Tiggrin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Tiggrin

Synonyms -

Cytological map position - 26D1--26D2

Function - extracellular matrix protein

Keywords - mesoderm, muscle attachment

Symbol - Tig

FlyBase ID: FBgn0011722

Genetic map position - 2-[20]

Classification - novel multidomain protein

Cellular location - extracellular

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Green, N., Odell, N., Zych, M., Clark, C., Wang, Z. H., Biersmith, B., Bajzek, C., Cook, K. R., Dushay, M. S. and Geisbrecht, E. (2016). A common suite of coagulation proteins function in Drosophila muscle attachment. Genetics [Epub ahead of print]. PubMed ID: 27585844
The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. The surprising discovery was made that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes thrombospondin (Tsp) and tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. It was further demonstrated that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, it is proposed that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment.
Zhang, C. U. and Cadigan, K. M. (2017). The matrix protein Tiggrin regulates plasmatocyte maturation in Drosophila larva. Development [Epub ahead of print]. PubMed ID: 28526755
The lymph gland (LG) is a major source of hematopoiesis during Drosophila development. In this tissue, prohemocytes differentiate into multiple lineages including macrophage-like plasmatocytes, which comprise the vast majority of mature hemocytes. Previous studies have uncovered genetic pathways that regulate prohemocyte maintenance and some cell fate choices between hemocyte lineages. However, less is known about how the plasmatocyte pool of the LG is established and matures. This study reports that Tiggrin, a matrix protein expressed in the LG, is a specific regulator of plasmatocyte maturation. Tiggrin mutants exhibit precocious maturation of plasmatocytes, while Tiggrin overexpression blocks this process, resulting in a buildup of intermediate progenitors (IPs) expressing prohemocyte and hemocyte markers. These IPs likely represent a transitory state in prohemocyte to plasmatocyte differentiation. It was also found that overexpression of Wee1 kinase, which slows G2/M progression, results in a phenotype similar to Tiggrin overexpression while String/Cdc25 expression phenocopies Tiggrin mutants. Further analysis revealed that Wee1 inhibits plasmatocyte maturation through up-regulation of Tiggrin transcription. These results elucidate connections between the extracellular matrix and cell cycle regulators in the regulation of hematopoiesis.

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:

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).

Integrins regulate responsiveness to Slit repellent signals

Integrins are concentrated within growth cones, but their contribution to axon extension and pathfinding is unclear. Genetic lesionof individual integrins does not stop growth cone extension or motility, but does increase axon defasciculation and axon tract displacement. In this study, a dosage-dependent phenotypic interaction is documented between genes for the integrins, their ligands, and the midline growth cone repellent, Slit, but not for the midline attractant, Netrin. Longitudinal tract axons in Drosophila embryos doubly heterozygous for slit and an integrin gene, encoding alphaPS1, alphaPS2, alphaPS3, or ßPS1, take ectopic trajectories across the midline of the CNS. Drosophila doubly heterozygous for slit and the genes encoding the integrin ligands Laminin A and Tiggrin reveal similar errors in midline axon guidance. It is proposed that the strength of adhesive signaling from integrins influences the threshold of response by growth cones to repellent axon guidance cues (Stevens, 2002).

Tiggrin is a secreted glycoprotein that contains an RGD motif and is considered to be a ligand of the PS2 integrin. Embryos homozygous for a loss of function allele of Tiggrin have a subtle Fas II phenotype reminiscent of integrin mutants. CNS axon tracts are wavy, and no midline axon guidance errors are seen. Labeling of the most lateral axon tract is interrupted between segments. Like the integrin genes, tig also has a semidominant interaction with slit. Fas II labeling of fascicles between segments is reduced. Midline guidance errors are seen in one in three segments (Stevens, 2002).

Drosophila Laminin is a trimer of three proteins: Laminin A, B1, and B2. Laminin is known to be a ligand of PS1 integrin and possibly other integrins as well. Mutants have not been isolated for the B1 and B2 chains; however, a loss of function allele for lanA encoding the A chain has been characterized. The Fas II phenotype of the lanA mutant is nearly wild type, revealing midline guidance errors in 4% of segments. When doubly heterozygous with sli, in sli/+;lanA/+ embryos, the frequency of midline crossovers is >30% (Stevens, 2002).

Does a change in lanA function also affect integrin function in CNS axon tract formation? lanA interaction with scb was examined because Laminin is not known to be a ligand of alphaPS3/4 (encoded by scb), and scb has a strong semidominant interaction with slit. Both lanA and scb reveal midline guidance errors when homozygous. However, in the scb/+;lanA/+ double heterozygote, midline guidance errors are not seen. Nevertheless, this genotype shares aspects of the integrin CNS phenotype: defasciculation and interruptions in Fas II labeling of the most lateral fascicle. This suggests function of both genes in a common or parallel pathway. If the interaction of scb and lanA is independent of the interaction of either gene with sli, then the phenotype of the triple heterozygote scb/sli;lanA/+ would reflect the addition of the scb/sli, scb/+;lanA/+, and sli/+;lanA/+ phenotypes. The degree of defasciculation and midline guidance errors in all axon tracts of the triple heterozygote appears to be additive. However, a narrowing of the CNS and the medial displacement of all axon tracts are also seen in the triple heterozygote. This phenotype is typical of mutants in genes required for midline guidance and is not a component of the integrin mutant phenotype. The synergistic interaction of these three genes suggests dosage-dependent function for each gene in common or parallel pathways (Stevens, 2002).

Thus, a reduced level of expression of the genes for four integrins (alphaPS1, alphaPS2, alphaPS3/4, and ßPS1) or two integrin ligands (Tiggrin and Laminin) increases the probability that CNS axons make pathfinding errors when slit expression is reduced. Expression of the integrins Tiggrin and Laminin A has been demonstrated in the CNS. Integrin expression is not localized and may be expressed in both glia and neurons. Overexpression of alphaPS3 or Laminin A in motoneurons affects axon guidance. Loss of function of the integrins disrupts axon fasciculation and longitudinal axon fascicle placement in the embryonic nerve cord but does not clearly affect axon guidance. These observations have been extended in this study, with different alleles of the integrins, demonstrating a similar function for alphaPS3/4, and revealing axon fascicle phenotypes for loss of function of integrin ligands Tiggrin and Laminin A. The mutant phenotypes share common elements: mild phenotypes show wavy axon tracts and reduced Fas II labeling between segments, whereas severe phenotypes include defasciculation and fascicle displacement, including midline axon guidance errors. The integrins have different extracellular ligands. Therefore, the integrins contribute similarly to axon tract integrity, independent of the ligand that they bind (Stevens, 2002).

Integrin phenotypes in the CNS do not demonstrate a direct role for integrins in growth cone guidance. In contrast, perturbation of midline growth cone repellent signals results in a medial narrowing of the CNS and ectopic midline crossing of longitudinally projecting axons, rather than defasciculation and displacement of axon tracts. One feature of integrin and tiggrin phenotypes shared with robo and dock mutant phenotypes is a thinning or loss of Fas II labeling in the most lateral axon fascicle. This fascicle expresses Fas II late in embryogenesis. This phenotype may reflect impaired or delayed development of independent fascicles in the nerve cord, as implicated by studies of robo function (Stevens, 2002).

The semidominant interaction of all integrins, Tiggrin, and Laminin A with slit is more prevalent than might be expected if the integrins play a specialized role in Slit signaling. scab also has a dramatic semidominant interaction with dock (Nck), which functions in diverse axon guidance events. scb/dock double heterozygotes have disrupted longitudinal, commissural, and peripheral axon tracts. A similar genetic test suggests that ßPS integrin modulates RhoA activity and axon stability in the mushroom body. These diverse phenotypes reflect an adhesive function of the integrins that reduces the responsiveness of growth cones to guidance signals. Independent evidence suggests that this occurs in the growth cone, but a role for integrin in the glia that emit guidance signals cannot be discounted (Stevens, 2002).


cDNA clone length - 6,558 bp

Bases in 5' UTR - 149


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).

Tiggrin: | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 11 November 2016

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