved protein that is dramatically up-regulated during the programmed death of moth skeletal muscle.

We used the abdominal intersegmental muscles (ISMs) of the moth Manduca sexta as a source of transcripts that are dramatically up-regulated during programmed cell death. One of these transcripts, Small Cytoplasmic Leucine-Rich Repeat Protein (SCLP), encodes a protein of approximately 24 kD that contains four perfect and two imperfect leucine-rich repeat (LRR) motifs. DNA sequence database analysis suggests that SCLP is a phylogenetically-conserved gene of unknown function. Both Northern and Western blots demonstrated that SCLP is expressed in the ISMs at all stages examined, but increases greater than 10-fold when the cells become committed to die. This increase in expression is regulated by the same change in the circulating ecdysteroid titer that controls death. Low levels of SCLP expression are also seen in flight muscle and fat body, but not in ovary, male sexual accessory gland, or Malpighian tubules. Immunohistochemical analysis demonstrates that SCLP is a cytoplasmic protein. Western blot analysis of proteins from the fly Drosophila suggests that an SCLP-related protein is expressed at the larval and pupal stages, but not in embryos or adults. Targeted expression of moth SCLP to a variety of different tissues in Drosophila using the Gal4/UAS P element system failed to generate an overt phenotype. These data are interpreted as suggesting that whereas SCLP presumably plays an important role in programmed cell death of muscle, perhaps by acting as an adaptor protein, its expression is insufficient to initiate death by itself.

A 220-kDa Activator Complex of the 26 S Proteasome in Insects and Humans: A ROLE IN TYPE II PROGRAMMED INSECT MUSCLE CELL DEATH AND CROSS-ACTIVATION OF PROTEASOMES FROM DIFFERENT SPECIES

The S10b (SUG2) ATPase cDNA has been cloned by reverse transcription-polymerase chain reaction/rapid amplification of cDNA ends from mRNA of intersegmental muscles of the tobacco horn moth (Manduca sexta). The S10b ATPase is a component of the 26 S proteasome, and its concentration and that of its mRNA increase dramatically during development in a manner similar to other ATPases of the 19 S regulator of the 26 S proteasome. The S10b and S6' (TBP1) ATPases are also present in a complex of approximately 220 kDa in intersegmental muscles. The 220-kDa complex markedly activates (2-10-fold) the 26 S proteasome, even when bound to anti-S10b antibodies immobilized on Sepharose, and increases in concentration approximately 5-fold like the 26 S proteasome in the intersegmental muscles in preparation for the programmed death of the muscle cells. A similar activator complex is present in human brain and placenta. Free activator complexes cross-activate: the Manduca complex activates rat skeletal muscle 26 S proteasomes, and the placental complex activates Manduca 26 S proteasomes. The placental activator complex contains S10b and S6', but not p27. This 220-kDa activator complex has been evolutionarily conserved between species from insect to man and may have a fundamental role in proteasome regulation.

Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation.

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C(2)C(12) myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved "switches" to block muscle differentiation and induce death.

Fast locomotion in caterpillars

The maximum forward crawling speeds of caterpillars are limited by the hydraulic design of the body and the peristaltic mode of operation of the segmental muscles. High speed locomotory manoeuvers can be achieved by reversing the direction of the normal peristaltic wave (from posterior-anterior to anterior-posterior) although the penalty is a dramatically reduced duty factor of the legs and potential instability. This study describes the suite of reverse gaits available to caterpillars, from reverse walking (the kinematic inverse of normal forward walking), through to reverse galloping (in which all the legs save the claspers are wrenched free of the ground with each step) to recoil-and-roll, a unique form of locomotion in which the insect free-wheels backwards at high-speed. These reverse forms of locomotion are produced primarily in response to threat, involve bilateral activation of the intersegmental muscles and are relatively simple in terms of neural control. The ecological roles of high-speed locomotion are considered in the light of potential predators and the normal habitat and terrain.

Heartbeat and body movement: roles in gas exchange in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae

The objective of the present article is to analyse the role of heartbeat and body movements in respiratory function in Galleria mellonella L. (Lepidoptera: Pyralidae) pupae with clear gas exchange cycles. We did not find any direct evidence of co-ordination between heartbeat and respiration or body movements and respiration. Cyclic CO 2 output was independent of abdominal movements and heart pulsations during the entire pupal development. In young pupae, heart activity periods were independent of abdominal movements. In middle-aged pupae, there appeared a clear trend towards the coincidence of heartbeat periods with the periods of abdominal movements. In late pupae, the heart pulses and the contractions of body skeletal muscles were mostly synchronized. It may be suggested that the contractions of intersegmental somatic abdominal muscles support the heartbeat, and, in this way, the hemolymph flow is accelerated. The heart and circulatory action may facilitate gaseous diffusion in the tracheae.

Respecified larval proleg and body wall muscles circulate hemolymph in developing wings of Manduca sexta pupae.

Most larval external muscles in Manduca sexta degenerate at pupation, with the exception of the accessory planta retractor muscles (APRMs) in proleg-bearing abdominal segment 3 and their homologs in non-proleg-bearing abdominal segment 2. In pupae, these APRMs exhibit a rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for long bouts, without externally visible movements. On the basis of indirect evidence, it was proposed previously that APRM contractions during the pupal motor pattern circulate hemolymph in the developing wings and legs. This hypothesis was tested in the present study by making simultaneous electromyographic recordings of APRM activity and contact thermographic recordings of hemolymph flow in pupal wings. APRM contractions and hemolymph flow were strictly correlated during the pupal motor pattern. The proposed circulatory mechanism was further supported by the findings that unilateral ablation of APRMs or mechanical uncoupling of the wings from the abdomen essentially abolished wing hemolymph flow on the manipulated side of the body. Rhythmic contractions of intersegmental muscles, which sometimes accompany the pupal motor pattern, had a negligible effect on hemolymph flow. The conversion of larval proleg and body wall muscles to a circulatory function in pupae represents a particularly dramatic example of functional respecification during metamorphosis.

Activator complexes containing the proteasomal regulatory ATPases S10b (SUG2) and S6 (TBP1) in different tissues and organisms.

Each 19S regulator of the 26S proteasome contains six ATPase subunits as well as many (>14) non-ATPase protein subunits. The ATPase subunits have been detected in other complexes which may regulate transcription and possibly other cellular processes. The S10b (yeast SUG2 or human p42) and the S6' (TBP1) ATPases have been found in an activator complex (modulator) prepared from bovine red cells. We have identified and partially characterised a similar activator from different human tissues (from soluble extracts of human brain, placenta and human embryonic kidney cells) and an insect: an activator is present in soluble extracts of abdominal intersegmental muscle from Manduca sexta. Activation is ATP and concentration dependent. There is no stimulation of human red cell-derived 20S proteasome by the Manduca activator ruling out 11S regulator in the preparations. Additionally, cross-species activation occurs: the Manduca activator increases the activity of rat skeletal muscle 26S proteasomes and the human placental activator similarly increases the activity of 26S proteasomes prepared from muscles from Manduca sexta. Finally, there is no evidence for other ATPases in the activator complex.

Segment-specific retention of a larval neuromuscular system and its role in a new, rhythmic, pupal motor pattern in Manduca sexta.

At pupation in Manduca sexta, accessory planta retractor muscles and their motoneurons degenerate in segment-specific patterns. Accessory planta retractor muscles in abdominal segments 2 and 3 survive in reduced form through the pupal stage and degenerate after adult emergence. Electromyographic and electrophysiological recordings show that these accessory planta retractor muscles participate in a new, rhythmic 'pupal motor pattern' in which all four muscles contract synchronously at approximately 4 s intervals for extended bouts. Accessory planta retractor muscle contractions are driven by synaptic activation of accessory planta retractor motoneurons and are often accompanied by rhythmic activity in intersegmental muscles and spiracular closer muscles. The pupal motor pattern is influenced by descending neural input although isolated abdominal ganglia can produce a pupal motor pattern-like rhythm. The robust pupal motor pattern first seen after pupal ecdysis weakens during the second half of pupal life. Anemometric recordings indicate that the intersegmental muscle and spiracular closer muscle component of the pupal motor pattern produces ventilation. Accessory planta retractor muscle contractions lift the flexible abdominal floor, to which the developing wings and legs adhere tightly. We hypothesize that, by a bellows-like action, the accessory planta retractor muscle contractions circulate hemolymph in the appendages. Morphometric analysis shows that dendritic regression is similar in accessory planta retractor motoneurons with different pupal fates, and that accessory planta retractor motoneurons begin to participate in the pupal motor pattern while their dendrites are regressed.

Importance of the intersegmental trunk muscles for the stability of the lumbar spine. A biomechanical study in vitro.

A biomechanical study was performed to determine the consequences of a simulation of muscle forces on the loads imposed on the functional spinal units. No biomechanical study has investigated the effect of incorporation of agonist and antagonist muscle forces on the loading of functional spinal units. Spinal disorders and low back pain are increasingly becoming a worldwide problem. Traditional conservative therapies are intended to strengthen the muscles of the trunk using a judicious regimen of physical exercises. Eighteen whole, fresh-frozen human cadaveric lumbar spine specimens (L2-S2; average age, 53.4 years) were tested in a spine tester using pure flexion-extension, lateral bending, and axial moments. The effects of coactivation of psoas and multifidus muscles on L4-L5 mobility were simulated in vitro by applying two pairs of corresponding force vectors to L4. The segmental stability was defined by the correlation of an applied moment to the resultant deformation as shown in load-displacement curves, and the range of motion was defined as the angular deformation at maximum load. The coactivation of muscles was accompanied by a 20% decrease in the range of motion (i.e., a significant increase in stability) during lateral bending and axial moments. Application of flexion-extension moments and muscle coactivation resulted in a 13% increase in the sagittal range of motion. The action of the intersegmental agonist and antagonist muscles biomechanically increases the overall stiffness (stability) of the intervertebral joints in axial torque and lateral bending, whereas it may destabilize the segment in flexion.

Point Of View: Importance of the Intersegmental Trunk Muscles for the Stability of the Lumbar Spine

Point Of View: Importance of the Intersegmental Trunk Muscles for the Stability of the Lumbar Spine

Laminectomy and functional impairment of the lumbar spine: the importance of muscle forces in flexible and rigid instrumented stabilization--a biomechanical study in vitro.

Laminectomy is the accepted treatment for spinal canal stenosis in cases where conservative treatment has failed. Opinions diverge on the resulting clinical instability and the necessity of instrumented stabilization. The present biomechanical study was performed to determine the functional impairment following laminectomy and the stabilizing effect of flexible and rigid devices. This was the first time that the effects of agonist and antagonist intersegmental lumbar muscle forces acting on intact, unstable and instrumentally stabilized functional spinal units have been investigated. Six human cadaveric lumbar spines were tested in a spine tester. The coactivation of agonist and antagonist muscle forces resulted in increased stability under the load conditions of bending and rotation; a slight increase in the range of motion was noted during flexion. The functional impairment following laminectomy was corrected by ligamentoplasty and by means of muscle forces. Ligamentoplasty appears to be an alternative to decompression with spondylodesis, especially in patients with well-developed muscles.

Intracellular Proteinases of Invertebrates: Calcium-Dependent and Proteasome/Ubiquitin-Dependent Systems

Cytosolic proteinases carry out a variety of regulatory functions by controlling protein levels and/or activities within cells. Calcium-dependent and ubiquitin/proteasome-dependent pathways are common to all eukaryotes. The former pathway consists of a diverse group of Ca(2+)-dependent cysteine proteinases (CDPs; calpains in vertebrate tissues). The latter pathway is highly conserved and consists of ubiquitin, ubiquitin-conjugating enzymes, deubiquitinases, and the proteasome. This review summarizes the biochemical properties and genetics of invertebrate CDPs and proteasomes and their roles in programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, gametogenesis and fertilization, development and pattern formation, cell-cell recognition, signal transduction and learning, and photoreceptor light adaptation. These pathways carry out bulk protein degradation in the programmed death of the intersegmental and flight muscles of insects and of individuals in a colonial ascidian; molt-induced atrophy of crustacean claw muscle; and responses of brine shrimp, mussels, and insects to environmental stress. Selective proteolysis occurs in response to specific signals, such as in modulating protein kinase A activity in sea hare and fruit fly associated with learning; gametogenesis, differentiation, and development in sponge, echinoderms, nematode, ascidian, and insects; and in light adaptation of photoreceptors in the eyes of squid, insects, and crustaceans. Proteolytic activities and specificities are regulated through proteinase gene expression (CDP isozymes and proteasomal subunits), allosteric regulators, and posttranslational modifications, as well as through specific targeting of protein substrates by a diverse assemblage of ubiquitin-conjugases and deubiquitinases. Thus, the regulation of intracellular proteolysis approaches the complexity and versatility of transcriptional and translational mechanisms.

The 26S-proteasome: regulation and substrate recognition.

There is extensive reprogramming of the ATPase regulators of the 26S proteasome before the programmed elimination of the abdominal intersegmental muscles (ISM) after eclosion in Manduca sexta [1]. This extensive ATPase reprogramming only occurs in ISM which are destined to die and not in flight muscle (FM). The MS73 ATPase also increases in the proleg retractor muscles which die at a developmentally different stage to ISM. The non-ATPase regulator S5a shows a similar increase to the ATPase regulators. We have cloned the Manduca SUG2 ATPase and shown that this ATPase is a component of the 26S proteasome. This ATPase shows a similar increase in concentration to the other ATPases in 26S proteasomes before muscle death. The SUG2 ATPase is also associated with other smaller complexes besides the 26S proteasome which act as activators of the 26S proteasome. Finally, in a yeast two-hybrid genetic screen we have identified a protein in human brain which interacts with the MS73 ATPase (and human S6). The interacting protein contains 6 ankyrin repeats and is co-immunoprecipitated with anti-MS73 antiserum after in vitro transcription/translation. The ankyrin repeat protein may interact with the MS73 ATPase as part of the substrate recognition process by the 26S proteasome. Many proteins degraded by the 26S proteasome contain ankyrin repeats, e.g. IkB and some cyclins: binding through ankyrin repeats to an ATPase regulator may complement protein ubiquitination and S5a binding as recognition signals by the 26S proteasome.

Allelic variation of the polyubiquitin gene in the tobacco hawkmoth, Manduca sexta, and its regulation by heat shock and programmed cell death

The intersegmental muscles (ISMs) of the tobacco hawkmoth, Manduca sexta, undergo programmed cell death (PCD) following adult eclosion in response to a decline in the circulating titer of the hormone 20-hydroxyecdysone. The ability of the ISMs to die requires de novo gene expression and a number of cDNAs representing differentially expressed genes have been isolated from condemned cells. One of the genes that is dramatically up-regulated with ISM death is polyubiquitin, which has been shown in many organisms to function as a heat shock protein and as an essential mediator of proteolysis. Northern blot analysis of ISM RNA samples pooled from multiple individuals demonstrated the presence of several polyubiquitin transcripts. In this study, we sought to determine: 1) if these transcripts were the product of multiple genes or multiple alleles, and 2) if all polyubiquitin genes/alleles in the moth are regulated by both heat shock and the endocrine signals that regulate death. Data from Southern blot analysis suggested that the Manduca genome has a single polyubiquitin gene that is represented by multiple alleles. Transcript analysis supported the hypothesis that all polyubiquitin alleles are regulated by both heat shock and the hormonal cues that regulate muscle death. Polyubiquitin transcripts accumulated to much higher levels and had longer half-lives following hormonal induction relative to that seen in response to heat shock. These data suggest that there are multiple polyubiquitin alleles in the laboratory population of Manduca, all of which share common regulatory sequences that drive expression to meet the needs for proteolysis involved in both heat stress and death.