Agnes Ayme-Southgate

The Myofibrillar Protein, Projectin, is Highly Conserved Across Insect Evolution Except for Its PEVK Domain

All striated muscles respond to stretch by a delayed increase in tension. This physiological response, known as stretch activation, is, however, predominantly found in vertebrate cardiac muscle and insect asynchronous flight muscles. Stretch activation relies on an elastic third filament system composed of giant proteins known as titin in vertebrates or kettin and projectin in insects. The projectin insect protein functions jointly as a “scaffold and ruler” system during myofibril assembly and as an elastic protein during stretch activation. An evolutionary analysis of the projectin molecule could potentially provide insight into how distinct protein regions may have evolved in response to different evolutionary constraints. We mined candidate genes in representative insect species from Hemiptera to Diptera, from published and novel genome sequence data, and carried out a detailed molecular and phylogenetic analysis. The general domain organization of projectin is highly conserved, as are the protein sequences of its two repeated regions—the immunoglobulin type C and fibronectin type III domains. The conservation in structure and sequence is consistent with the proposed function of projectin as a scaffold and ruler. In contrast, the amino acid sequences of the elastic PEVK domains are noticeably divergent, although their length and overall unusual amino acid makeup are conserved. These patterns suggest that the PEVK region working as an unstructured domain can still maintain its dynamic, and even its three-dimensional, properties, without the need for strict amino acid conservation. Phylogenetic analysis of the projectin proteins also supports a reclassification of the Hymenoptera in relation to Diptera and Coleoptera.

Drosophila Projectin: A Look at Protein Structure and Sarcomeric Assembly

The large projectin protein is found in all Drosophila muscles; however, it shows a dual sarcomeric localization depending on the muscle type. In larval and adult synchronous muscles, projectin is found localized over the A-band. Initial in vitro binding assays indicate interactions of several projectin regions with themselves and myosin heavy chain. These interactions might be critical for the assembly of projectin over the myosin filament during embryonic myofibrillogenesis and larval growth. On the other hand, projectin localizes over the I-Z-I region in indirect flight muscles. Correspondingly, projectin is found in association with forming Z-bands during pupation and colocalizes with alpha-actinin and kettin.

In indirect flight muscles Drosophila projectin has a short PEVK domain, and its NH2-terminus is embedded at the Z-band

Insect indirect flight muscles (IFM) contain a third filament system made up of elastic connecting or C-filaments. The giant protein projectin is the main, if not the only, component of these structures. In this study we found that projectin is oriented within the IFM sarcomere with its NH2−terminus embedded in the Z-bands. We demonstrate that this protein has an elastic region that can be detected by the movement of specific epitopes following stretch. One possible elastic region is the PEVK-like domain located close to the NH2−terminus. The amino acid length of this region is short, and 52% of its residues are P, E, V or K. We propose a model in which projectin extends from the Z-band to the lateral borders of the A-band. The PEVK-like domain and a series of Ig domains spanning the intervening I-band may provide the elastic properties of projectin.

Assembly of the giant protein projectin during myofibrillogenesis in Drosophila indirect flight muscles

BackgroundProjectin is a giant modular protein of Drosophila muscles and a key component of the elastic connecting filaments (C-filaments), which are involved in stretch activation in insect Indirect Flight Muscles. It is comparable in its structure to titin, which has been implicated as a scaffold during vertebrate myofibrillogenesis.MethodsWe performed immunofluorescence studies on Drosophila pupal tissue squashes and isolated myofibrils to identify the pattern of appearance and assembly for projectin and several other myofibrillar proteins, using both wild type and mutant fly stocks.Results and conclusionsIn the first step of assembly, projectin immunolocalization appears as random aggregates colocalizing with α-actinin, kettin and Z(210), as well as, F-actin. In the second step of assembly, all these proteins become localized within discrete bands, leading ultimately to the regularly spaced I-Z-I regions of myofibrils. This assembly process is not affected in myosin heavy chain mutants, indicating that the anchoring of projectin to the thick filament is not essential for the assembly of projectin into the developing myofibrils. In the actin null mutation, KM88, the early step involving the formation of the aggregates takes place despite the absence of the thin filaments. All tested Z-band proteins including projectin are present and are colocalized over the aggregates. This supports the idea that interactions of projectin with other Z-band associated proteins are sufficient for its initial assembly into the forming myofibrils. In KM88, though, mature Z-bands never form and projectin I-Z-I localization is lost at a later stage during pupal development. In contrast, treatment of adult myofibrils with calpain, which removes the Z-bands, does not lead to the release of projectin. This suggests that after the initial assembly with the Z-bands, projectin also establishes additional anchoring points along the thick and/or thin filaments. In conclusion, during pupation the initial assembly of projectin into the developing myofibril relies on early association with Z-band proteins, but in the mature myofibrils, projectin is also held in position by interactions with the thick and/or the thin filaments.

Projectin PEVK domain, splicing variants and domain structure in basal and derived insects

The third elastic filament of striated muscles consists of giant proteins: titin (in vertebrates) and kettin/projectin (in insects). In all three proteins, elasticity is at least partly associated with the so‐called PEVK domain. The projectin PEVK domains of diverse insects are highly divergent compared with an otherwise conserved protein organization. We present the characterization of the PEVK domain in two dragonflies and in human lice. A conserved segment at the end of the PEVK, the NH2‐terminal conserved segment‐1 (NTCS‐1), may serve as an anchor point for projectin to either myosin or actin, providing a mechanical link. The analysis of alternative splicing variants identifies the shortest PEVK isoform as the predominant form in the flight muscles of several insects, possibly contributing to myofibrillar stiffness.

Myofilament proteins in the synchronous flight muscles of Manduca sexta show both similarities and differences to Drosophila melanogaster

Insect flight muscles have been classified as either synchronous or asynchronous based on the coupling between excitation and contraction. In the moth Manduca sexta, the flight muscles are synchronous and do not display stretch activation, which is a property of asynchronous muscles. We annotated the M. sexta genes encoding the major myofibrillar proteins and analyzed their isoform pattern and expression. Comparison with the homologous genes in Drosophila melanogaster indicates both difference and similarities. For proteins such as myosin heavy chain, tropomyosin, and troponin I the availability and number of potential variants generated by alternative spicing is mostly conserved between the two insects. The exon usage associated with flight muscles indicates that some exon sets are similarly used in the two insects, whereas others diverge. For actin the number of individual genes is different and there is no evidence for a flight muscle specific isoform. In contrast for troponin C, the number of genes is similar, as well as the isoform composition in flight muscles despite the different calcium regulation. Both troponin I and tropomyosin can include COOH-terminal hydrophobic extensions similar to tropomyosinH and troponinH found in D. melanogaster and the honeybee respectively.

Projectin, the Elastic Protein of the C-Filaments

In adult insects, the highly specialized indirect flight muscles (abbreviated as IFMs) are powerful muscles adapted for the rapid repeated contractions necessary for flight. These muscles are referred to as asynchronous muscles, because they undergo multiple rounds of contraction for each single nerve impulse, a property made possible by the stretch-activation mechanism.1, 2, 3, 4, 5 The stretch-activation mechanism is explained as a “delayed increase in tension due to stretch” that activates the muscle and results in contraction. The IFMs are attached to the cuticle (exoskeleton) and because they are organized as two sets of nearly perpendicular muscles, their length oscillate in response to the stretch activation-contraction cycles. The stretch activation mechanism has been shown to be an intrinsic property of the myofibrillar apparatus,1 and is made possible by several special physiological adaptations, such as a high resting stiffness. To explain some of the IFMs’ properties, early models proposed the existence of an additional third filament system with elastic properties, which is usually referred to as the connecting or C-filament system. Electron microscopy studies of insect flight muscles revealed the presence of fine connections between the Z bands and the thick filaments.6, 7, 8, 9, 10, 11 In particular, electron microscopy of stretched myofibrils or purified Z disks of insect flight muscles have shown the presence of filaments extending or “projecting” from the Z band towards the myosin filaments and just overlapping the tip of the A band.12, 13 In honeybee IFMs, connecting filaments can be extended to well over ten times their normal rest length. When these muscles are stretched in rigor and then released, the recoil forces of the connecting filaments cause the sarcomere to shorten, leading to the crumpling of the thin filaments held in rigor.9 The search for component(s) of the C-filament system led to the identification and characterization of the protein, projectin. Saide unequivocally demonstrated by antibody staining and biochemical analysis that the third connecting filament of honeybee flight muscles is composed of projectin.12