BIOLOGICAL OVERVIEW

Tiggrin is a component of the extracellular matrix (ECM), found at sites for muscle insertion or attachment known as apodemes (Fogarty, 1994). The concentration of Tiggrin at these segmentally repeated sites gives embryos stained with anti-Tiggrin antibodies the striped appearance associated with Tigger, a tiger-like character introduced in the 1938 children's classic by A.A. Milne, The House at Pooh Corner.

A few words on other components of the ECM are in order, before reviewing Tiggrin. Other ECM proteins that like Tiggrin have been identified at Drosophila muscle insertions are collagen IV, papilin (a glycoprotein containing a TSP1 [properidin] domain). and glutactin (a glycoprotein containing carboxyesterase homology). Transcripts of Tenascin A (see Drosophila Tenascin-major), a member of the tenascin family, are also found in cells located at apodemes (Baumgartner, 1993). Dachsous, a cadherin, and Laminin A have both been associated with apodemes. Mutants of crocodile, coding for a forkhead domain transcription factor, lack certain apodemes. Action of stripe, coding for a EGR type zinc finger transcription factor, has been found to be both necessary and sufficient to initiate the developmental program of epidermal muscle attachment. Ectopic expression of Stripe in various epidermal cells transforms these into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. These markers include Goovin, Delilah, and beta1 tubulin (Becker, 1997).

The extracellular matrix itself is an interconnected network of glycoproteins, proteoglycans and glycosaminoglycans secreted and assembled by cells. At the apodeme sites the PS2 integrins function to maintain muscle attachments and Tiggrin has been shown to be is a ligand of the PS2 integrins. This suggests that Tiggrin in the ECM binds to the PS2 integrins and mediates PS2-ECM interactions. Along striated muscles Tiggrin and the PS2 integrins colocalize at Z-bands (Fogerty, 1994). PS1 (Multiple edematous wings) and PS2 (Inflated) integrins contain a common betaPS subunit (Myospheroid) that is associated with either an alphaPS1 or an alphaPS2 subunit. Thus, PS1 and PS2 integrins are respectively alphaPS1betaPS and alphaPS2betaPS heterodimers (Fogarty, 1994).

Three observations suggest that Tiggrin interacts with the PS2 integrins:

(1) Though Tiggrin does not show any strong sequence similarity to other proteins, it does contain the integrin recognition sequence RGD and this sequence is recognized by the PS2 integrins.

(2) Tiggrin is detected at muscle attachment sites that also contain PS2 integrins.

(3) In a cell culture assay, Tiggrin supports PS2-mediated cell spreading.

If all functions of the PS2 integrins are mediated by their interactions with Tiggrin then Tiggrin mutants would be expected to display the same range of phenotypes that are found in mutations that remove the PS2 integrins. This would include complete detachment of most muscles from their attachment sites, and central nervous system, wing and gut defects. More likely, Tiggrin may be only one of several ligands used by the PS2 integrins, and Tiggrin may have additional functions that are independent of its interactions with the PS2 integrins. Tiggrin mutant phenotypes are shown to be complex, affecting some but not all of the tissues that are defective in PS2 integrin mutants. In these tissues, Tiggrin mutants display novel phenotypes not described for PS2 integrin mutants (Bunch, 1998).

At segment borders where ends of multiple muscles attach to epidermal tendon cells, and where accumulations of Tiggrin protein are found in wild-type animals, the gaps between muscles increase from 7 mm in wild-type third instar larvae to 30 mm in Tiggrin mutant larvae. One model that could explain these gaps is that the PS2 integrins are involved in two adhesion sites when neighboring muscles make attachments to the same or neighboring epidermal tendon cells. The first site is the well-documented muscle-epidermal attachment. This attachment may or may not utilize Tiggrin. The second site involves muscle-muscle attachment. Muscle-muscle attachments have not been described in detail; however, experiments that genetically remove the epidermal tendon cells result in muscles that detach from the epidermis but remain attached to each other (Martin-Bermudo, 1996). A recently isolated mutant, rhea, also displays muscles that detach from the epidermis but remain attached to each other (Prout, 1997). This strongly suggests that muscle-muscle attachments exist in normal animals. The results reported here suggest that Tiggrin is required to maintain and/or establish these specialized muscle-muscle junctions (Bunch, 1998).

In contrast to the longitudinal and oblique bodywall muscles, transverse bodywall muscles appear to attach only to the epidermis, do not show strong Tiggrin staining and do not show defects in the Tiggrin mutants. This indicates that these two muscle attachment sites are different and is consistent with the model that Tiggrin is involved mainly with the muscle-muscle junctions and not so critical at the muscle-epidermal junctions. Prokop (1998a) has observed that these two muscle junctions are ultrastructurally quite distinct. The transverse muscles display a close apposition (30-40 nm) between muscle and epidermis; in contrast to this, at segment borders where longitudinal and oblique muscles converge at sites on the epidermis, large accumulations of tendon matrix, including Tiggrin, separate cells by several mm. In this model, the absence of Tiggrin results in muscles remaining attached to the epidermal cells but detaching from each other, resulting in the separated, but well-ordered, muscle termini. The PS2 integrins are involved in both attachments because inflated mutants (in which the alphaPS2 subunits are removed) result in completely detached and rounded up muscles. This model suggests that the PS2 integrins may use different ligands for the muscle-epidermal attachment. Tenascin major is an example of a potential ECM component that may carry out this function. It is found at attachment sites, has an RGD sequence and interacts with the PS2 integrins in cell spreading assays (Baumgartner, 1994). Other extracellular molecules known to function or locate to these attachment areas include Laminin, Slit, Masquerade, m-Spondin, Collagen IV and Groovin. By interacting directly or indirectly with the PS2 integrins and other cell surface receptors, these proteins may further support the muscle-epidermal attachment in the absence of Tiggrin (Bunch, 1998).

For Tiggrin to mediate a direct link between cells via the PS2 integrins would require two PS2 integrin-binding sites, but Tiggrin has only one RGD sequence. Biochemical data are consistent with Tiggrin forming extended rod-like homodimers or homotrimers that are approximately 180 nm in length (Fogerty, 1994). If two Tiggrin molecules dimerize in an anti-parallel fashion, this would place the RGD integrin-binding domain on each end of the rod and could serve as a direct link between PS2 integrins on adjacent cells. However, the 180 nm length of such a dimer or trimer is not consistent with the distance of several mm between neighboring muscles visualized by actin staining or at the EM level (Prokop, 1998a). Furthermore, Tiggrin is anchored to the ECM by interactions with other matrix components, as evidenced by the correct localization of Tiggrin in myospheroid mutants, which lack PS2 integrins (Fogerty, 1994). Therefore Tiggrin may provide a link to the ECM rather than a direct linkage between the PS2 integrins on neighboring cells (Bunch, 1998).

GENE STRUCTURE

Bases in 5' UTR - 149

PROTEIN STRUCTURE

Amino Acids - 2186

Structural Domains

Analysis of the Tiggrin sequence reveals 12 potential N-linked glycosylation sites. Some of these sites are glycosylated because Tiggrin's electrophoretic mobility is increased slightly following treatment with PNGase F, an enzyme that removes N-linked oligosaccharides. The adhesive recognition sequence RGD is found in Tiggrin near the C terminus. Two copies of a putative adhesive recognition sequence, Leu-Arg-Glu (LRE) are found. The predicted secondary structure of Tiggrin suggests that the protein can be divided into three major regions. A central repetitive domain of approximately 1,250 amino acids extends from residues 496 to 1,743 and is predicted to contain primarily a helical structure and very little beta sheet structure. This domain is flanked by (1) the N-terminal domain (residues 1-495), which contains the sole Cys and (2) the C-terminal domain (residues 1,744-2,186), which contains the RGD cell attachment motif. The central domain is composed of 16 contiguous repeats, except for an 81 amino acid spacer between repeats 14 and 15. Repeat 5 is the closest to the consensus sequence. Pairwise comparisons of individual repeats to repeat 5 show 25-42% amino acid identity and 55-61% similarity. To search for other proteins that might contain this type of repeat, the program Pro-fileMake was used to generate a profile sequence from the aligned repeats. A search of two protein databases failed to find significant matches to the repeat profile sequence. This suggests that the Tiggrin repeat is a novel protein motif. It is likely to have a high content of alpha-helix, with only 4 of the 16 repeats containing a proline residue that is incompatible with alpha-helix. While each Tiggrin repeat contains 4-5 potential heptad repeats of hydrophobic residues, such as occur in coiled-coil alpha-helical structures, these heptad repeats do not form a contiguous end-to-end sequence. Weak sequence similarities (<20% identity) exist between Tiggrin's region of tandem repeats and several filamentous proteins that contain coiled-coil alpha-helices, e.g. myosin, spectrin and dystrophin (Bunch, 1998).

date revised: 6 July 98

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