BIOLOGICAL OVERVIEW

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. It remains unknown how this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology. Drosophila Rho-kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Mizuno, 1999 and Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

The rok transcript is ubiquitous in all stages of embryogenesis and in imaginal discs. Drok² mutants die before developing into wandering 3^rd instar larvae. To investigate the function of rok in the morphogenesis of adult tissues, somatic Drok² clones generated by mitotic recombination were examined. Homozygous Drok² clones in the eye are of similar size as their wild-type siblings (twin spots), indicating no gross defects in proliferation or survival of Drok² mutant cells. However, about 50% of ommatidia exhibit an increase or decrease in photoreceptor numbers. A change of photoreceptor number has also been reported in eye clones homozygous for a hypomorphic RhoA allele. Of those ommatidia with the correct number of photoreceptors, 60% were misrotated, whereas in control clones all ommatidia were properly rotated. These phenotypes resemble those of planar cell polarity (PCP) mutants such as frizzled, dishevelled, and RhoA, suggesting that rok may also participate in the signaling cascade controlling PCP. Unlike fz and dsh, however, very few ommatidia showed aberrant chirality (Winter, 2001).

The function of rok in the wing was determined, where PCP is exhibited at a single cell level. In wild-type wings, each cell produces a single, distally oriented wing hair, while in Drok² adult wing clones, greater than 70% of the cells produce multiple hairs. In contrast to most known PCP genes, including fz and dsh, which exhibit both stereotypical orientation and multiple hair defects, most hairs within Drok² clones maintain a largely distal orientation (Winter, 2001).

To investigate the nature of the rok phenotype during the development of wing cell polarity, phalloidin was used to examine the F-actin distribution in Drok² clones during prehair initiation. Mutant clones were positively marked using the MARCM strategy. In control clones or in heterozygous tissue neighboring Drok² mutant clones, each wing cell generates a single F-actin bundle from its distal vertex, extending toward its distal neighbor. However, in Drok² clones, a majority of the cells generate more than one F-actin bundle. The F-actin-based prehairs always initiate at the cell periphery. Notably, the majority of the prehairs maintain a roughly distal orientation (in the case of multiple hairs, the vector average remains distal). The similarities between the pupal and adult clonal phenotypes indicate that the multiple hair phenotype of adult Drok² clones is likely the result of failure to restrict F-actin bundle assembly to a single site during prehair formation. The multiple hair/actin phenotype is seen only in mutant cells and never in neighboring heterozygous or wild-type cells, indicating that rok acts in a cell-autonomous manner (Winter, 2001).

The similarity of the Drok² clonal phenotype in the wing to aspects of the phenotypes of fz, dsh, and RhoA led to the hypothesis that Rok may act downstream of Fz/Dsh. To assess the genetic interactions among these genes, the multiple hair phenotype was quantitated in a defined region: the ventral surface of the proximal-anterior region of the wing. Use was made of the dsh¹ allele, which is defective for PCP function without affecting Wg signaling. In dsh¹ hemizygous males, an average of 16.8 cells with multiple hairs (12% of the cells) are present in this region. When Rok is overexpressed via a tubP-Drok transgene in the dsh¹ hemizygous background, the average number of cells exhibiting multiple hairs is reduced by more than 7-fold to 2.3 per wing region. tubP-Drok expression in a wild-type genetic background does not give rise to any obvious phenotypes. Suppression of the dsh¹ multiple hair phenotype by tubP-Drok expression could also be seen when F-actin-based prehairs were visualized in the pupal wing. In contrast to the suppression of the dsh¹ phenotype by overexpression of Rok, reduction of rok dosage by 50% (assuming that Drok² is null) results in a 2.5-fold increase in the number of multiple hair cells (Winter, 2001).

Genetic interactions between rok and fz were examined in a different assay. The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, since both overexpression and loss of function of these genes result in polarity defects in the eye and the wing. Overexpression of Fz 30 hr after puparium formation (APF) produces primarily a multiple hair phenotype that is suppressed by dsh¹ heterozygosity. Similarly, reducing the wild-type copy number of RhoA and rok by half suppresses the phenotype by 2- to 2.5-fold. Taken together, these experiments suggest that Rok functions downstream of Fz/Dsh in restricting the number of F-actin-based prehairs (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh¹ multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh¹ mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh¹ phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh¹ background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqh^AX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh¹ phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).

The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh¹ mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh¹ mutants (Winter, 2001).

Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip⁰²⁹⁵⁷, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip⁰²⁹⁵⁷ wing cells possess multiple F-actin prehairs (Winter, 2001).

Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).

Reduction of ck activity potently suppresses the dsh¹ multiple hair phenotype. This result contrasts with the result that zip¹ enhances the dsh¹ multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).

Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh¹, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).

To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok² clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh¹, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh¹ mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh¹. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

Mammalian and C. elegans Rho-kinase/ROCK have been implicated in regulating actin stress fiber formation in fibroblasts (Leung, 1996; Amano, 1997; Ishizaki, 1997) or changes in hypodermal cell shape during embryonic development (Wissmann, 1997). How Rho-kinase/ROCK is regulated by an extracellular signal in a physiological process is unknown. Drosophila Rho-kinase has been shown in this study to be required for restricting the number of sites of F-actin bundle assembly in response to activation of the Fz/Dsh pathway. Together with the previously established genetic link between Fz/Dsh and RhoA, a molecular pathway from Fz/Dsh to the regulation of actin structure has been established. Proteins that physically mediate the interaction between Fz and Dsh, and between Dsh and RhoA, remain to be identified (Winter, 2001 and references therein).

The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).

Several other genes have been implicated in regulating wing hair number based on their loss-of-function phenotypes, including inturned (in), fuzzy(fy), and multiple wing hair (mwh). These gene products are thought to participate in a distinct branch of the polarity signaling pathway for the following reasons: (1) they each have significant effects on both hair orientation and hair number, they are unlikely to function downstream of Rok; (2) expression of the phosphomimetic Sqh-E20E21, or the nonactivatable Sqh-A20A21, in mwh or in mutant backgrounds does not modify their phenotypes (Winter, 2001); (3) while Rok functions in the eye as well as in the wing, In, Fy, and Mwh have no apparent role in PCP signaling in the eye (Winter, 2001).

The misorientation of ommatidia in Drok² eye clones is also consistent with its functioning in the Fz/Dsh/RhoA pathway. It is conceivable that Rok may act as an effector of RhoA in regulating the process of ommatidial rotation. Alternatively, loss of Rok function could lead to a disruption of the actin cytoskeleton and a consequent loss of the appropriate cell -- cell contacts that support intercellular signaling essential for ommatidial rotation. Although rok mutants exhibit misrotation of ommatidia, they acquire the proper chiral form. Determination of ommatidial rotation may reflect a Rok-dependent subset of PCP signaling in the eye that is, in some way, molecularly analogous to the subset of PCP signaling in the wing, as reflected in the regulation of hair number (Winter, 2001).

Biochemical and cell culture studies have identified many potential substrates for Rho-kinase/ROCK (reviewed in Amano, 2000) The relative contribution of phosphorylation of these different substrates in mediating various functions of Rho-kinase/ROCK is unclear. Strong genetic evidence has been provided that phosphorylation of MRLC at key residues (Ser-21 in Drosophila, equivalent to Ser-19 in mammals) is a critical output for Rok signaling. Phosphomimetic MRLC expression not only suppresses the multiple wing hair phenotype, but also lethality, of Drok² mutants. Biochemical and immunostaining experiments further demonstrate that Rok modulates MRLC phosphorylation in vivo. Previous work has suggested that Rok regulates phosphorylation of MRLC, both by direct phosphorylation and also by phosphorylation and inhibition of the myosin binding subunit of myosin phosphatase (MBS: see Drosophila Myosin binding subunit) (Amano, 2000). However, at present the relative contribution of direct phosphorylation by Rok of MRLC, versus phosphorylation of MBS that indirectly affects MRLC phosphorylation is not known (Winter, 2001).

The direct genetic interactions between dsh¹ and various sqh transgenes further substantiates the notion that regulation of MRLC phosphorylation is not only a major output of Rok signaling, but also of Fz/Dsh signaling to the actin cytoskeleton in regulating hair number (Winter, 2001).

By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).

It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).

In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).

Recent evidence suggests that vertebrate homologs of Dsh and Fz regulate cell morphogenesis using a pathway(s) analogous to that of the Drosophila PCP pathway. The findings described in this study of Rok raise the possibility that the Fz/Dsh cytoskeletal pathway may also be at work in regulating cell polarity in vertebrates, including the stereocilia formation in the inner ear (Winter, 2001).

GENE STRUCTURE

cDNA clone length - 6259

Bases in 5' UTR - 1022

Exons - 10

Bases in 3' UTR - 1064

PROTEIN STRUCTURE

Amino Acids - 1390

Structural Domains

The Drosophila homolog of Rho-kinase/ROK alpha, Rok, which has conserved the basic structural feature of Rho-kinase/ROK alpha consisting of the N-terminal kinase, central coiled-coil and C-terminal pleckstrin homology (PH) domains, has been identified (Mizuno, 1999).

Rok structure has been compared with that of rat ROK-beta. Both proteins are composed of a conserved N-terminal domain, a serine/threonine kinase domain, a large coiled-coil domain with an embedded Rho-binding domain, and a C-terminal Plekstrin-homology (PH) domain split by a Cys-rich domain (Winter, 2001).

date revised: 16 April 2001

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