TI - Results Armadillo in junctions and polarity . AB - The classical view of Arm is that it has two non-overlapping functions as a nuclear transcriptional activator of Wg signaling and as a structural component of adherens junctions . These two functions give two different phenotypes in Drosophila embryos where loss of junctions leads to cuticle disintegration and loss of nuclear signaling leads to patterning defects ( compare Fig 1 C to I ) . Many studies , however , have suggested the possibility that Arm's activity at the junction is also actively regulated by signaling , meaning that junctional Arm is not exclusively a static structural component[26] . To test this hypothesis , we mutated two threonines to alanines in Arm ( T111A and T121A , hereafter referred to as ArmAA ) that are required in vitro to stabilize its binding to alpha-catenin[28] . We looked at Drosophila cuticles to analyze the effect of ArmAA on both the adherens junctions and on Wg signaling in the nucleus . In strong loss-of-function armO43A01 maternal and zygotic ( M/Z ) mutants ( hereafter referred to as armO43A01 (M/Z) ) no intact cuticle is made because loss of Arm leads to a drastic loss of cell cell adhesion in the epidermis .lt @@@@@ gt armO43A01 (M/Z) embryos eventually disintegrate and only small pieces of tissue remain ( Fig 1C ) . We found that low levels of ArmAA expression restored some cuticular integrity that is completely lost in armO43A01 (M/Z) embryos , suggesting restoration of some cell cell adhesion in the epidermis ( Fig 1D ) [29] . This phenotype was similar to expression of an alpha-catenin/E-cadherin fusion protein in armO43A01 (M/Z) mutants , which restores adherens junctions by bypassing the need for Arm to bridge the alpha-catenin and E-cadherin interaction . However , expression of this fusion protein does not restore Arm's nuclear signaling function as indicated by a lack of anterior-posterior patterning , or more specifically , a loss of naked cuticle and a lawn of denticles phenotype[30] ( Fig 1D and E ) . Increasing ArmAA expression levels restored some nuclear Wg signaling activity , as indicated by partial restoration of anterior-posterior patterning , which is shown here by the presence of both naked cuticle and cuticle with denticles ( Fig 1F and 1G ) . More importantly , increased ArmAA expression revealed a striking new phenotype characterized by the random polarization of denticles ( Fig 2C ) . Previously observed arm loss-of-function phenotypes do not appear to have this level of denticle disorganization ( Fig 1I-J , Fig 2B and Table 1 ) . Two pieces of evidence indicated that this denticle disorganization phenotype was due to a lack of functional Arm at the adherens junction , and that ArmAA was competent to transduce the Wg signal in the nucleus . First , in armXM19 (M/Z) mutants , the truncated ArmXM19 protein does not activate the Wg signal in the nucleus , but does retain function in the adherens junction[31] . Expression of ArmAA in armXM19 (M/Z) mutants lead to an essentially wild-type cuticle in terms of both patterning and denticle organization , ( Fig 1H and I ) indicating that ArmAA must fulfill the required role of Arm in transducing the nuclear Wg signal . Second , ArmAA must be competent in transducing the nuclear Wg signal , because a drastic increase in its levels through the removal of the negative Wg pathway regulator zw3 ( in addition to loss of endogenous arm function , or an armXM19 , zw3 (M/Z) double mutant ) leads to a completely naked cuticle , a hallmark of constitutive Wg signaling activation ( Fig 1K-M ) [29] . Therefore , we conclude that phosphorylaTION of Thr111 and Thr121 in Arm is likely to be required for the proper function of Arm at the adherens junction , but not in the nucleus , and perturbation of this PHOSphorylation leads to disorganized planar polarity of the denticles . To further demonstrate that this disorganized denticle phenotype was a defect in planar organization ; we compared the orientation of denticles in various genotypes . As described above , wild-type denticles are highly organized and almost always point toward the anterior or posterior of the embryo ( Fig 1B and 2A ) . Therefore , denticles that point away or toward the midline ( the dorso-ventral direction ) are mispatterned . We approached this problem both qualitatively by scoring the phenotype ( Fig. 2 shows denticles colored according to their orientation with blue and orange representing D/V oriented denticles ; Table 2 : . . . . . . . . 1-12 offers a summary of various genotypes scored qualitatively ) and quantitatively by counting denticles that are mispolarized ( Table 1 ) . These experiments showed that in wild-type and weak arm loss-of-function mutants , most denticles point correctly ( 945+-15% and 855+-18% respectively ) . In contrast , in armO43A01 (M/Z) embryos expressing ArmAA , only about half of the denticles point correctly ( 480+-56 ) . To determine the cell biological basis of this defect , we investigated the epithelia that will produce denticles . Immunofluorescence studies during late embryogenesis revealed that in wild-type embryos , ventral epidermal cells that will produce denticles adopt different cell shapes than those that will produce naked cuticle . Denticle-producing cells are small and rectangular , whereas naked cuticle cells are large and amorphous . Further , the Actin foci that will eventually become denticles are almost always localized to the posterior margin of the cell ( Fig 3A-D and [14] ) . Interestingly , we found that Arm is asymmetrically localized predominantly to the dorsal or ventral ( D/V ) sides of the denticle-producing cells ( Fig 3B and [14] ) . In ArmAA embryos , the Actin foci are localized randomly ( Fig 3E ) and the D/V polarization of Arm is disrupted ( Fig 3F ) . Taken together , the denticle disorganization , the failure of epidermal cells to take on the correct shapes , the improper asymmetric membrane polarization of Arm , and the failure of Actin foci to be asymmetrically localized to the posterior margin of the epidermal cells in ArmAA expressing embryos indicates that threonine phosphoryration of Arm is likely to be required for proper planar polarization in the epidermis .