Thomas A. Bunch

Thrombospondin-mediated adhesion is essential for the formation of the myotendinous junction in Drosophila

Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. We show that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. We propose a model in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space.

Molecular Pathways: The Role of Primary Cilia in Cancer Progression and Therapeutics with a Focus on Hedgehog Signaling

Abnormal Hedgehog (Hh) pathway activity has been reported in many cancers, including basal cell carcinomas, medulloblastomas, rhabdomyosarcomas, glioblastomas, and breast and prostate cancers. For this reason, the Hh pathway is a flourishing area for development of anticancer drugs such as Hh ligand antagonists (e.g., 5E1 and robotnikinin), Smo inhibitors (e.g., GDC-0449 and IPI-926), and Gli transcriptional activity inhibitors (e.g., GANT58 and GANT61). It is now clear that primary cilia are required for activation of the Hh pathway in normal vertebrate cells. It is in the primary cilium that both positive and negative effectors of the Hh pathway are processed by posttranslational modifications. In many cancers, preliminary results suggest that primary cilia are lost. As drugs that inhibit different steps of the Hh pathway are developed, it will be important to consider how these drugs will function in the context of primary cilia in the tumor environment. Here, we discuss why some of the Hh inhibitors may be ineffective if primary cilia are lost on cancer cells. Understanding the relationships between clinical inhibitors of the Hh pathway and the presence or absence of primary cilia may turn out to be critical for targeting these therapeutics to the correct population of patients and improving their efficacy. Further work is needed in this area to maximize the potential of these exciting therapeutic targets. Clin Cancer Res; 18(9); 2429–35. ©2012 AACR.

Splice Variants of the Drosophila PS2 Integrins Differentially Interact with RGD-containing Fragments of the Extracellular Proteins Tiggrin, Ten-m, and D-Laminin α2

Two new potential ligands of theDrosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin α2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the αPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the αPS2 m8 splice variant. The βPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the β splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the βPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure.

A DM domain protein from a coral, Acropora millepora, homologous to proteins important for sex determination

SUMMARY The identification and functional studies of DM domain‐containing proteins Doublesex, MAB‐3, and DMRT1 indicated that flies, nematodes, and humans share at least some of the molecular mechanisms of sex determination. We identified a gene, AmDM1, from the coral Acropora millepora that encodes a homologous DM domain‐containing protein. Molecular analyses show that the AmDM1 primary transcript is processed to generate four different messenger RNAs. Alternative use of two polyadenylation sites produces transcripts that vary only in the 3′ untranslated regions, whereas alternative splicing generates transcripts with and without the region coding for the DM domain. All the transcripts include a second motif, the DMA domain, which is found in a number of other proteins containing a DM domain. Hermaphroditic A. millepora differentiates sexual cells seasonally before the spring spawn, and Northern blot analysis shows that the AmDM1 transcripts are present at higher levels during sexual differentiation. The non‐DM domain‐containing messages are also present at significant levels in late embryos, but DM domain transcripts are extremely rare at this stage. These data suggest that the association of DM domain proteins and sexual determination or differentiation predates the separation of the Cnidaria from the rest of the Metazoa.

Integrin αIIbβ3 Activation in Chinese Hamster Ovary Cells and Platelets Increases Clustering Rather than Affinity

Integrin αIIbβ3 affinity regulation by talin binding to the cytoplasmic tail of β3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of αIIbβ3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn2+, a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of β3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets.

Functional Properties of Alternatively Spliced Forms of the Drosophila PS2 Integrin α Subunit

The Drosophila αPS2 protein is encoded by two alternatively spliced transcripts. The respective αPS2 proteins differ by the presence of 25 amino acids in the αPS2 (C) protein, not found in the αPS2 (m8) subunit, in a region thought to be important for ligand binding. We examined the functional properties of Drosophila S2 cells transformed with genes expressing either of these proteins, in association with a Pre subunit. Both PS2 integrins support cell spreading on vertebrate vitronectin or, to a lesser extent, on fibronectin. Interestingly, the PS2(C) form promotes spreading more efficiently on vitronectin than does the PS2(m8) form, with an opposite relative efficiency seen for fibronectin. Also, the two forms of PS2 show different requirements for divalent cations in order to mediate efficient cell spreading. These divalent cations are not required to maintain the association of α and β sub-units. Spreading of both cell types is similarly RGD sensitive, and both PS2 integrins appear to associate with th...

Differences in Regulation of Drosophila and Vertebrate Integrin Affinity by Talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talins role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talins integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.

Drosophila cell adhesion molecules.

Publisher Summary This chapter describes Drosophila cell adhesion molecules. The primary reason for the interest in Drosophila cell adhesion proteins is the ability to undertake sophisticated genetic analyses of function in situ. Analysis in situ is especially important when examining the function during development, where cell culture systems are of limited value. Drosophila has become a major experimental system for understanding the fundamental mechanisms of pattern formation and development. Cells transformed with wild-type or altered genes for adhesion proteins have been instrumental in dissecting the molecular and cell biology of vertebrate adhesion and similar approaches are available to the Drosophila biologist. Typically, these studies utilize the S2 Drosophila cell line that normally grows as round, nonadhering cells. One of the lessons emerging from mushrooming molecular anatomy database is that all metazoans are composed of similar proteins or at least the proteins generated from the combinations of similar domains. Cell adhesion proteins are no exception to this generalization. In some proteins, such as the integrin β subunit, nearly all the functional domains appear to be identical with those of the vertebrate homologs.

Amino acid changes in Drosophila αPS2βPS integrins that affect ligand affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila αPS2βPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the αvβ3 ligand-mimetic antibody WOW-1. The specificity of αPS2βPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the βPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the αPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the αPS2 GFFNR → GFANA mutation. A mutation in the βPS I domain (βPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by αPS2βPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin β subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the βPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because βPS-ΔPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.

Role of the PS integrins in Drosophila development.

The PS1 and PS2 integrins of Drosophila are heterodimers of αPS1βPS and αPS2βPS subunits, respectively, with very strong structural similarity to vertebrate integrins. Cell transfection experiments indicate that the PS integrins are receptors for extracellular matrix components and are functionally analogous to vertebrate integrins. Matrix ligands from Drosophila tissues have been identified for PS1 and PS2 integrins, using transformed cells and a cell‐spreading assay. Mutations in all three subunit genes have been identified, and the phenotypes of mutants indicate that PS integrins are required for the proper morphogenesis of a number of embryonic tissues. Using methods to produce genetic mosaics and transformation of integrin transgenes into whole animals, integrin functions in adult morphogenesis also have been examined. In the pupal wing, integrins are critically required to maintain the connection between dorsal and ventral epithelia, and recent results suggest that in early pupal development, the integrins are acting as specific receptors, as opposed to a non‐specific cell‐matrix glue.