Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. This study analyzes a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, it is shown that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. The results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system (Matusek, 2006).

Proteins of the formin family play key roles in the regulation of the cytoskeleton. Although the connection between formins and microtubules is less well understood, formins are implicated in a large number of actin-based processes, including cell polarization, division, movement, stress fiber formation and vesicular trafficking. Recent work suggests that formins catalyze the assembly of unbranched actin structures and nucleate actin filaments directly (Matusek, 2006).

Although all members of this highly conserved family are defined by the presence of the formin homology domain 2 (FH2), which is both necessary and sufficient to nucleate actin in vitro, formins are multi-domain proteins that contain several other conserved sequences as well. The FH2 domain is usually flanked on the N-terminal side by a proline-rich FH1 domain that can serve as a docking site for the G-actin-binding protein profilin. In addition, the FH1 domain also binds WW domains and SH3 domains, including those of the Src family. A distinct formin subfamily, the DRF group (Diaphanous related formins: see Drosophila Diaphanous) has the ability to interact with an activated Rho GTPase through an N-terminal GTPase-binding domain (GBD). This binding alleviates the autoinhibitory interaction between the GBD and the short C-terminal DAD domain (Diaphanous autoregulatory domain) (Alberts, 2001). Crystallographic analysis of the N-terminal part of mouse Dia1 provides a structural basis of this regulation and has identified several other functional domains within this part of the protein, including a dimerization domain (DD), (Otomo, 2005a; Rose, 2005). In addition, the crystal structure of C-terminal formin domains reveals that the FH2 domain also forms a dimer required for actin nucleation and processive filament capping (Otomo, 2005b; Shimada, 2004; Xu, 2004), strongly suggesting that native formins act in dimeric forms (Matusek, 2006).

Recently, a novel formin subtype, DAAM (Dishevelled-associated activator of morphogenesis), has been identified and implicated in planar cell polarity signaling during Xenopus gastrulation (Habas, 2001). The polarized orientation of cells within the plane of a tissue, or planar cell polarization (PCP), is an important aspect of cellular differentiation and is often necessary to the formation of functional organs. Genes controlling PCP have been extensively studied in Drosophila, revealing a crucial role for frizzled (fz) signaling through dishevelled (dsh) during the course of PCP establishment. The downstream components of the Fz/PCP pathway include RhoA, Drok, the JNK cascade and several other genes, most of which act tissue specifically. Many of the genes involved in PCP signaling are also required for polarized morphogenetic cell movements such as convergent extension during early vertebrate embryogenesis suggested that the novel FH2 protein Daam1 is required for convergent extension is Xenopus embryos and that Daam1 might function as a bridging factor between Dsh and RhoA. Moreover, Wnt/Fz-mediated activation of RhoA appeared to depend on Dvl (a Dsh homolog) and Daam1 (Habas, 2001). However, in contrast to this model, previous work has provided evidence that formins act as Rho effectors downstream of the Rho GTPases (Matusek, 2006).

To gain further insights into the function of this novel class of FH2 proteins, the single Drosophila member of the DAAM family was analyzed. Phenotypic analysis of DAAM mutants showed that it plays either no role or possibly a redundant role in PCP establishment in Drosophila. However, evidence was found that DAAM is involved in the regulation of the actin cytoskeleton in several different tissues, including the tracheal system. The Drosophila tracheal network is one of the best characterized model systems of branching morphogenesis. During the first phase of tracheal development, the primordial cells invaginate in each embryonic hemisegment from the epidermis, migrate and undergo cell shape changes to form the primary branches. Subsequently, some tracheal branches fuse with an adjacent branch to build up a continuous tubular network. This process is mediated by fusion cells located at the branch tips, which recognize each other in the adjacent hemisegments and become doughnut shaped, forming a lumen that connects the two branches. Before the end of embryogenesis, tracheal cells secrete a cuticle on their apical, luminal surface that protects the larvae from dehydration and infection. The tracheal cuticle is distinguished from the epidermal cuticle by the presence of cuticle ridges, often called taenidial folds that are thought to prevent the collapse of tracheal tubes while allowing them to expand and contract along their length (Matusek, 2006).

Drosophila DAAM is required to organize an array of parallel running actin cables beneath the apical surface of the tracheal cells that define the taenidial fold pattern of the cuticle. The actin ring pattern corresponds exactly to that of the taenidial fold pattern, and it is proposed that the actin rings organized by DAAM define the position of taenidial fold formation. The genetic interaction and epistasis data are consistent with a model that DAAM activity is regulated by RhoA. In addition, DAAM works together with the non-receptor tyrosine kinases Src42A and Tec29 to regulate the actin cytoskeleton of the Drosophila tracheal system (Matusek, 2006).

The basic structure of the insect tracheal system is a highly conserved tubular network in every species. The most important function of this network is to allow oxygen flow to target cells. Thus, tracheal tubes need to be both rigid enough, to ensure continuous air transport, and flexible enough along the axis of the tubes, to prevent the break down of the tube system when body parts or segments move relative to each other. These requirements are mainly ensured by the tracheal cuticle, which covers the luminal surface of the tubes and displays cuticle ridges (making the overall structure similar to the corrugated hose of a vacuum cleaner). Analysis of DAAM mutants provides the first direct evidence that this hypothesis is correct. The data demonstrate that in the absence of DAAM the taenidial fold pattern is severely disrupted, often leading to the collapse of the tubes and to discontinuities in the tubular network. In addition, the analysis revealed that the remarkably ordered cuticle pattern, displayed in the wild-type trachea tubes, depends on DAAM-mediated apical actin organization. Apical actin is organized into parallel-running actin cables, much the same way teanidial folds run in the cuticle. Significantly, the formation of these actin bundles precedes the onset of cuticle secretion, and the number and phasing of the actin rings correspond exactly to that of the taenidial folds in the cuticle. Thus these studies revealed a novel aspect of apical actin organization in the tracheal cells that has not been appreciated before (Matusek, 2006).

The DAAM gene encodes a novel member of the formin family of proteins, involved in actin nucleation and polymerization. Consistent with this, DAAM is colocalized with apical actin in the tracheal cells, and the activated form of DAAM is able to induce actin assembly when expressed in tracheal cells and in other cell types. In DAAM mutant tracheal cells, apical actin is still detected, albeit at a somewhat lower level than in wild type, but the bundles formed in the mutant are much shorter and thinner than in wild type, and often appear to be crosslinked to each other. Most strikingly, global actin organization is almost completely lost, although some local order can still be detected. Remarkably, the cuticle pattern in mutant tracheal cells still follows the underlying aberrant actin pattern. Overall, in DAAM mutants, both the tracheal cuticle and the apical actin pattern resemble a mosaic of locally ordered patches that failed to be coordinated and aligned with each other and the axis of the tracheal tubes. It is thus proposed that the apical actin bundles play a key role in patterning the tracheal cuticle by defining the place of taenidial fold formation. Regarding the function of DAAM, the results suggest that the major role of this formin in the tracheal cells is to organize the actin filaments into parallel running actin rings or spirals instead of simply executing the well characterized formin function related to actin assembly. However, whether this is a direct effect on actin organization, and thus represents a novel formin function, needs to be further elucidated. An alternative model could be that DAAM is primarily required for actin polymerization but tightly coupled to an actin 'organizing' protein. In such scenario, the polymerization activity should be a redundant requirement, whereas the link to the organizing protein would be a DAAM-specific function, thereby explaining the presence of unorganized actin bundles in DAAM mutant tracheal cells (Matusek, 2006).

In the case of the main tracheal airways, which are multicellular along their periphery, it is striking that in wild type the run of the actin bundles is perfectly coordinated across cell boundaries. In addition, the run is always perpendicular to that of the tube axis. How does DAAM ensure the coordination of these two aspects of actin organization? Because the DAAM protein and the apical actin cables are both found at the level of the adherens junctions, it is possible that DAAM regulates the coordination of the actin cables at the cell boundaries through a direct interaction with junctional protein complexes. However, other explanations are also possible, and further experiments will be required to elucidate the molecular mechanism of this regulatory function. The fact that actin cables normally run perpendicular to the tube axis seems to suggest that tracheal cells are able to sense a global orientation cue and align their actin bundles accordingly. The nature and source of this cue is unknown, as is the mechanism by which DAAM is involved in the read-out of this signal. Nevertheless, it is interesting that in DAAM and btl-Gal4/UAS-C-DAAM mutant trachea, the main pattern of the cuticle phenotype is often changing from one segment to the other, suggesting that the effect of the 'global' orientation cue is limited to metameric units (Matusek, 2006).

Sequence comparisons of FH2 proteins suggest a close phylogenetic relationship between the DRF, FRL and DAAM subfamilies (Higgs, 2005). Members of these three subfamilies have a high level of conservation in the FH2 domain, and importantly, also in the region of the GBD and DAD domains, suggesting that the FRL and DAAM family formins are also regulated by autoinhibition and RhoGTPases, like the DRFs. Further evidence is presented in support of this view. First, DAAM and RhoA display a strong genetic interaction. Second, C-DAAM (an N-terminally truncated form of DAAM) behaves like an activated form much the same way DRF family formins behave. Third, epistasis experiments with C-DAAM and RhoA suggest that RhoA acts upstream of DAAM. Thus, the data support the model in which DAAM, at least in the Drosophila tracheal system, is regulated by autoinhibition that can be relieved by RhoGTPases (Matusek, 2006).

This conclusion, however, contradicts the observation that human DAAM1 is required for Wnt/Fz/Dvl dependent RhoA activation in cultured cells and that xDaam1 appears to mediate Wnt-11 dependent RhoA activation in Xenopus embryos (Habas, 2001). These results suggested that DAAM functions upstream of RhoA in non-canonical Wnt/Fz-PCP signaling. An explanation for these distinct conclusions might be related to the fact that DAAM, in contrast to xDaam1, does not appear to be required for Fz/Dsh-PCP signaling. Hence, it is possible that the Drosophila ortholog is regulated in the same way as the DRF formins, while the vertebrate family members can be regulated in a different way, once bound by Dsh/Dvl and recruited into PCP signaling complexes (Matusek, 2006).

Genetic interactions with the hypomorphic DAAM^Ex1 allele identified two non-receptor tyrosine kinases, Src42A and Tec29, as strong interacting partners. Although both of these kinases play multiple roles during embryogenesis, single mutants for both affect the tracheal cuticle pattern in a similar way to DAAM. These results suggest that DAAM and the Src family kinases work together to regulate the actin cytoskeleton and cuticle pattern in tracheal cells. Although it is not known whether DAAM physically binds Src42A and/or Tec29, it has been established that the FH1 region of DRFs and other formins can bind SH3 domains, including those of the Src family kinases. In agreement with these data that DAAM acts upstream of Src42A and Tec29 in tracheal cells, cytoskeleton remodeling and SRF activation mediated by mouse Dia1 and mouse Dia2 requires Src activity. Moreover, a recent report suggests that RhoD and human DIA2C regulate endosome dynamics through Src activation, proposing that Src activity is stimulated via human DIA2C dependent recruitment to early endosomes (Gasman, 2003). Similarly, the Limb deformity protein (a formin) interacts with Src on the perinuclear membranes of primary mouse fibroblasts. Based on these examples, it is speculated that in Drosophila tracheal cells the RhoA/DAAM/Src module may not only be required to organize apical actin bundles, but additionally it might represent a link to secretory vesicles and to the regulation of exocytosis. Future studies will be required to test this hypothesis, and to unravel the mechanisms whereby DAAM family formins and Src family kinases contribute to cytoskeletal remodeling in the Drosophila tracheal system and in other tissues (Matusek, 2006).

GENE STRUCTURE

cDNA clone length - 5369 (isoform b)

Bases in 5' UTR - 1168

Exons - 8

Bases in 3' UTR - 739

PROTEIN STRUCTURE

Amino Acids - 1153 (isoform b)

Structural Domains

A recently published phylogenetic analysis (based on sequence comparisons of 101 FH2 domains from 26 eukaryotic species) has concluded that metazoan formins can be grouped into seven major subclasses (Higgs, 2005). This analysis suggested that the DAAM subfamily clearly represents a distinct class. This is consistent with the previous finding that the DAAM subfamily exhibits extensive sequence similarity both within and outside of their highly conserved FH1 and FH2 domains (Habas, 2001). At present, however, very little is known about the in vivo function of the DAAM subfamily. Sequence analysis revealed the presence of a single DAAM ortholog in the Drosophila genome corresponding to the annotated gene CG14622. Although FlyBase predicts that this gene might code for several different transcript classes, this work focused on the predicted CG14622-RB transcript that appears to be encoded by the RE67944 EST clone. Sequencing of this full-length cDNA clone indicated a protein consisting of 1153 amino acids. Further analysis revealed that the ORF contains several conserved domains including an FH1, FH2, GBD and a putative DAD domain characteristic of formins. Based on the overall homology level and the phylogenetic analysis of FH2 domains (Higgs, 2005), the DAAM subfamily appears to be most related to the Dia subfamily, raising the possibility that similar to the DRFs, DAAM formins are also regulated by an autoinhibitory mechanism that can be relieved upon RhoGTP binding (Matusek, 2006).

date revised: 11 September 2006

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