BIOLOGICAL OVERVIEW

scab was initially described in a study of mutations that affect the pattern of the larval cuticle (Nusslein-Volhard, 1984). The defect in dorsal closure that describes the effects of scab mutation is similar to that seen in myospheroid mutant embryos. Myospheroid (also known as beta PS) is the dimerization partner of two previously characterized alpha integrins: alphaPS1 (Multiple edematous wings) and alphaPS2 (Inflated). Dorsal closure defect is not seen in null mutations of these two alpha integrins, indicating that some other alpha integrin must team up with betaPS during dorsal closure.

In a search for the presumed missing integrin, attention was focussed on a 90kDa band associated with immunoprecipitates of Myospheroid, resulting from the application of an anti-betaPS antiserum. This 90 kDa protein forms a non-covalent, divalent cation-dependent complex with Myospheroid. The 90 kDa protein binds well to both lentil lectin and Concanavilin A beads, suggesting that it is a glycoprotein. The protein was purified by immunoprecipitation, lecitin binding, elution and SDS gel electrophoresis and subjected to tryptic digestion; the resulting peptides were then sequenced. Degenerate primers based on the amino acid sequences were used to identify the cDNA coding for the 90 kDa protein. The sequence revealed an alpha integrin subunit that has been designated alphaPS3 (Stark, 1997). Thus, the cloning of scab, revealing as it does the gene coding for the missing integrin, completes a picture of integrin activity with the discovery of a third alpha integrin partnering Myospheroid (Stark, 1997).

Additional defects in scab embryos correspond to sites of Scab expression. The defect in tracheal development in scb embryos, and the corresponding defects in embryos lacking both maternal and zygotic Myospheroid indicate that Scab, in conjunction with Myospheroid, plays a role during the migration and fusion of tissue in tracheal development. Scab expression is also seen in the salivary glands, beginning as they invaginate from the surface of the embryo and continuing as they form a tubular structure surrounding a central lumen. After dorsal closure, expression is clearly present in some cells of the dorsal vessel. Pericardial cells lie at the edge of the epidermis during dorsal closure and later contribute to the formation of the dorsal vessel. Defects are observed in the localization of the pericardial cells in scb mutant embryos, and a similar, although more severe defect has now been identified in embryos lacking both maternal and zygotic Myospheroid. The defect in mys embryos is strikingly similar to that reported for embryos missing the laminin A chain (Yarnitzky, 1995). This supports the hypothesis that alphaPS3 and betaPS both play a role in the movement and migration of the pericardial cells during dorsal vessel formation and suggests that laminin may be a ligand during this process (Stark, 1997).

Volado is a new memory mutant of Drosophila. The locus encodes two isoforms of a new alpha-integrin, a molecule that dynamically mediates cell adhesion and signal transduction. Volado, now termed Scab, is expressed preferentially in mushroom body cells, which are neurons known to mediate olfactory learning in insects. The Vol antigen is found concentrated in the mushroom body parikarya and calyces, peduncles and alpha, beta and gamma lobes. The calyces, peduncles and lobes contain the mushroom body dedrites, axons and axon terminal, respectively. Volado proteins are concentrated in the mushroom body neuropil, the brain area that contains mushroom body processes in synaptic contact with other neurons. The distribution of the two Vol isoforms are globally coexpressed. Enriched expression is also observed in the ellipsoid body, a region of the central complex, thought to be involved in the coordination of motor behaviors. Volado mutants display impaired olfactory memories within 3 min of training, indicating that the integrin is required for short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. Thus the Volado integrin is essential for the physiological processes underlying memory. A model is proposed in which integrins act as dynamic regulators of synapse structure or are involved in the signaling events underlying short-term memory formation. It is envisioned that release of a modulatory neurotransmitter on a mushroom body neuron might mobilize the intracellular events altering the binding of integrins displayed at another synapse made by that cell. For example, protein kinase C and Ras activation are both known to activate integrin binding. Activation of either could produce a rapid (within minutes) alteration in the structure and efficacy of that synapse. The modulation of integrin affinity for ligands might also underlie the construction or pruning of existing synapses, or the activation of silent synapses during learning or memory encoding (Grotewiel, 1998).

Integrins are necessary for the development and maintenance of the glial layers in the Drosophila peripheral nerve

Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors, play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia have not been determined in any animal. This study shows that, in the peripheral nerves of Drosophila melanogaster, two integrin complexes (αPS2βPS and αPS3βPS) are expressed in the different glial layers and form adhesion complexes with integrin-linked kinase and Talin. Knockdown of the common beta subunit (βPS) using inducible RNAi in all glial cells results in lethality and glial defects. Analysis of integrin complex function in specific glial layers showed that loss of βPS in the outermost layer (the perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated degradation of the ECM. Knockdown of βPS integrin in the innermost wrapping glia causes a loss of glial processes around axons. Together, these data suggest that integrins are employed in different glial layers to mediate the development and maintenance of the protective glial sheath in Drosophila peripheral nerves (Xie, 2011).

Peripheral nerves in vertebrates and Drosophila are organized in similar ways, with central axons wrapped by an inner class of glia that are surrounded in turn by layers of external glia. The glia and the surrounding ECM establish a tubular sheath to protect axons from physical damage and pathogens. This study shows that at least two integrin heterodimers are expressed in these different glial layers and are localized at focal adhesions with Ilk and Talin. αPS2βPS integrin is prevalent in the PG and the αPS3βPS integrin is more prevalent in the wrapping glia (WG). Since βPS2 integrin is expressed mostly in the outermost PG and can bind ligands that contain the tripeptide RGD sequence, it most likely functions by binding to ECM ligands in the NL. The majority of αPS3 integrin is expressed by the internal SPG and WG and αPS3 integrin has been shown to interact with laminins in Drosophila. However, it is possible that the integrin complex in the WG might have other ligands and might mediate direct cell-cell interactions, since a pronounced basal lamina associated with the peripheral axons was not detected and Mmp2 expression had no effect in the internal regions of the peripheral nerves (Xie, 2011).

Peripherial glia (PG) form the outermost glial layer in the Drosophila nervous system but their origin and function are not well understood, even though they were identified some time ago. Drosophila PG are structurally similar to their vertebrate counterparts and have been proposed to have similar roles. Vertebrate perineurial cells and the associated collagen fibers provide important mechanical support to nerves and their development might rely on ECM-mediated signals, as β1 integrin is found in the perineurium. However, little is known about the role of integrins in perineurial cells (Xie, 2011).

The current results show that Drosophila PG express αPS2 and βPS integrin subunits (and to a lesser extent αPS3), which colocalize with both Ilk and Talin. Knocking down integrins disrupts perineurial wrapping, but it was not possible to distinguish whether the PG failed to initiate wrapping or failed to maintain their processes around the nerves to accommodate the growing nerve surface. However, degradation of the neural lamella (NL) by Mmp2 overexpression generates a PG wrapping phenotype similar to that of βPS RNAi. Thus, binding of βPS integrin to ligands in the ECM mediates the radial spread of PG around the tubular structure of the nerve and suggests that PG retract their membrane when integrin-ECM interaction is interrupted. Moreover, knockdown of Talin in the PG produces a similar wrapping defect. This suggests that, in the PG, integrin adhesion complexes mediate the connection between the extracellular NL and the intracellular actin cytoskeleton and are required for the initiation or maintenance of glial ensheathment (Xie, 2011).

The function of the PG in the peripheral nerve is not well understood. The PG do not generate an impermeable barrier and it is the subperineurial glia (SPG) layer that creates the blood-nerve barrier. Loss of PG ensheathment did not result in paralysis or lethality, which are signs of a disrupted blood-brain barrier. Larger molecules (~500 kDa) are blocked by the NL or the PG, perhaps mirroring the protective function of the vertebrate perineurium against pathogens. However, in Mmp2-overexpressing larvae, the affected nerves become thin and are difficult to retain intact during tissue preparation. This suggests that the NL and PG provide important mechanical support as is seen with the perineurium in mammalian nerves (Xie, 2011).

In the center of Drosophila peripheral nerves, the WG embed axons in bundles or individually within single membrane wraps, similar to the non-myelinating Schwann cells of vertebrate peripheral nerves. The current results show that WG predominantly express αPS3 and βPS integrin subunits in complexes positive for Ilk and Talin along the WG membrane. When βPS expression is knocked down, the complexity of the glial processes between the associated axons is greatly reduced. Only a few long processes and small membrane protrusions are observed around the axons, suggesting that the WG might be retracting their processes in the absence of βPS integrin. The role of integrins appears to be conserved between WG and Schwann cells in vertebrates. For example, myelinating Schwann cells lacking β1 integrin or Ilk do not extend membrane processes around axons, resulting in impaired radial sorting. The role of integrins in non-myelinating Schwann cells is not known but the current results suggest that integrins have similar functions in Drosophila and vertebrates in mediating the glial ensheathment of peripheral axons (Xie, 2011).

No clear ECM has been observed in the internal regions of Drosophila nerves by immunofluorescence analysis or transmission electron microscopy. Therefore, the integrin complex could promote WG sheath formation by mediating direct cell-cell adhesion between the glial membrane and its associated axon, or between glial membranes. A potential candidate for an integrin-interacting protein expressed on axons or glia is Neuroglian, the Drosophila L1 (Nrcam) homolog. L1 is an Ig domain transmembrane protein that is known to bind RGD-dependent integrins. Loss of the integrin-binding domain of L1 results in wrapping defects in both myelinating and non-myelinating Schwann cells. However, the presence of low levels of ECM cannot be ruled out given the weak laminin immunolabeling that was observed. Even though Mmp2 expression in the WG had no effect, it is still possible that the integrin-mediated adhesion is Mmp2 resistant. To resolve this issue, further ultrastructural and genetic studies will be required (Xie, 2011).

GENE STRUCTURE

A splice variant of Scab contains an alternative 5' end replacing the first 63 amino acids.

PROTEIN STRUCTURE

Amino Acids - 1064

Structural Domains

The Scab protein has a 24 amino acid N-terminal signal sequence followed by a large (1033 amino acid) extracellular domain, a single transmembrane domain (23 amino acid) and a short (35 amino acid) cytoplasmic tail. The extracellular domain contains seven repeats common to all alpha integrin subunits. The alternate 5' splice has a 25 amino acid signal sequence followed by 38 amino acids and terminates in the first repeat. The last three repeats each contained a consensus divalent cation-binding motif Dx(D/N)x(D/N)GxxD for alpha integrins. A degenerate site is seen at position 262, between alpha repeats 3 and 4, the Gxx having been deleted. This site occurs exactly at the N terminus of the 90 kDa protein purified from Schneider L2 cells. A protein lacking the predicted structures from the first 261 amino acids would be unlikely to function as a normal integrin since a number of the conserved repeats would be eliminated. This may have been an artifact of the protein purification process, or may reflect some unusual protein variant. The 20 amino acids starting at position 195 match a motif found in a series of zinc- or cobalt-dependent enzymes. The histidine in this motif has been hypothesized to function in metal ion binding. Although functional evidence for this motif is not available, it may indicate novel divalent cation-dependence for alphaPS3. Immunoprecipitation and sucrose density gradients show that this integrin belongs to the group of alpha integrins that are cleaved external to the transmembrane domain into a light and heavy chain. The greatest homology to the consensus cleavage site (K/R)R(E/D)hydrophobic is the RRDL at position 928. A consensus cleavage site is seen in this position in alignments with human integrins alpha6, alpha7, alpha3, Scab and C. elegans F54G8.3 and nearby in human aIIb integrin. This site occurs between the conserved cysteines implicated in the disulfide bonding of the heavy and light chains, and is subsequent to the last of the tryptic sequences obtained from the putative heavy chain in Schneider L2 cells, all of which fall between amino acids 262 and 848. These lines of evidence are consistent with cleavage occurring at this site in Scab. 14 cysteine residues are present; 12 are in conserved locations, two are in novel sites and two residues, which are usually found, are absent. Nine potential N-linked glycosylation sites (NxT/S) are found in the extracellular domain. The highly conserved KxGFF(K/N)R is found just inside the putative transmembrane domain. The remainder of the cytoplasmic domain contains no striking similarity to any known protein. The sequence of Scab has no close vertebrate homolog (Stark, 1997).

date revised: 25 August 2018

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