Dishevelled Associated Activator of Morphogenesis


Activated DAAM interferes with cuticle patterning in tracheal cells and transforms the cuticle pattern of the fusion cells

Truncated formins consisting only of the C-terminal FH1 and FH2 domains behave as constitutively active forms in many contexts. To test whether DAAM activity is regulated in a similar manner, UAS-C-DAAM (carrying a truncated DAAM encoding the C-terminal 637 amino acids including the FH1 and FH2 domains transgenic flies) were generated. Strikingly, C-DAAM expressed in the trachea (with the btl-Gal4 driver) caused distinct phenotypic effects in the fusion cells and the regular tracheal cells (Matusek, 2006).

After the cuticle is laid down in the tracheal system, fusion cells are easily distinguished from others because these narrow, doughnut-shaped cells have a characteristic granulated cuticle pattern that is very distinct from the parallel running apical ridges. The same difference can be found at the level of actin organization, as apical actin is also concentrated into a granular pattern. Significantly, anti-DAAM staining in wild type revealed that fusion cells, unlike the normal tracheal cells, do not express DAAM. When C-DAAM is expressed in fusion cells it induces a dramatic change in the actin organization and cuticle pattern, causing strong actin accumulation at the apical surface. However, most of the actin is found in largely unorganized bundles, although the typical granular pattern is barely visible. In addition, such fusion cells are often wider than their wild-type counterparts, and their granular cuticle pattern is transformed towards a stripy pattern, partly resembling the taenidial folds of normal tracheal cells. However, when full-length DAAM was expressed in the fusion cells (or elsewhere in the tracheal system) no phenotypic effects were detected (Matusek, 2006).

Several conclusions can be drawn from these data. First, C-DAAM behaves as an activated formin, consistent with its domain structure and showing that the activity of DAAM is regulated by N-terminal sequences like the DRF subfamily. Second, although actin cables polymerized by C-DAAM failed to organize into such perfect rings as found in wild-type tracheal cells, C-DAAM plays an instructive role in cuticle secretion by polymerizing actin cables that are sufficient to change the cuticle pattern in a DAAM non-expressing cell towards the pattern characteristic of DAAM-expressing cells. Third, the mere presence of DAAM is not sufficient to change the cuticle pattern of fusion cells, only the activated form can do that, suggesting that fusion cells lack the activator(s) of this formin (Matusek, 2006).

Overexpression of C-DAAM in normal tracheal cells (where the endogenous wild-type DAAM is expressed) resulted in severe impairment of taenidial fold formation and actin organization in a qualitatively similar manner to the absence of DAAM. Recent results indicated that formins normally act in dimeric or multimeric forms. It has also been demonstrated that homotypic protein interaction of the mammalian formin, Fhos, is mediated by the FH2 domain, and an FH1-FH2 (C-DAAM equivalent) Fhos fragment is able to bind the full-length protein. Therefore, it was possible that the btl-Gal4/UAS-C-DAAM phenotype is, at least partly, caused by an antimorphic effect of C-DAAM by interfering with the wild-type protein through dimerization. In that case, it is expected that increasing wild-type DAAM levels will suppress the btl-Gal4/UAS-C-DAAM phenotype (Matusek, 2006).

To test this hypothesis, btl-Gal4 driven co-expression of FL-DAAM and C-DAAM was examined. This caused a strong suppression of the btl-Gal4/UAS-C-DAAM phenotype in regular tracheal cells, but not in fusion cells. These data indicate that C-DAAM indeed behaves as an antimorph in regular tracheal cells. In fusion cells, however, the lack of suppression by expressing FL-DAAM is consistent with DAAM having no endogenous function in these cells. Because the UAS-Gal4 system usually induces a robust expression, C-DAAM is likely to be in excess when compared with endogenous DAAM in btl-Gal4/UAS-C-DAAM tracheal cells. Thus, the tracheal phenotype in the regular tracheal cells is possibly the sum of two effects: an antimorphic effect through interference with the endogenous protein and a neomorphic effect through an excess of activated C-DAAM, which increases actin polymerization rate but fails to organize actin bundles properly. An alternative hypothesis to explain that C-DAAM overexpression leads to similar phenotypic effects to the loss of DAAM, could be that the amount of F-actin produced by C-DAAM exceeds the actin organizing capacity of endogenous DAAM. Since higher levels of apical actin could not be detected in these tracheal cells, the first hypothesis is favored (Matusek, 2006).

The precise mechanism by which C-DAAM impairs actin organization and cuticle patterning in tracheal cells is unclear. It is remarkable that although activated DAAM is lacking most of the formin regulatory domains, it was specific and only disrupted cuticle patterning without affecting other biological processes in the trachea. By comparison, expression of a constitutively activated form of dia (DiaCA), the most closely related Drosophila formin to DAAM, destroys the normal branching pattern of the tracheal tubes. In addition, dorsal trunk fusion is often blocked and tracheal cuticle is not secreted. Thus, it appears that DiaCA has an earlier effect on trachea development than C-DAAM. Together, these results suggest that DAAM carries a highly specific trachea function that is restricted to the assembly and organization of apical actin cables, which direct cuticle patterning, with most of the functional specificity coming from the C-terminal FH1-FH2 domains (Matusek, 2006).

DAAM interacts with RhoA and Src family non-receptor tyrosine kinases

To gain insights into the regulation of DAAM and to identify potential partners that work together with DAAM to organize apical actin in tracheal cells, a genetic interaction test was carried out with a panel of cytoskeleton regulators known to be expressed in the Drosophila respiratory system. The weak hypomorphic DAAMEx1 allele was used that exhibits a mild phenotype, and thus appears to be the most appropriate for dominant genetic interaction assays. This approach identified RhoA, and two Src family non-receptor tyrosine kinases, Src42A and Tec29, as strong enhancers of the DAAMEx1 phenotype. To test whether these interactors have tracheal cuticle patterning phenotypes on their own, the trachea of their homozygous mutant embryos and larvae were examined. In agreement with the observation that btl-Gal4 driven expression of dominant-negative RhoA (RhoAN19) blocks lumen formation, it was found that absence of RhoA arrests tracheal development before dorsal trunk fusion takes place. Therefore, it was not possible to assess directly the effect of RhoA single mutants on apical actin organization and/or cuticle structure. However, although some of the very few hatching Src42A and Tec29 mutant larvae displayed a tracheal necrosis phenotype, others displayed a superficially normal-looking tracheal tree, where cuticle secretion has been completed. These larvae die as first instar, and, strikingly, exhibit an abnormal taenidial fold pattern similar to that of DAAM mutant larvae. Although the Tec295610 and Src42AE1 tracheal phenotypes are weaker than that of the DAAM null allele, consistent with the conclusion that apical actin organization determines the taenidial pattern, these mutations also affect apical actin bundles in the tracheal system. Thus, these results indicate that Src42A and Tec29 are involved in taenidial fold patterning and during this process probably act together with DAAM. Significantly, the FH1 domain is known to bind SH3 proteins, and the Src family kinases have been implicated in several forming-related processes. Additionally, it was found that the RhoA, Src42A and Tec29 mutations not only enhance the tracheal cuticle defects caused by DAAM, but also decreased the viability of the semilethal DAAMEx1 allele, suggesting that these proteins may work together in other tissues as well. The observation that RhoA is an enhancer of DAAMEx1 further supports the model that DAAM family formins are regulated by Rho GTPases, similar to the regulation of the DRF subfamily. These data are also consistent with the previous report demonstrating that RhoA is a direct binding partner of human DAAM1 (Habas, 2001). Surprisingly, however, DAAMEx1 does not show a genetic interaction with dsh, the Drosophila homologue of Dvl, another protein found to bind human DAAM1 (Habas, 2001). Although this result does not exclude the possibility that DAAM activation in the tracheal cells normally requires Dsh binding, a detailed analysis of the trachea function of wg signaling, including dsh, failed to reveal a tracheal cuticle defect, suggesting that Dsh is not involved in DAAM regulation in the trachea (Matusek, 2006).

To further investigate the regulatory relationship between DAAM and its genetic interactors, whether the btl-Gal4/UAS-C-DAAM phenotype is sensitive to the gene dose of RhoA, Src42A and Tec29 was examined. This epistasis analysis indicated that while RhoA did not modify the effect of C-DAAM expression, Src42A and Tec29 dominantly suppressed the respective cuticle defects. These data are consistent with the model that RhoA is an upstream regulator of DAAM, whereas the non-receptor tyrosine kinases act downstream of or in parallel to it. Hence, these results further support the view that the DAAM family formins are regulated by Rho GTPases. With respect to non-receptor tyrosine kinases, the data support similar conclusions to those of previous reports, demonstrating that mouse Dia1 and mouse Dia2 act upstream of Src in the regulation of actin dynamics, and that human DIA2C is required for Src activation (Gasman, 2003) during the process of endosome regulation (Matusek, 2006).

Dishevelled Associated Activator of Morphogenesis: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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