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Vol. 19, Issue 8, 3589-3598, August 2008

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Differences in Regulation of Drosophila and Vertebrate Integrin Affinity by Talin

Teresa L. Helsten^*, Thomas A. Bunch, Hisashi Kato^*, Jun Yamanouchi^*, Sharon H. Choi^*, Alison L. Jannuzi, Chloe C. Féral^*, Mark H. Ginsberg^*, Danny L. Brower^,,, and Sanford J. Shattil^*

*Department of Medicine, University of California, San Diego, La Jolla, CA 92093; and Departments of Molecular and Cellular Biology and Biochemistry, Arizona Cancer Center, Tucson, AZ 85724

Submitted January 28, 2008; Revised April 23, 2008; Accepted May 15, 2008
Monitoring Editor: Jean E. Schwarzbauer

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
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 talin's role in regulating Drosophila PS2βPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting PS2βPS 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 PS2βPS with those of human IIbβ3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of PS2βPS with those of IIbβ3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type IIbβ3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila PS2βPS affinity because of structural features inherent in the PS2βPS extracellular and/or transmembrane domains.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Integrin adhesion receptors couple the extracellular matrix with the actin cytoskeleton, allowing transmission of both mechanical force and biochemical signals across the plasma membrane (Hynes, 2002). The cytoskeletal protein talin, a product of the talin 1 (TLN1) gene, provides a key physical linkage between integrin β cytoplasmic domains and F-actin (Critchley, 2004; Wiesner et al., 2005). Talin's N-terminal head region contains a FERM domain with a PTB subdomain capable of interacting with several proteins, including integrin β cytoplasmic domains (Wegener et al., 2007). Talin's C-terminal rod domain contains binding sites for several cytoskeletal proteins, including actin (Calderwood, 2004; Critchley, 2004). Loss of talin in worms, flies, and mice leads to lethal phenotypes that closely resemble those caused by loss or mutation of integrins, indicating that talin's function overlaps that of integrins in the developing organism (Monkley et al., 2000; Brown et al., 2002; Brower, 2003; Cram et al., 2003; Wiesner et al., 2005). In fact, talin is essential for the normal development of organisms ranging in complexity from the multicellular slime mold Dictyostelium discoideum to vertebrates (Nuckolls et al., 1992; Albiges-Rizo et al., 1995; Bolton et al., 1997; Priddle et al., 1998; Tsujioka et al., 1999; Monkley et al., 2000; Cram et al., 2003). In vertebrates, it is also clear that talin plays a key role in regulating the affinity of integrins for adhesive ligands (Calderwood et al., 1999, 2002; Tadokoro et al., 2003; Ginsberg et al., 2005; Wegener et al., 2007). Although it is conceivable that the integrin–actin linkage and integrin affinity modulation functions of talin have evolved in concert, it is also possible that they developed independently.

In Drosophila melanogaster, talin null mutant embryos demonstrate normal localization of integrin PS2βPS to muscle ends, but failure of PS2βPS clustering into focal adhesion-like structures (Brown et al., 2002). At the onset of contraction, the muscles detach from their tendon cell anchorage points and PS2βPS detaches from the actin cytoskeleton but remains attached to extracellular matrix ligands (Brown et al., 2002). This suggests that talin is essential for maintenance of an integrin-actin linkage strong enough to resist mechanical force above a certain threshold. This idea is supported by experiments with optical tweezers showing that talin is required for maintenance of a 2 pN slip bond between fibronectin and the cytoskeleton (Giannone et al., 2003; Jiang et al., 2003). A role for talin in affinity modulation of PS2βPS has been speculated upon, but not measured directly (Brown et al., 2002; Tanentzapf and Brown, 2006).

Until recently, integrin affinity modulation in Drosophila has only been inferred from sophisticated genetic manipulation and indirect measurements using cell adhesion and spreading assays. We have developed a ligand-mimetic antibody Fab fragment, TWOW-1, that is selective for high-affinity PS2βPS and provides a facile means to assess PS2βPS affinity in cultured cells (Bunch et al., 2006). The binding selectivity of TWOW-1 is due in part to the fact that the H-CDR3 region of this Fab fragment contains a 53-amino acid RGD tract derived from tiggrin, a natural Drosophila matrix ligand for PS2βPS (Fogerty et al., 1994). Using TWOW-1, we have shown that PS2βPS affinity is indeed increased or decreased by certain integrin mutations that would be predicted to do so by structure-function analyses of vertebrate integrins (Bunch et al., 2006; Devenport et al., 2007). Given that the heterodimeric structure of integrins and the major amino acid sequence motifs of both integrins and talin are highly conserved between Drosophila and mammals (Burke, 1999; Hynes and Zhao, 2000; Senetar and McCann, 2005), it is reasonable to hypothesize that, as in vertebrates, talin modulates the affinity of Drosophila integrins. Here TWOW-1 was used to test this hypothesis and we demonstrate that this is not the case. Unlike vertebrate integrins, PS2βPS appears to be relatively resistant to affinity modulation by talin, even when its cytoplasmic domains containing putative talin-binding sites or its transmembrane domains are replaced with those of human IIbβ3. On the other hand, overexpression of the Drosophila talin head domain is capable of activating IIbβ3 in a manner similar to that of the human talin head domain. Taken together, these results suggest that, in contrast to talin's linkage function, the regulatory role of talin in integrin affinity modulation may be a relatively more recent evolutionary development in higher organisms.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Antibodies
TWOW-1 is a ligand-mimetic Fab specific for Drosophila PS2βPS that was produced and purified as reported (Bunch et al., 2006). It was used at final concentrations of 2–120 µg/ml as indicated. PAC-1 IgM ascites (Shattil et al., 1985) and PAC-1 Fab (Abrams et al., 1994) are ligand-mimetic antibodies specific for human IIbβ3. Surface expression of integrins was quantified by flow cytometry using antibodies CF.2C7 (sometimes biotinylated) for Drosophila PS2 (Brower et al., 1984), CF.6G11 for Drosophila βPS (Brower et al., 1984) or D57 for IIbβ3 (O'Toole et al., 1994). Talin expression was detected using the mAb J10 for Drosophila talin (Brown et al., 2002) or 8d4 for Chinese hamster ovary (CHO) cell and human talin (Sigma, St. Louis, MO). The secondary antibody for all flow cytometry experiments was either R-phycoerythrin conjugated goat anti-mouse IgG (H+L; Biosource, Camarillo, CA) or Alexa-488–conjugated goat anti-mouse IgG (H+L; Molecular Probes, Eugene, OR), except for PAC-1 IgM, which was detected using R-phycoerythrin–conjugated goat anti-mouse IgM (Biosource). Biotinylated antibodies were detected using R-phycoerythrin-streptavidin (Molecular Probes).

Cell Lines and Culture
Drosophila S2/M3 cells stably expressing the PS2 and βPS integrin subunits under the control of the Drosophila HSP70 heat-shock promoter have been described (Bunch and Brower, 1992; Zavortink et al., 1993). For all cell culture experiments, the PS2C (canonical) and βPS4A isoforms were used (Graner et al., 1998). In some experiments, these integrin-expressing S2/M3 cells also stably expressed Drosophila talin head-GFP chimeras (wild-type or R367A mutants) under the control of the yeast Gal4 UAS (Tanentzapf et al., 2006). All Drosophila cells were grown in Shields and Sang M3 medium supplemented with 12.5% heat-inactivated fetal bovine serum and 2 x 10^–7 M methotrexate. For all experiments other than cell-spreading assays, S2/M3 cells were first cleared of accumulated matrix and other surface proteins by dispase/collagenase (Roche Applied Science, Indianapolis, IN). The cells were simultaneously heat shocked at 37°C to induce expression of the integrin transgenes (Jannuzi et al., 2002). CHO cells stably expressing human IIbβ3 or constitutively active IIbβ3 (D723R) have been described (Frojmovic et al., 1991; Hughes et al., 1996). CHO cells stably expressing Drosophila PS2βPS were generated as described below. All CHO cells were grown in DMEM supplemented with 10% fetal bovine serum.

Plasmids and Transfection
PS2 and βPS cDNAs were excised from the Drosophila HSP70 promoter-driven expression vectors used in the S2/M3 cell system and subcloned into the mammalian expression vector pcDNA3.1. These constructs were cotransfected into CHO cells in a 1:1 weight ratio using Lipofectamine (Invitrogen, Carlsbad, CA). After selection with G418, stable transformants expressing high levels of PS2βPS integrin were obtained by single-cell sorting using antibody CF.2C7. The βPS/pcDNA3.1 expression vector was used as the template for creation of a βPS double mutant (I830A/K832A) by standard PCR-based site-directed mutagenesis. This vector was transiently transfected along with wild-type PS2 into CHO cells. After 72 h, surface expression of the mutant integrin was measured by flow cytometry using anti-PS2 antibody CF.2C7 and was found to be comparable to that of wild-type PS2βPS; in contrast, several βPS cytoplasmic domain truncation mutants failed to express in the CHO cell system.

Mammalian expression vectors encoding chimeric integrins with the extracellular domains of Drosophila PS2 or βPS and the intracellular (and in some cases the transmembrane) domains of human IIb or β3, respectively, were generated using standard overlap extension PCR. The following oligonucleotide primers were used to create the chimeras containing the fly transmembrane domain: PS2 forward 5'-ACG AGA AGC TGG TGA AGA AGT CCT ATC TGC-3', PS2 reverse 5'-GAA GCC GCA CTT GTA GAG CAG CCA GAC-3', PS2-IIb forward 5'-GTC TGG CTG CTC TAC AAG GTC GGC TTC TTC AAG CGG AAC-3', IIb reverse 5'-AGA GCG GCC GCG CAC CAT CAC TCC CCC TCT TCA TCA TCT TC-3', βPS forward 5'-CAA TAT TAT CTT CGC CGT CAC TGC CAG-3', βPS reverse 5'-GAG CAG CTT CCA CAG CAG GAG AAT G-3', βPS-β3 forward 5'-C ATT CTC CTG CTG TGG AAA CTC CTC ATC ACC ATC CAC GAC-3', and β3 reverse 5'-AGA GCG GCC GCA AGA TCT TAA GTG CCC CGG TAC GTG ATA TTG-3'. For the constructs with the human transmembrane domains, the primers are the same as above other than the following: PS2 reverse 5'-ATC GGG CAC CTG AAG CGG TTC CGG TTC-3', PS2-IIb forward 5'-CCG CTT CAG GTG CCC GAT GCC ATT CCA ATC TGG TGG GTG C-3', βPS forward 5'-TCC GGT CAT GGT ACC TGC GAA TGC GGT-3', βPS reverse 5'-ATG AAA ACC TTG GCC GGA CAC TCC TT-3', and βPS-β3 forward 5'-AGA GCG GCC GCA AGA TCT TAA GTG CCC CGG TAC GTG ATA TTG-3'. Chimeric integrins with the extracellular and transmembrane domains of human IIb or β3 and the intracellular domains of Drosophila PS or βPS were constructed with following oligonucleotide primers: IIb forward 5'-CCT CCT GTC AAC CCT CTC AA-3', IIb reverse 5'-CCA CAT GGC CAG GAC CAG G-3', IIb-PS forward 5'-CTG GTC CTG GCC ATG TGG AAG TGC GGC TTC TTT AAC CGC-3', PS reverse 5'-TTT CTT AAG CTG GCA CTC TAC AGG TGC TCG TC-3', β3 forward 5'-GCA ATG GGA CCT TTG AGT GT-3', β3 reverse 5'-CCA GAT GAG CAG GGC GGC-3', β3-βPS forward 5'-GCC GCC CTG CTC ATC TGG AAG CTG CTC ACT ACG ATC CAC G-3', βPS reverse 5'-TTT CCT GCA GGG CGA ATC TAT TTG CCC GCA TAC ATG-3'. These constructs were placed under the control of the CMV promoter in pcDNA3.1 and sequences were confirmed by direct DNA sequencing.

Plasmids expressing a chimeric integrin subunit were transfected into CHO cells in a 1:1 weight ratio with either the corresponding Drosophila wild-type or chimeric integrin subunit. For talin head overexpression experiments, chimeric and wild-type integrin constructs were transiently transfected into CHO cells along with a green fluorescent protein (GFP)-murine talin head F2–F3 domain chimera (or empty vector) and harvested 48 h later for analysis of TWOW-1 binding by flow cytometry. For talin knockdown experiments, chimeric integrins were stably transfected into the CHO cells. After selection with G418, stable transformants expressing high levels of the chimeric integrins were obtained by single-cell sorting using antibody CF.2C7.

A mammalian expression vector encoding GFP-Drosophila talin head domain chimera was created by cloning the Drosophila talin head domain (amino acid residues 1-470) using reverse-transcribed RNA obtained from S2/M3 cells. The talin head coding sequence was subcloned in-frame into pEGFP-N1 (Clontech, Palo Alto, CA) to create GFP-Drosophila talin head under the control of the CMV promoter. A GFP-murine talin head F2-F3 fragment (amino acids 206-405) was described previously (Calderwood et al., 2002). GFP-talin head chimeras were transfected into CHO cells expressing either IIbβ3 or PS2βPS using Lipofectamine. Expression was confirmed by GFP fluorescence and Western blotting of cell lysates with a monoclonal anti-enhanced GFP (EGFP) antibody (Clontech).

RNA Interference
Knockdown of talin and βPS integrins by RNA interference (RNAi) in Drosophila S2/M3 cells was performed as described (Worby et al., 2001) with exceptions as noted. Total genomic DNA was isolated from S2/M3 cells (QIAamp DNA minikit, Qiagen, Chatsworth, CA) and used as the template for PCR amplification of single-stranded DNA fragments 400-600 nucleotides (nt) in length that correspond to exons of the rhea (talin) and myospheroid (βPS) genes as follows: DT273 exon 5 of talin, DT996 exon 9 of talin, and Mys6010 exon 5 of βPS. The oligonucleotide primers used to amplify these segments of DNA are as follows, where the first 20 nt of each is the T7 RNA polymerase recognition sequence: DT273 forward 5'-TAA TAC GAC TCA CTA TAG GGC GCA CCA AGG GAA TCG AGA-3'; DT273 reverse 5'-TAA TAC GAC TCA CTA TAG GGC CAC TGA CTC TTC CAC CAT-3'; DT996 forward 5'-TAA TAC GAC TCA CTA TAG GGC GCT CTA TTG AAT GGC GTG-3'; DT996 reverse 5'- TAA TAC GAC TCA CTA TAG GGC GTT GGT GCC ACA ACT TTG-3'; Mys6010 forward 5'-TAA TAC GAC TCA CTA TAG GGC GAG GTG AAG AAT GCC ACA G-3'; Mys6010 reverse 5'-TAA TAC GAC TCA CTA TAG GGC AAC CAC ATT GGA TGA ATC G-3'. Cells were harvested 5 d after the addition of double-stranded RNA to the cell culture medium because preliminary experiments showed maximum reduction in levels of the target protein at this time, as determined by Western blotting or flow cytometry. For cell-spreading experiments using Drosophila S2/M3 cells, double-stranded RNAs targeting talin were prepared using the following oligonucleotide primer pairs, where the first 27 nt of each is the T7 RNA polymerase recognition sequence: talin exon 2 forward 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA CCA GCG AAT ATG GAC TGT TTA-3'; talin exon 2 reverse 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA TTT CAT CGT CCG TCT TTA GTT TC-5'; talin 3' untranslated region forward 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA CTA TAT GCC TCT AC-3'; talin 3' untranslated region reverse 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA GCA ACT GCA TAC ACG ACT CG-3'; GFP forward 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA ATG GTG AGC AAG GGC-3'; GFP reverse 5'-GAA TTA ATA CGA CTC ACT ATA GGG AGA CAG TTA TTA CTT GTA CAG-3'. For these constructs, near complete talin knockdown was confirmed by Western blotting at 72 h after transfection. Knockdown of talin in CHO cells was performed using short hairpin RNA (shRNA; Paddison et al., 2002). The plasmid encoding shRNA targeting murine talin1 (MT749), in contrast to its mismatched control shRNA, has been shown to effectively knockdown CHO cell talin, with a maximum effect at 72 h (Tadokoro et al., 2003). Therefore, CHO cells stably expressing IIbβ3 (D723R) were cotransfected with PS2, βPS, pEGFP-C1 (Clontech) and either MT749 or its mismatched control using Lipofectamine. In experiments with CHO cells stably expressing chimeric or wild-type integrins, cells were similarly transfected with MT749 or mismatched control shRNA and analyzed 72 h later by flow cytometry or Western blotting.

Flow Cytometry
Surface expression and affinity of integrins was monitored by flow cytometry as described for S2/M3 cells (Bunch et al., 2006) and CHO cells (Tadokoro et al., 2003). Analyses were gated on high-GFP–expressing cells as a marker for transfection. Nonspecific binding of ligand-mimetic antibodies to PS2βPS (TWOW-1) or IIbβ3 (PAC-1) was taken as binding in the presence of 10 mM EDTA or 2 mM RGDS and was subtracted from total binding to obtain specific binding. In experiments designed to test the effect of metabolic energy depletion on integrin affinity, harvested cells were incubated in buffer containing 0.2% sodium azide and 4 mg/ml 2-deoxy-D-glucose (Sigma) for 30 min at room temperature before staining with ligand-mimetic antibodies. Staining and washes were performed in the absence of glucose, whereas controls were processed in glucose-containing buffer. For measurement of intracellular talin expression, cells were fixed for 30 min in 0.5% paraformaldehyde, which was then neutralized for 5 min with 0.2 M glycine in 0.5 M Tris, and 0.2% sodium azide, pH 7.4. After two washes with PBS, cells were permeabilized with 0.1% saponin in PBS/1.0% bovine serum albumin (BSA), pH 7.4, and talin staining was achieved with antibody J10 (2%) or 8d4 (10 µg/ml). Nonimmune Ig of appropriate isotype was used as a negative control. After addition of fluorophore-conjugated secondary antibody and further washing with buffer containing 0.025% saponin, talin expression was quantified by flow cytometry (Tadokoro et al., 2003).

Cell Spreading
S2/M3 cells, including those stably expressing PS2βPS, were treated with RNAi constructs targeting talin or GFP (a negative control) in Ultra Low Attachment 24-well plates (Corning Glass Works, Corning, NY). Two different double-stranded RNAs targeting talin were used: one corresponding to exon 2 and the other corresponding to the 3'-untranslated region. After 3 d, the cells were plated on tissue culture plates in usual growth medium containing serum and allowed to spread for 4–5 h before being photographed to assess cell spreading. This spreading is integrin-mediated (parental S2/M3 cells without PS2βPS integrins do not spread under these conditions) and depends upon ligands provided by the serum in the medium or synthesized by the cells (Bunch and Brower, 1992; Jannuzi et al., 2002). All photographs were coded, and mixed, and the percentages of spread cells was determined by a blinded observer. For each experiment, three representative fields were photographed and scored. Results are the averages of the three experiments ± SEM.

Affinity Chromatography with Recombinant Integrin Cytoplasmic Tails
Expression and purification of recombinant human integrin tail constructs bound to His-bind resin (Novagen, Madison, WI) and affinity chromatography using these constructs have been described (Pfaff et al., 1998; Arias-Salgado et al., 2003). The recombinant Drosophila βPS tail and its IYK/AYA mutant were similarly prepared using the appropriate βPS/pcDNA3.1 vectors as templates for PCR amplification of the coding sequence corresponding to residues K-800 to K-846. The following oligonucleotide primers were used: forward 5'-GCT ATC TGG AAG CTT CTC ACT ACG ATC-3'; and reverse 5'-CTT TTC ATC GGA TCC AAT CTA TTT GCC CGC-3'. As a source of talin, human platelets or S2/M3 cells were solubilized in buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM sodium vanadate, 0.5 mM sodium fluoride, 1 mM leupeptin, and complete protease inhibitor cocktail (Roche Applied Science). After clarification, 750 µg of platelet lysate or 2.5 mg of S2/M3 lysate were incubated with 100 µl of resin for 4 h at room temperature, and after washing, bound proteins were subjected to SDS-PAGE. Then talin was detected on Western blots with antibody 8d4 or J10. The amount of cytoplasmic tail proteins bound to the resin was monitored by Coomassie brilliant blue staining of the polyacrylamide gels (Pfaff et al., 1998; Arias-Salgado et al., 2003).

TWOW-1 Binding to Imaginal Discs
TWOW-1 binding to heads from late third instar larvae was carried out in BES-buffered Tyrode's (137 mM NaCl, 2.9 mM KCl, 20 mM BES, 0.1% glucose, pH 7.5) containing 1 mg/ml BSA, 0.01 mM Ca²⁺, and 1 mM Mg²⁺. Where indicated, the buffer also contained either 1 mM MnCl₂ or 5 mM EDTA. The heads were blocked in BES-Tyrode's plus BSA for 10 min before incubation with TWOW-1 (120 µg/ml) for 20 min at room temperature. Samples were washed three times in buffer then incubated with secondary antibody (0.02 mg/ml AlexaFluor 568 goat anti-mouse immunoglobulin; Molecular Probes) for 20 min at room temperature. After three washes, they were transferred in steps of increasing glycerol concentration to 30% 0.1 mM Tris, pH 8.2, and 70% glycerol. Wing imaginal discs were dissected and mounted in VectaShield Mounting Medium (Vector Laboratories, Burlingame, CA) before visualization on a Zeiss Universal Microscope using an AxioCam MRm digital camera with AxioVisionAC v4.2 software (Zeiss, Thornwood, NY).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In vertebrates, talin modulates the adhesive ligand binding affinity of many β1, β2, and β3 integrins (Ginsberg et al., 2005). Here we sought to determine whether talin is also capable of modulating the affinity of Drosophila PS2βPS, whose β cytoplasmic domain is closely related to vertebrate β cytoplasmic domains, β1 in particular (Burke, 1999; Hynes and Zhao, 2000). The affinity state of PS2βPS was monitored with TWOW-1, a ligand-mimetic Fab fragment selective for high-affinity PS2βPS (Bunch et al., 2006). For comparison, in some experiments the affinity of human IIbβ3 was monitored with the ligand-mimetic PAC-1 Fab (Abrams et al., 1994). Because both TWOW-1 and PAC-1 Fabs are monovalent, they are sensitive to changes in integrin affinity but not to changes in integrin valency or clustering (Abrams et al., 1994; Hato et al., 1998).

In initial studies, TWOW-1 was found to bind specifically to Drosophila S2/M3 cells stably expressing PS2βPS, as assessed by flow cytometry. In contrast, S2/M3 cells expressing no significant amounts of PS2βPS had negligible TWOW-1 binding (not shown). Addition of 1 mM MnCl₂ caused a further increase in TWOW-1 binding to S2/M3 cells expressing PS2βPS (Figure 1A, compare the black and hatched bars on the far left). MnCl₂ was used here because it is an extrinsic activator of Drosophila and vertebrate integrins (Takagi et al., 2002; Litvinov et al., 2004; Bunch et al., 2006) and because cellular agonists that activate PS2βPS have yet to be identified, unlike the case of platelets where physiological agonists, such as thrombin, activate IIbβ3 (Shattil and Newman, 2004). With vertebrate integrins, both the basal activation state and agonist-induced activation are blocked by RNAi-mediated talin knockdown (Tadokoro et al., 2003). Therefore, talin in the S2/M3 cells was knocked down with either of two independent double-stranded RNAs derived from Drosophila talin (DT273 and DT996). DT273 or DT996 caused a specific 85–90% reduction in talin expression, as assessed by intracellular staining with an anti-talin antibody and flow cytometry (Figure 1B) or Western blotting (not shown). Despite talin knockdown, neither basal nor MnCl₂-induced TWOW-1 binding were affected (Figure 1A). As a control, a double-stranded RNA (Mys6010) designed to knock down βPS caused a marked reduction in PS2βPS expression and TWOW-1 binding (Figure 1, A and B). Although it is theoretically possible that the small amount of residual talin in the talin knockdown cells was sufficient to support the basal activation state of PS2βPS, it is notable that knockdown of vertebrate talin by only 70% abolishes vertebrate integrin activation (Tadokoro et al., 2003), and talin knockdown significantly affected the ability of the cells to spread. Thus, Drosophila talin does not appear to be required for regulation of basal PS2βPS affinity in Drosophila S2/M3 cells.

View larger version (33K): [in this window] [in a new window]   Figure 1. Talin knockdown does not affect PS2βPS affinity in Drosophila S2/M3 cells. S2/M3 cells stably expressing PS2βPS were transfected with double-stranded RNA constructs targeting talin (DT273 and DT996) or the βPS integrin subunit (Mys6010). Cells were harvested 5 d later and analyzed by flow cytometry. (A) Specific binding of the ligand-mimetic Fab fragment, TWOW-1 (10 µg/ml). In some cases, TWOW-1 binding was performed in the presence of 1 mM MnCl2. Results are expressed as means ± SEM; n = 3 independent experiments. (B) Knockdown of talin significantly reduces intracellular talin expression, but does not affect surface expression of PS2βPS, and vice versa. Protein expression, measured by flow cytometry as detailed in Materials and Methods, is reported as percentage of that observed in control cells not subjected to knockdown. Data represent means ± SEM; n = 8–14.

View larger version (34K): [in this window] [in a new window]   Figure 2. Talin knockdown does not affect PS2βPS activation in CHO cells. CHO cells stably expressing the constitutively active human integrin mutant IIbβ3 (D723R) were transiently cotransfected with plasmids encoding GFP, PS2, βPS, shRNA targeting talin (MT749) or a mismatched shRNA control. Seventy-two hours later, transfected cells were identified by GFP fluorescence and specific binding of TWOW-1 Fab or PAC-1 Fab was measured by flow cytometry. MT749, in contrast to its mismatched control, significantly reduced intracellular talin expression, but did not affect surface expression of PS2βPS (not shown). Results are means ± SEM; n = 4.

View larger version (22K): [in this window] [in a new window]   Figure 3. Activation of PS2βPS is dependent on cellular metabolic energy. S2/M3 cells stably expressing PS2βPS (A) or CHO cells transiently expressing PS2βPS and stably expressing human IIbβ3 (D723R) (B) were depleted of metabolic ATP by incubation with 2-deoxy-D-glucose and sodium azide for 30 min. Specific TWOW-1 or PAC-1 Fab binding was then measured. Results are means ± SEM; n = 4.

View larger version (30K): [in this window] [in a new window]   Figure 4. Talin head overexpression does not increase activation of PS2βPS. (A) TWOW-1 (30 µg/ml) binding to S2/M3 cells stably expressing PS2βPS and GFP-Drosophila talin head (TH wild-type or TH (R367A) mutant). Where indicated, TWOW-1 binding was assessed in the presence of 1 mM MnCl2. GFP was used as a marker for transfection and an analysis gate for TWOW-1 binding was set on cells with high or no GFP expression to assess transfected (GFP-TH) or untransfected (No TH) cells, respectively. Data are means ± SEM; n = 3. (B) CHO cells stably expressing PS2βPS or IIbβ3 were transiently transfected with either a GFP Drosophila talin head chimera or a GFP murine talin head (F2-F3 fragment) chimera. After 48 h, specific TWOW-1 or PAC-1 IgM binding was measured. Preliminary experiments showed that transient expression of the Drosophila or murine talin head constructs in CHO cells caused a 70–100% increase in the surface expression of PS2βPS, as determined using two separate anti-integrin antibodies, but there was no such effect on surface expression of IIbβ3. Although the mechanism for this effect is not known, TWOW-1 binding was therefore normalized for PS2βPS expression in this series of experiments. Data are means ± SEM; n = 5–6.

View larger version (63K): [in this window] [in a new window]   Figure 5. Talin binding to β integrin cytoplasmic tails is not species-specific. Affinity chromatography was performed as described in Materials and Methods using recombinant cytoplasmic tail mimics of human β1, β3, a random peptide derived from β3 (rβ3), IIb, and Drosophila βPS. Lysates from human platelets (A) or Drosophila S2/M3 cells (B) were used as a source of human talin (hTalin) or Drosophila talin (dTalin). Binding of talin to integrin tails was assessed by Western blotting. Input amounts of tail mimics were monitored by staining with Coomassie brilliant blue. These experiments are representative of three so performed.

View larger version (40K): [in this window] [in a new window]   Figure 6. Mutations that abrogate talin binding to integrin β cytoplasmic tails do not affect PS2βPS integrin activation state. (A) Affinity chromatography was performed as in Figure 5 using human β1, Drosophila wild-type βPS, and the βPS (I830A/K832A) mutant (designated IYK/AYA), all incubated with Drosophila S2/M3 cell lysates. For clarity, the amino acid sequences of the wild-type and mutant βPS tails are shown, with the positions of the mutated amino acids underlined. This experiment demonstrates that the IYK to AYA mutations abolish talin binding to βPS. (B) CHO cells were transiently transfected with GFP, PS2, and either wild-type βPS or βPS (I830A/K832A). Then TWOW-1 (10 µg/ml) binding was determined. Differences in the results obtained for βPS and βPS (I830A/K832A) were not statistically significant. Also, there were no differences in integrin surface expression as measured by flow cytometry (not shown). Results are mean ± SEM; n = 3.

Amino acid sequences within both the (Knezevic et al., 1996; Yuan et al., 2006) and β (Patil et al., 1999; Vinogradova et al., 2002; Garcia-Alvarez et al., 2003; Wegener et al., 2007) cytoplasmic domains of vertebrate integrins appear capable of supporting talin interactions. These sequences include the β subunit membrane-proximal region and NPxY motifs, which are highly conserved in Drosophila βPS (Figure 7). In addition, the transmembrane domains of vertebrate integrin subunits are involved in transmitting talin-mediated conformational changes to the integrin extracellular domains (Li et al., 2003; Ginsberg et al., 2005; Luo et al., 2005; Partridge et al., 2005). In flies, these structure–function relationships are less well understood. However, mutations in either the membrane-proximal region or the first NPxY motif of a dimeric βPS mimic abolish both recruitment of the talin head and the dominant negative muscle detachment phenotype of the βPS mimic (Tanentzapf et al., 2006). Moreover, mys^XR04, which lacks NPxY motifs (Jannuzi et al., 2002), is unable to recruit talin head, but retains the ability to recruit full-length talin, albeit at reduced levels (Tanentzapf and Brown, 2006). To determine whether differences between Drosophila and vertebrate extracellular, transmembrane or cytoplasmic domains might help to explain the relative resistance of PS2βPS to affinity modulation by talin, we generated chimeric Drosophila/human integrin and β subunits. The first set of chimeras studied contained the PS2 and βPS extracellular domains and either the transmembrane or cytoplasmic domains of PS2βPS or IIbβ3 (Figure 7, A and B). Chimeric integrins were expressed in CHO cells with or without the murine GFP-talin head F2-F3 fragment, and TWOW-1 binding was quantified. Although expression of GFP-talin F2-F3 increased PAC-1 binding to IIbβ3 in CHO cells as expected (not shown), GFP-talin F2-F3 failed to increase TWOW-1 binding to any of these chimeric integrins, even ones that contained the transmembrane and cytoplasmic domains of IIb and β3 (Figure 7D). Furthermore, shRNA-mediated knockdown of talin had no effect on TWOW-1 binding to these chimeric integrins (Figure 7E).

View larger version (46K): [in this window] [in a new window]   Figure 7. Influence of integrin domains on talin activation of chimeric Drosophila/human integrins. (A) The amino acid sequences of the cytoplasmic domains of PS2βPS and IIbβ3. Clustal W (version 1.83) multiple sequence alignments of the cytoplasmic tail domains of the respective fly and human integrin subunits are shown. Regions in gray boxes are thought to be important for talin binding (to varying degrees), in particular the membrane-proximal portions of both and β integrin subunits and the region in β involving the first NxxY (NPXY) motif (see text for references). Clustal W symbols: * = identical, = conserved, = semiconserved. (B and C) The chimeric integrins studied in this experiment. For clarity, domains from PS2βPS are denoted D (open bars) and those from IIbβ3 are denoted H (gray bars). For example, a chimeric integrin designated D/D/H contains and β subunits with the fly extracellular and transmembrane domains and the human cytoplasmic domains. In D and F, CHO cells were transiently transfected with wild-type PS2βPS (designated D/D/D), wild-type IIbβ3 (H/H/H), or a chimeric integrin as indicated. They were cotransfected with either GFP as a marker of transfection (No TH) or a murine GFP-tagged talin head F2–F3 fragment (TH). After 48 h, cells were incubated in the absence or presence of MnCl2 and TWOW-1 (D) or PAC-1 (F) binding was measured by flow cytometry (n = 3). In E and G, CHO cells were cotransfected with the indicated wild-type or chimeric integrins and with MT749 to knockdown talin or a mismatch control shRNA as in Figure 2. Then TWOW-1 (E) or PAC-1 (G) binding was quantified by flow cytometry (E, n = 3; G n = 4). TWOW-1 and PAC-1 binding are expressed as specific binding normalized to integrin expression, the latter measured in parallel by flow cytometry. In E and G, MT749, but not its mismatched control, significantly reduced intracellular talin expression (not shown). Data represent means ± SEM.

Certain lethal muscle or wing phenotypes in the fly resulting from PS2βPS gain- or loss-of-function mutations have been attributed to an increase or decrease in integrin affinity for ligands (Martin-Bermudo et al., 1998; Brower, 2003; Tanentzapf and Brown, 2006; Devenport et al., 2007). More recently TWOW-1 binding experiments in S2/M3 cells have supported these contentions, demonstrating that PS2βPS integrin affinity is influenced by activating mutations in the PS2 cytoplasmic tail or the βPS I-like domain (Bunch et al., 2006). However, it must be emphasized that changes in PS2βPS affinity in fly embryos have been inferred rather than measured directly. In an attempt to evaluate PS2βPS affinity in a relevant Drosophila tissue, TWOW-1 binding to wing imaginal discs was determined. Imaginal discs (and other tissues) from late third instar larvae were dissected and incubated with TWOW-1 in BES-Tyrode's buffer with Ca²⁺ and Mg²⁺, or in the same buffer plus Mn²⁺ to activate integrins. Imaginal discs from this stage express PS2βPS primarily on the ventral epithelium of the presumptive wing, making this an excellent tissue to assess binding specific for PS2βPS. Analysis of cDNAs from imaginal discs suggests that they express primarily the PS2m8 isoform at this time (Brown et al., 1989), and experiments with S2 cells have shown that this variant has low affinity for TWOW-1 in the absence of activation (Bunch et al., 2007). Wing discs incubated without MnCl₂ showed very little TWOW-1 binding (Figure 8A). However if MnCl₂ was present during the incubation, increased TWOW-1 binding was observed on the ventral cells (Figure 8B). In tissues such as imaginal discs, some TWOW-1 binding to PS2βPS may be obscured by the presence of a competing endogenous ligand, such as tiggrin. Nonetheless, the current results suggest that at least a portion of PS2βPS on this epithelium is in a low-affinity state just before metamorphosis.

View larger version (52K): [in this window] [in a new window]   Figure 8. Drosophila PS2βPS is not constitutively active in imaginal discs. Posterior halves of Drosophila wing imaginal discs from late third instar larvae, stained for TWOW-1 binding in the absence (A) or presence of MnCl2 (B). TWOW-1 binding to PS2βPS expressing ventral cells (arrows) is seen only when integrins are activated by MnCl2, except for slight staining seen in the wing pouch (asterisk in A), where integrin density is greatest. For both images the initial exposures and any contrast and brightness adjustments are identical. Bar, 50 µm.

	ACKNOWLEDGMENTS

 
This work is dedicated to the memory of our esteemed colleague, Danny Brower, whose untimely death occurred during the final stages of preparation of this manuscript. We thank Nick Brown and Guy Tanentzapf (Wellcome Trust/Cancer Research UK) for antibodies and the UAS-talin head constructs; Candace George, Jian Kang, Barry Moran, and Ararat Ablooglu for technical assistance; James Feramisco for the use of his imaging facility at UCSD; and Barb Carolus and Debbie Sakiestewa of the Arizona Cancer Center Flow Cytometry Service. This work was supported by grants from the National Institutes of Health (NIH) to S.J.S. (R01HL56595, PO1HL47900) and D.L.B. and T.A.B. (R01GM42474). T.L.H. was supported in part by an NIH training grant (T32AR007608).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-01-0085) on May 28, 2008.

Deceased.

Address correspondence to: Teresa L. Helsten (thelsten{at}ucsd.edu)

Abbreviations used: CHO, Chinese hamster ovary; GFP, green fluorescence protein; shRNA, short hairpin RNA; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RNAi, RNA interference.

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