Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly

Abstract

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.

Figure 1. Sequence alterations caused by inflated mutations

(A and B) Residues that are altered in inflated mutations are indicated by showing the side chains in red of the equivalent residues of αV, within the structure αVβ3 (αV in blue, β3 in grey) bound to the cyclo-RGD ligand (orange) (Xiong et al., 2002). Green dots are divalent cations. In (B) the β subunit has been removed and the α subunit β-propeller turned to show the face that contacts the β subunit. (C) Portions of a ClustalW alignment between αPS2, αV, and closely related α subunits from C. elegans and human showing conservation of the residues that are mutated. The if³⁵ mutation changes a cup tetrapeptide repeat within the core of the β-propeller. The if^C2B mutation changes the ligand binding pocket. The if¹⁷ allele changes the second of four cation binding sites. The divalent cation coordination consensus is outlined on the alignment. The if²¹ allele changes a GAP repeat located in the core of the β-propeller.

Figure 2. The if^C2B mutation causes αPS2βPS to detach from the ECM

Embryos were triple labelled with antibodies against αPS2 (green), talin (red), and myosin (blue). (A) In wild type embryos, talin and αPS2 colocalize at MAS. (B) Talin localization is dependent on integrins. Talin fails to be recruited to muscle ends in inflated null mutant (if^B4) embryos. (C) In if^C2B mutant embryos, talin colocalizes with αPS2 at MAS, and even at the end of detached muscles (arrowheads). Scale bar 50μm.

Figure 3. LamininW localization to basement membranes is dependent on the αPS2 RGD binding site

(A-E) Embryos were labelled with antibodies against the α chain of lamininW (red, white) and the epidermal septate junction marker, fasiclin III (blue), scale bar 50μm. (A). In wild type, lamininW localized to the ECM between adjacent muscles in a V-shaped pattern. (B) Dependence of lamininW localization on integrins. Embryos that lacked βPS (mys^XG43), and therefore lacked all βPS-containing heterodimers, failed to localize lamininW. LamininW localization was not altered in if¹⁷ (C) and if^SEF (D) mutant embryos. (E) LamininW was absent from muscle attachment sites in if^C2B mutant embryos.

Figure 4. Integrin is not required for the expression of the lamininW α chain

(A and B) In situ hybridization against wing blister mRNA shows that the laminin α chain was expressed normally in embryos lacking βPS.

Figure 5. αPS2 transport to the plasma membrane is defective in inflated mutants that display cytoskeletal detachment

(A-C) Phalloidin staining of F-actin highlights the muscles. (A) Wild type embryo, (B) muscle detachment in if^C2B mutant embryos at stage 16, (C) actin detachment in stage 17 if¹⁷ mutant embryos. (D-J, L) Embryos were labelled with antibodies against αPS2. (D and G) In wild type embryos the αPS2 subunit is localized exclusively to MAS and the levels of αPS2 increase between stage 16 and stage 17. (E and F) At stage 16 the levels of αPS2 localized to muscle ends in if³⁵ and if¹⁷mutant embryos were reduced compared to wild type. (H, I, and J) The αPS2 subunit accumulates in perinuclear regions inside the muscle and tendon cells in if¹⁷, if³⁵ and if²¹mutants, as does the (K) endoplasmic reticulum component protein disulfide isomerase, tagged with green fluorescent protein (ER-GFP; (Bobinnec et al., 2003). (L and L’) if³⁵ mutant embryos were double labelled with αPS2 and βPS antibodies. The βPS subunit also fills the inside of muscle and tendon cells. Scale bar 50μm.

Figure 6. Muscle detachment versus cytoskeletal detachment phenotypes observed in different inflated mutant alleles

Each panel shows muscles with the membrane labelled by expression of green fluorescent protein fused to the myristylation signal from Src (Src-GFP, green), and F-actin by phalloidin staining (red). (A, B) wild type embryos at stage 16 and 17, respectively. (C) In if^C2B embryos the muscles start detaching at stage 16, resulting in rounded up muscles with the membrane (white arrow) in close proximity to actin (red arrow). (D) In if²¹ mutant embryos (and if¹⁷, if³⁵ and if^SEF, not shown), the muscle actin (red arrows) contracted away from the attached muscle ends labelled with Src-GFP (white arrows). Scale bar 50μm.

Figure 7. Mutations in αPS2 cause similar defects when expressed in S2 cells

(A) S2 cells expressing βPS plus the m8 or C form of αPS2 from wild type or containing the if^C2B, if¹⁷, and if²¹ mutations were stained with an αPS2 specific monoclonal antibody, and immunofluorescence was quantitated by flow cytometry. Cells expressing the same integrins containing the activating mutation GFFNR>GFANA in the cytoplasmic domain of the αPS2 subunits were similarly stained. Shown are the average mean fluorescent intensities (MFI) and standard errors for three experiments. Each experiment analyzed 10,000 cells. The MFI of untransfected S2 cells was 10.6 +/− 0.6 (not shown). (B) The wild type and mutant cells were spread on 500 ng/ml RBB-Tigg, a fragment of tiggrin, or 5 μg/ml DLAM-RGD, a fragment of the lamininW α chain, and the percentage of the total cells that spread was visually determined. (C) Binding of the soluble monovalent αPS2βPS integrin ligand, TWOW-1, to wild type and mutant receptors. Binding is expressed as a ratio of specific TWOW-1 immunofluorscence over total integrin (as detected with the antibody used in A). Results are the average and standard error for three experiments.

Figure 8. Reduced levels of PS2 integrin recruit wild type levels of talin

Embryos were labelled with antibodies against αPS2 (green) and talin (red). (A and B) The amount of αPS2 transported to muscle ends was reduced in if³⁵ mutants (compare A’ to B’), however talin at MAS was not changed (A” and B”). Scale bar 50μm (C) Magnification of if³⁵ MAS at stage 17, scale bar 25μm. Talin preferentially localized to muscle ends and not with the αPS2 and βPS that is trapped inside the cell. (D) Quantification of average ratios of talin to αPS2 immunofluorescence at direct and indirect MAS from wild type and if³⁵ mutant embryos. An example of a direct MAS is indicated by an arrowhead in B’and B”. The arrows in B’ and B” point to an indirect MAS.

Figure 9. Talin separates from integrins when PS2 integrin levels are reduced at muscle attachment sites

To determine the point at which the link between integrins and the cytoskeleton was failing in if¹⁷, if²¹, and if³⁵ muscles, stage 17 mutant embryos were labelled with antibodies against αPS2, talin, myosin and βPS as indicated. (A) In wild type embryos, talin and αPS2 colocalized at muscle ends. (B) In if¹⁷ mutant embryos, talin and myosin detached from the muscle ends, and separated from αPS2 that remains at attachment sites (arrowheads). (C) In if³⁵ mutant embryos talin separated from both integrin subunits and followed the detaching cytoskeleton (arrowheads). A”” to C”” show enlarged segments of panels A to C, respectively. (B””) highlights the separation between integrin (green arrow) and talin at the muscle ends (red arrows). Scale bars 50μm.

Figure 10. Cytoskeletal detachment in if^SEF embryos is caused by a late loss of αPS2 expression in muscles

(A) Stage 17 if^SEF embryonic muscles displayed a cytoskeletal detachment phenotype. Embryos were stained with the indicated antibodies. (B-G) Embryos labelled with αPS2 antibodies. (B-C) The levels of αPS2 increased from stage 16 to stage 17 in wild type muscles. (E) The levels of αPS2 in stage 16 if^SEF muscles were similar to wild type, but at stage 17 (F) αPS2 levels have dropped dramatically in if^SEF muscles. (D and G) αPS2 levels are similar in the visceral muscle layer that surrounds the midgut (arrowheads) in wild type and if^SEF embryos, but reduced at MAS (arrows) in the if^SEF embryo. (H-K) The level of inflated mRNA (encoding αPS2), as revealed by in situ hybridisation, was reduced in the muscles (arrow) and tendon cells (black arrowhead) relative to the visceral mesoderm (white arrowhead) in early stage 16 if^SEF embryos (J,K) relative to wild type (H,I). Scale bars 50μm.

Figure 11. Muscle expression of both αPS2 and βPS are reduced in if^SEF mutants

The levels of αPS2 (green) in stage 17 if^SEF embryonic muscles (B and B’) is severely reduced compared to wild type (A and A’). βPS, which partners with αPS2 in muscle and αPS1 in the tendon cells, is also reduced from the muscles but remains in the tendon cells, paired with αPS1 (A” vs. B”). This shows that the if^SEF allele cause a loss of the αPS2βPS heteodimer rather than a loss of the epitope recognised by the αPS2 antibody. Scale bar 50μm.

Figure 12. Talin remained localized to muscle attachment sites lacking αPS2 in if^SEF mutants

Embryos were labelled with αPS2, talin and myosin antibodies. (A) In wild type stage 17 embryos, the confocal settings were adjusted so that the levels of talin were approximately equal to the levels of αPS2 at MAS. (B) Using the same confocal settings, αPS2 was barely detectable at stage 17 if^SEF MAS, while the levels of talin remained approximately equivalent to wild type. (C) Quantification of αPS2 and talin immunofluorescence. The average ratio of talin to αPS2 immunofluorescence is represented. Scale bar 50μm.