InteractiveFly: GeneBrief

crinkled: Biological Overview | References


Gene name - crinkled

Synonyms - myoVIIA

Cytological map position - 35B8-35B9

Function - bimolecular motor protein

Keywords - cytoskeleton, necessary for auditory organ development, interacts with Wingless pathway mediated aspects of epidermal denticle patterning, wing development

Symbol - ck

FlyBase ID: FBgn0000317

Genetic map position - chr2L:15,044,963-15,057,848

Classification - Myosin and Kinesin motor domain; MyTH4 domain' FERM central domain

Cellular location - cytoplasmic


NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Orme, M. H., et al. (2016). The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles. Nat Commun 7: 10972. PubMed ID: 26960254
Summary:
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. This study found that the Drosophila unconventional myosin Crinkled (Ck) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. The data indicate that CK acts as substrate adaptor, recruiting Shaggy46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, these results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling event.
Li, T., Giagtzoglou, N., Eberl, D., Nagarkar-Jaiswal, S., Cai, T., Godt, D., Groves, A.K. and Bellen, H.J. (2016). The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals. Elife [Epub ahead of print]. PubMed ID: 27331610
Summary:
Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, this study identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. It was shown that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. These observations reveal a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs.


BIOLOGICAL OVERVIEW

The specification of the body plan in vertebrates and invertebrates is controlled by a variety of cell signaling pathways, but how signaling output is translated into morphogenesis is an ongoing question. This study describes genetic interactions between the Wingless (Wg) signaling pathway and a nonmuscle myosin heavy chain, encoded by the crinkled (ck) locus in Drosophila. In a screen for mutations that modify wg loss-of-function phenotypes, multiple independent alleles of ck were isolated. These ck mutations dramatically alter the morphology of the hook-shaped denticles that decorate the ventral surface of the wg mutant larval cuticle. In an otherwise wild-type background, ck mutations do not significantly alter denticle morphology, suggesting a specific interaction with Wg-mediated aspects of epidermal patterning. This study shows that changing the level of Wg activity changes the structure of actin bundles during denticle formation in ck mutants. It was further found that regulation of the Wg target gene, shaven-baby (svb), and of its transcriptional targets, miniature (m) and forked (f), modulates this ck-dependent process. It is concluded that Ck acts in concert with Wg targets to orchestrate the proper shaping of denticles in the Drosophila embryonic epidermis (Bejsovec, 2012).

The ventral epidermis of Drosophila embryos is a well-established system for studying cell fate specification. At the end of embryogenesis, epidermal cells secrete a patterned array of cuticular structures that reflect the cell identities acquired in the epidermis at earlier stages of development. On the ventral surface of the larval abdomen, eight segmental belts of hook-shaped denticles alternate with expanses of flat, or naked, cuticle. Each belt contains roughly six rows of denticles, with each row displaying a characteristic size, shape and polarity. These distinct morphologies indicate unique positional values, at least some of which are imparted by signal transduction from the highly conserved Wg/Wnt growth factor pathway. During early embryogenesis, a cascade of transcription factors leads to activation of wg gene expression in segmental stripes that lie within the zone of cells that will secrete naked cuticle. Ectopic overexpression of wg across the segment, or hyperactivation of downstream components in the Wg signaling pathway, eliminates the denticle belts. Conversely, loss of wg activity causes all ventral epidermal cells to secrete denticles. The diversity of denticles is also reduced in wg null mutants, with most resembling the large denticles typical of the fifth row of the wild-type belt. Thus Wg signaling controls not only the segmental specification of naked cuticle expanses, but also generates the diversity of cell fates that give rise to the uniquely shaped denticles within the denticle belt (Bejsovec, 2012).

Denticles are formed by bundles of actin that accumulate apically and push out the apical membrane as they elongate. Incipient denticles first can be visualized as apical actin condensations in the ventral epidermal cells of stage 13 embryos, at roughly 10 hours after egg-laying (AEL). These actin condensations form preferentially along the posterior edge of the columnar epithelial cells, and over the next 2 hours become increasingly more organized and begin to elongate; during this elongation phase, microtubules become enriched at the base of the denticle and also within the core of the growing denticle. The mechanism by which the distinctive shapes of the denticles are specified is not well understood, but it requires Wg signaling between 4 and 6 hours AEL (Bejsovec, 2012).

This early phase of Wg activity stabilizes expression of engrailed (en) and its target, hedgehog (hh), in the adjacent row of cells. Wg and Hh signaling together control the expression of Serrate and rhomboid, which activate the Notch and EGF pathways, respectively, in defined rows within the segment; these gene activities are required to specify the diverse denticle types characteristic of a wild-type denticle belt (Bejsovec, 2012).

The organization of the actin-based denticle precursors and their transition to cuticular elements is directed by a set of structural proteins whose expression is controlled by the Wg-regulated transcription factor, Shaven-baby (Svb). Wg signaling represses svb, restricting its ventral expression to the domain of cells fated to secrete denticles. Ectopic svb expression in the naked region of the embryonic epidermis drives formation of apical actin extensions and subsequent production of ectopic denticles. A number of downstream targets of Svb have been identified; these include genes such as singed (sn) and forked, which encode known actin-remodeling proteins, and miniature, which encodes a membrane-anchored extracellular protein thought to mediate interaction between the cell membrane and the cuticle. However, the question remains as to how these structural proteins are deployed to form the distinct morphologies characteristic of each row of denticles. This study shows that the cytoplasmic myosin, Crinkled, interacts genetically with the Wg signaling pathway and plays a role in organizing the final shapes of the denticles during epidermal development (Bejsovec, 2012).

A genetic screen for modifiers of wg mutant phenotypes revealed an unexpected interaction between Wg signaling and the cytoplasmic myosinVIIA homolog, Ck, in shaping the denticles at late stages of embryonic development. Like other myosins, Ck/myosinVIIA has a typical actin-binding/ATPase head domain that mediates movement along actin filaments. However, the carboxy-terminus of Ck/myosinVIIA is unique in containing an SH3 domain and two FERM domains, which are shared by band 4.1, ezrin, radixin, moesin -- a family of proteins that link the actin cytoskeleton to membrane spanning proteins. These motifs are consistent with a role near the plasma membrane, possibly interacting with cell-surface receptors and/or adherens junctions. This raises the possibility that Ck may be involved in the association between the actin bundles of the incipient denticles and the apical membrane, where it could link the actin cytoskeleton to extracellular components of the cuticle through transmembrane proteins such as Miniature. Either loss or gain of function for the Wg target gene svb alters denticle morphology in the ck mutant background. Therefore, it is proposed that Ck myosin may help to distribute the products of some Svb target genes, such as Miniature, and thus facilitate the final morphology of the developing denticle. Genetic data suggest that wild-type Ck provides a buffering mechanism for the incorrect Svb target levels that accumulate in a wg null mutant (Bejsovec, 2012).

Wg signaling is most commonly associated with specifying the naked cuticle cell fate, but its other role in generating diverse denticle morphologies allowed requirements for Ck function in this process to be detected. The morphogenesis role requires lower levels of Wg signaling, as evidenced by weak mutations such as wgPE2 that can generate diversity but cannot specify naked cuticle cell fate. The finding that levels of svb and its target genes influence denticle morphology suggests that Wg signaling may generate denticle diversity in conjunction with Notch and EGF signaling by producing subtly graded differences in svb expression that are below the limits of current detection methods. Temperature shift experiments suggest that this is a continuing, independent role for Wg signaling, as it is detected after the point at which Wg input regulates the pattern of Serrate and rhomboid expression. It is proposed that late Wg signaling helps titrate the synthesis of svb target gene products to optimal levels required for shaping the denticles. The ck mutant provides a sensitized background that may allow further investigation of this possibility. The enhancement of denticle morphology defects along the dorsolateral edges of the denticle field also suggests input from dorsoventral patterning pathways, such as Dpp signaling (Bejsovec, 2012).

MyosinVIIA in humans is known to play a crucial role in hearing (reviewed by Hasson, 1999; Maniak, 2001; Petit, 2001; Dror, 2009). Stereocilia on the hair cells of the inner ear transduce the mechanical stimulation of sound waves into electrical impulses. Stereocilia are stabilized by bundles of actin filaments and microtubules, much like the denticles and bristles that decorate the fly epidermis. Mutations in the human myosinVIIA are associated with Usher syndrome, the most common hereditary deafness/blindness disorder, which results in disorganized stereocilia that cannot transduce sound. The precise role of myosinVIIA in organizing and maintaining these structures is as yet unknown. However, ck mutants in the fly also are deaf, and show morphological changes in the auditory sensory structures (Todi, 2005), suggesting that the fly is a powerful model system for exploring this aspect of myosinVIIA function. Indeed, the connection between Ck and Miniature may also be relevant to human hearing disorders. Mutations in α-tectorin, a human protein that shares functional domains with Miniature and organizes extracellular matrix in the cochlea, are associated with hereditary hearing loss (Bejsovec, 2012).

Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development

Myosin VIIA (MyoVIIA) is an unconventional myosin necessary for vertebrate audition. Human auditory transduction occurs in sensory hair cells with a staircase-like arrangement of apical protrusions called stereocilia. In these hair cells, MyoVIIA maintains stereocilia organization. Severe mutations in the Drosophila MyoVIIA orthologue, crinkled (ck), are semi-lethal and lead to deafness by disrupting antennal auditory organ (Johnston's Organ, JO) organization. ck/MyoVIIA mutations result in apical detachment of auditory transduction units (scolopidia) from the cuticle that transmits antennal vibrations as mechanical stimuli to JO. Using flies expressing GFP-tagged NompA, a protein required for auditory organ organization in Drosophila, the role of ck/MyoVIIA in JO development and maintenance was examined through confocal microscopy and extracellular electrophysiology. This study shows that ck/MyoVIIA is necessary early in the developing antenna for initial apical attachment of the scolopidia to the articulating joint. ck/MyoVIIA is also necessary to maintain scolopidial attachment throughout adulthood. Moreover, in the adult JO, ck/MyoVIIA genetically interacts with the non-muscle myosin II (through its regulatory light chain protein and the myosin binding subunit of myosin II phosphatase). Such genetic interactions have not previously been observed in scolopidia. These factors are therefore candidates for modulating MyoVIIA activity in vertebrates. These findings indicate that MyoVIIA plays evolutionarily conserved roles in auditory organ development and maintenance in invertebrates and vertebrates, enhancing understanding of auditory organ development and function, as well as providing significant clues for future research (Todi, 2008).

Mutations in MyoVIIA lead to inner ear transduction anomalies in vertebrates (Ernest, 2000; Gibson, 1995; Weil, 1995). In humans, MyoVIIA mutations cause syndromic and non-syndromic deafness (Liu, 1997a; Liu, 1997b; Weil, 1997). MyoVIIA is expressed by inner ear hair cells 9182663 (Hasson, 1997; Hasson, 1995), where it is important for establishing and maintaining stereocilia organization and for proper auditory transduction 9435277 (Self, 1998; Kros, 2002). MyoVIIA has been proposed to function in stereocilia cohesion and organization by interacting with a number of proteins, yet its precise function in vertebrate hair cells remains unknown (Todi, 2008).

The Drosophila auditory organ, Johnston's Organ (JO) is evolutionarily related to the vertebrate inner ear (Todi, 2004 and references therein). Auditory transduction in Drosophila occurs at the anteriorly placed antennae, each comprising three segments (a1–a3) and a feathery arista. Acoustic stimuli impact the arista to rotate a3 in relation to a2 at the a2/a3 joint, where a3 inserts into a2 (see Schematic of the antenna and JO.). a2/a3 movements stretch JO mechano-sensitive units (scolopidia) within a2. Each scolopidium consists of neurons with ciliated dendrites and support cells. Ciliated dendrites are enclosed by the scolopale space, an ionically separate area produced by the scolopale cell and supported by actin-rich rods with interspersed microtubules (Todi, 2004). Dendrites are apically encapsulated by the dendritic cap, an extracellular structure that anchors the scolopidium into the a2/a3 joint. NompA, a putatively filamentous protein secreted by the scolopale cell, is the only dendritic cap component reported to date (Chung, 2001). The cap cell apically envelopes the scolopale cell and aids in apical attachment (Todi, 2008).

Drosophila antennae develop from the antennal imaginal discs, clusters of undifferentiated cells in the larva. These discs comprise several concentric folds, the central-most one becoming the distal-most arista and sequentially peripheral ones leading to a3, a2 and a1, respectively. During and after head eversion from the pupal thorax the discs evaginate, increase in size and migrate towards their final position. Neuronal staining with the monoclonal antibody 22C10 indicates that JO is formed from three groups of cells that are first detected in the presumptive a2 a few hours before head eversion (Todi, 2008).

The Drosophila orthologue of myoVIIA is encoded by crinkled (ck). Previous work has showed that ck/MyoVIIA is necessary for adult JO organization and function (Todi, 2005). Mutations in ck/MyoVIIA lead to deafness as a result of scolopidial detachment from the a2/a3 joint. This detachment leads to JO disorganization, most likely due to a ck/MyoVIIA role in forming the dendritic cap, which is malformed in mutant flies 15886106 (Todi, 2005). Yet, whether ck/MyoVIIA is developmentally important for JO organization has not been explored. Since cellular export of the dendritic cap component protein, NompA, appears unaffected by mutations in ck/MyoVIIA, this study used GFP-NompA to follow ck/MyoVIIA involvement in JO organization during development. Additionally, proteins were investigated that may affect ck/MyoVIIA to elucidate function in the JO by implementing the power of Drosophila genetics (Todi, 2008).

This study demonstrates that ck/MyoVIIA is acutely required early in the apical attachment of scolopidia, either by directly facilitating dendritic cap extension through the epithelial layer, or anchoring the cap during tensioning and elongation. Furthermore, a continuing requirement was shown for ck/MyoVIIA to maintain attachments throughout adulthood. Finally, evidence is presented that in the JO, ck/MyoVIIA genetically interacts with the non-muscle myosin light chain regulatory protein (Sqh) and the myosin binding subunit of myosin phosphatase (DMBS). The data suggest that the mechanism of ck/MyoVIIA in JO differs from a previously characterized PCP pathway, at least at the level of the components examined (Drok, zip/MyoII. Instead, novel genetic interactions between ck/MyoVIIA and myosin regulatory proteins appear to operate in JO. Whether these tissue-specific interactions reflect deployment of alternative forms of PCP or whether the JO function of ck/MyoVIIA is unrelated to PCP functions remains to be established. The data are consistent with the idea that similar genetic pathways may affect auditory transduction in vertebrates, and draw attention to the complexity of myosin regulatory mechanisms in different cell types that still require dissection in Drosophila and vertebrate models of deafness (Todi, 2008).

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II (Zipper in Drosophila) plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). Genetic interaction studies were confirmed and extended to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, it was demonstrated that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms -- by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development (Franke, 2010).

The array of phenotypes caused by the directed expression of an allelic series of myosin II truncation constructs shows a variety of new roles for zip/MyoII in wing morphogenesis and confirms expected roles. Perturbing zip/MyoII function resulted in: ectopic and/or expanded vein and crossveins, the formation of multiple wing hairs instead of a single hair, the branching of individual wing hairs and the loss of adhesion between the dorsal and ventral wing epithelial cell sheets. An expected role of myosin II in cytokinesis was very likely the cause of reduced cell proliferation. Truncation allele expression in the thorax indicates these new roles for myosin II are not restricted to wing morphogenesis and likely extend to the correct morphogenesis of actin-based protrusions (hairs or setae; bristles or microchaetae and macrochaetae) throughout the fly (Franke, 2010).

During wing development, expression of these dominant negative truncation alleles cause wing blister phenotypes, which are also caused by mutations in integrin genes. Nonmuscle myosin II is known to participate in integrin-based adhesion of individual migrating cells to extracellular matrix and ablation of nonmuscle myosin IIA heavy chain caused cell-cell adhesion defects in early mouse embryogenesis. Nonmuscle myosin II also functions in the correct lateral arrangement and regulation of cells within a single epithelia. In Drosophila embryonic myofibril formation, correct nonmuscle myosin II localization requires a PS2 integrin. This study extends the role of nonmuscle myosin II in cell adhesion to include an essential role in the apical adhesion of two different epithelial cell sheets to one another. The loss of zip/MyoII function in one of the two wing epithelia sheets was sufficient to abolish adhesion, indicating that zip/MyoII function is necessary in both cell sheets. An accumulation of zip/MyoII at the dorsal-ventral compartment boundary in wing imaginal discs has been reported and may be important for myosin's role in the adhesion between these two cell sheets. Understanding how zip/MyoII functions and coordinates with known adhesion molecules in cell sheet adhesion is an important area for further investigation (Franke, 2010).

Multiple wing hair phenotypes were most easily quantified when GFP-zip/MyoII-Rod(ΔNterm58) was expressed. Like numerous other proteins, zip/MyoII appears to have a direct role in the production of a single hair from the distal vertex of each wing cell. Branching phenotypes were observed in wing hairs, setae and bristles (micro- and macrochaetae) when GFP-zip/MyoII-Rod(ΔNterm58), zip/MyoII-Rod or GFP-zip/MyoII-Neck-Rod was expressed. Branching phenotypes are not generally observed in planar cell polarity mutants and show that zip/MyoII plays a distinct, yet important role in the correct morphology of different actin-based cellular protrusions. Branching of individual hairs or bristles has been characterized in furry and tricornered mutants and can also result from drug treatments (cytochalasin D or latrunculin A) that affect the actin cytoskeleton. As bristles and wing hairs use different assembly strategies of actin bundles to generate their morphology, the simplest explanation of these results is that zip/MyoII plays an early role in these processes. Consistent with these findings, zip/MyoII mutants have defects in shaping and positioning of actin-based protrusions in the embryonic epidermis. Proteins known to contribute to planar polarity (e.g., the proximal and distal proteins Flamingo, Frizzled, Dishelveled, Diego, Strabismus, also known as Van Gogh, and Prickle) all appear to function upstream of the actin cytoskeleton and there are no reports of a direct physical interactions between these proteins and the actin cytoskeleton. As a consequence it is suspected that zip/MyoII functions downstream of planar polarity patterning. How it coordinates with proteins known to be involved in hair morphogenesis will require further investigation (Franke, 2010).

Ectopic and/or expanded vein and crossvein wing patterning phenotypes were observed with the expression of each truncation allele and in the absence of other phenotypes indicating that vein and crossvein positioning defects result directly from zip/MyoII perturbation. The most obvious defects observed were in the positioning of the posterior crossvein, resulting in both ectopic crossveins as well as expanded crossvein tissue. While several loci have been identified that affect vein and crossvein patterning, only a few give rise to ectopic or expanded tissue. Mutant forms of the Dachsous and fat protocadherins have been shown to shift the relative position of the anterior and posterior crossveins with respect to one another but do not cause ectopic tissue. Thus these findings suggest a potentially novel role for zip/MyoII in tissue patterning (Franke, 2010).

The truncation alleles developed in this study will be useful for the analysis of myosin function elsewhere in development. Indeed the current studies show roles for zip/MyoII in movements that contribute to other regions of the adult fly epidermis al well (Franke, 2010).

This study found that full-length zip/MyoII is required for correct function and localization in Drosophila, consistent with findings in S. pombe, which showed that truncations of its myosin heavy chains are not capable of rescue. This contrasts findings in S. cerevisiae where the tail region of myosin II can functionally substitute for full-length protein. The dependence of GFP-zip/MyoII-Neck-Rod on full-length zip/MyoII for localization could occur through two, not necessarily mutually exclusive, mechanisms. First, an individual GFP-zip/MyoII-Neck-Rod protein could heterodimerize with an endogenous, wild-type zip/MyoII heavy chain. Second, GFP-zip/MyoII-Neck-Rod proteins could form homodimers, which associate with endogenous zip/MyoII homodimers, and assemble into bipolar filaments. The results indicate that GFP-zip/MyoII-Neck-Rod heavy chains predominantly form homodimers (Franke, 2010).

The observation that zip/MyoII-HMM(ΔCterm407)-GFP does not induce a multiple wing hair or branching phenotype may provide insights into how zip/MyoII contributes to these processes. One possibility is that zip/MyoII rod-induced aggregates, which recruit endogenous zip/MyoII, may also recruit proteins important for the establishment of PCP thereby altering their subcellular localization. Immunostaining imaginal discs for known PCP proteins that express rod-containing zip/MyoII fragments could help address this (Franke, 2010).

The results demonstrate that nonmuscle myosin II function can be dominantly perturbed by two distinct mechanisms. Previous findings in D. discoidium, yeast, tissue culture cells and Drosophila have demonstrated that myosin II function can be perturbed by expression of truncation constructs. Expression of GFP-zip/MyoII-Rod(ΔNterm58) and zip/MyoII-Rod resulted in the formation of very large intracellular aggregates. Aggregation is consistent with in vitro findings that the light meromyosin region (LMM) is insoluble at physiological ionic strength. Consistent with this study, it has been shown that the formation of myosin II rod aggregates in D. discoidium were intracellular and could contain endogenous, full-length myosin II. Thus, expression of the rod domain recruits full-length nonmuscle myosin II into intracellular aggregates, thereby depleting the total cellular pool of functional nonmuscle myosin II (Franke, 2010).

The second mechanism results in the titration of free light chains from endogenous, full-length nonmuscle myosin II thereby depleting the total cellular pool of nonmuscle myosin II that can be regulated through light chains. A construct capable of function through both mechanisms (one containing both the tail and neck domains) is expected to be the most potent nonmuscle myosin II dominant negative. The results are consistent with this -- GFP-zip/MyoII-Neck-Rod consistently generated the most severe phenotypes. Moreover, these observations suggest that both mechanisms function to simply titrate endogenous, wild-type zip/MyoII heavy chain and are therefore comparable to loss of function alleles (Franke, 2010).

The C-terminal and N-terminal antisera enable semi-quantitative analysis of the expression of a truncation allele to endogenous zip/MyoII. Western blot analysis of whole 3rd instar larvae may be misleading as it does not provide a cellular context for comparing expression levels. Comparing the relative amount of fluorescence between a control region (endogenous zip/MyoII) and an experimental region (endogenous zip/MyoII and rod truncation allele; the current results likely provides a more accurate means for comparing expression -- expression of GFP-zip/MyoII-Neck-Rod is approximately two to three times that of zip/MyoII (Franke, 2010).

That GFP-zip/MyoII expression caused a mild wing phenotype is likely the consequence of heavy chain overexpression without comparable light chain expression. When GFP-zip/MyoII was placed in a heterozygous zipper background the penetrance of phenotypes decreased. The most parsimonious explanation is that the total amount of heavy chain (endogenous plus transgene) was reduced due to the heterozygous background, which helped alleviate the imbalance of heavy and light chains (Franke, 2010).

Having distinct truncation alleles that perturb zip/MyoII function to different extents enables one to screen for enhancers and suppressors of zip/MyoII in desired processes. Expressing of GFP-zip/MyoII-Rod(ΔNterm58) identified both genetic enhancers and suppressors of the multiple wing hair phenotype with components of the PCP pathway (Franke, 2010).

Previously, zip/MyoII was shown to genetically interact with dsh. The current results extend previous findings to include interactions with other PCP pathway genes (Fz and ck/MyoVIIA). The genetic interaction with Fz suggests that in addition to having a direct role in the production of a single wing hair, zip/MyoII may also participate in wing hair polarity. The genetic studies with ck/MyoVIIA show that it acts antagonistically to zip/MyoII with respect to the multiple wing hair phenotype and to other processes in wing morphogenesis resulting in more severe wing phenotypes (e.g., wing blisters). A role for myosin VIIs in cell adhesion has been demonstrated in different organisms. The mechanism(s) by which these heavy chains function in these different processes will require further investigation (Franke, 2010).

Each truncation allele fulfilled criteria of being specific zip/MyoII dominant-negatives: each caused phenotypes in a dose-dependent manner and these phenotypes were overlapping among the different truncation alleles. The allelic series of myosin II truncation constructs generated allow nonmuscle myosin II function to be variably perturbed in a cell, tissue and temporally specific, and therefore, conditional manner. Thus, these tools now make it possible to interrogate zip/MyoII function during any desired time interval or developmental process in Drosophila (Franke, 2010).

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy. This study shows that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals (see JO function is disrupted by ck/myoVIIA mutations). This study demonstrates that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity (Todi, 2005; full text of article).

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA

Myosin VIIs provide motor function for a wide range of eukaryotic processes. Mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. A mutation in ck was first identified by Bridges in the 1930s, but the allele was lost (Bridges and Brehme, 1944). The Ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult 'escapers; that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. A homology model was generated for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, it was established that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, these data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability (Kiehart, 2004; full text of article).

Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism

Mutations of myosin VIIA cause deafness in various species from human and mice to Zebrafish and Drosophila. This study analyzed the kinetic mechanism of the ATPase cycle of Drosophila myosin VIIA by using a single-headed construct with the entire neck domain. The steady-state ATPase activity (0.06 s-1) was markedly activated by actin to yield V(max) and K(ATPase) of 1.72 s-1) and 3.2 microm, respectively. The most intriguing finding is that the ATP hydrolysis predominantly takes place in the actin-bound form (actin-attached hydrolysis) for the actomyosin VIIA ATPase reaction. The ATP hydrolysis rate was much faster for the actin-attached form than the dissociated form, in contrast to other myosins reported so far. Both the ATP hydrolysis step and the phosphate release step were significantly faster than the entire ATPase cycle rate, thus not rate-determining. The rate of ADP dissociation from actomyosin VIIA was 1.86 s-1), which was comparable with the overall ATPase cycle rate, thus assigned to be a rate-determining step. The results suggest that Drosophila myosin VIIA spends the majority of the ATPase cycle in an actomyosin.ADP form, a strong actin binding state. The duty ratio calculated from the kinetic model was approximately 0.9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure (Watanabe, 2006; full text of article).

Dimerized Drosophila myosin VIIa: a processive motor

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. The molecular mechanism of Drosophila myosin VIIa was studied using transient kinetics and single-molecule motility assays. Myosin VIIa moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is approximately 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin VIIa is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate-limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin VIIa (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin VIIa to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter (Yang, 2006; full text of article).

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

The actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton (Turner, 2008).

Native nonmuscle myosin II stability and light chain binding in Drosophila

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. This study characterizes the properties, relationships and associations that each subunit has with one another in Drosophila. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. The stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. Heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion it was found that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are presentFour myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) are identified as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach six previously uncharacterized genes were characterized that contain IQ-motifs and may be essential light chain binding partners (Franke, 2006).

In Drosophila, the stability of the two nonmuscle myosin II light chains in the absence of their heavy chain binding partner is markedly different. zip/MyoII deficient embryos had significantly reduced levels of sqh/RLC, but wild-type levels of mlc-c/ELC. reduced due to mutations in its sqh/RLC binding partner, mlc-c/ELC also remains at wild type levels. Despite this difference in behavior, it was found that by far, the major binding protein for both light chains in soluble extracts from wild type embryos is the zip/MyoII heavy chain. The simplest explanation for these observations is that sqh/RLC protein is unstable in the absence of zip/MyoII heavy chain protein, whereas mlc-c/ELC is stable, similar to findings in D. discoideum. All three myosin II polypeptides were analyzed when either the heavy chain or RLC was overexpressed and no increase was found in polypeptide levels of those subunits not targeted for overexpression. The findings for the turnover of polypeptide subunits are similar to that of other protein complexes ranging from spectrin to cytochromes (Franke, 2006).

In sqh/RLC mutant animals, zip/MyoII levels are reduced, but not to the same extent as sqh/RLC. Unlike sqh/RLC, zip/MyoII heavy chains are partially stable and form aggregates in the absence of sqh/RLC. Staining demonstrated that mlc-c/ELC, whose levels are unaltered in sqh/RLC mutant animals, associated with aggregated heavy chains. Aggregation and/or associations may cause the partial stabilization of heavy chains in sqh/RLC mutants. Analysis of heavy chain levels in sqh/RLC and mlc-c/ELC double mutants would address this, but such analysis is currently not possible, because no mutant mlcc/ ELC alleles are reported and the interpretation may be compromised by mlc-c/ELC binding to other proteins. Overall, the data support a model in which transcriptional or translational feedback does not regulate the level of each polypeptide. Instead, the ability of each polypeptide to persist in mutant backgrounds is dependent on both the inherent stability of each polypeptide and its association with its binding partner(s) (Franke, 2006).

The experiments confirm that sqh/RLC association with zip/MyoII heavy chain is required to prevent heavy chain aggregation, demonstrating that exposure of the hydrophobic helix comprising the distal IQ-motif in zip/MyoII is sufficient to cause aggregation in Drosophila. These results are consistent with studies on slime mold (D. discoidium( and scallop (Pecten maximus) myosin IIs that show removal of the RLC promotes aggregation of myosin II heads. Interestingly, (and in contrast), in D. discoidium cells lacking ELC, myosin II heavy chain localization appears normal. sqh/RLC primarily associates with zip/MyoII heavy chain at sites where nonmuscle myosin II is believed to function. A small fraction of the sqh/RLC localizes to aggregates, but this is likely to result from the recruitment of native, nonmuscle myosin II into heavy chain aggregates. In contrast, mlc-c/ELC associates with heavy chain at sites of aggregation demonstrating that mlc-c/ELC association is not sufficient to prevent aggregation (Franke, 2006).

In lysates from wild-type embryos it was found that the majority of both mlc-c/ELC (>90%) and sqh/RLC (>95%) is bound to zip/MyoII. More quantitative experiments are necessary to determine if a substantial difference between the amount of sqh/RLC and mlc-c/ELC not associated with zip/MyoII heavy chain exists. In S. pombe, cdc4/ELC protein levels are far in excess (>7-fold) of both heavy chains and RLC, suggesting that the amount of ELC not associated with nonmuscle myosin II heavy chain varies in different organisms (Franke, 2006).

In several organisms, the nonmuscle ELC has been shown to bind proteins other than nonmuscle myosin II heavy chain. zip/MyoII is an abundant, ubiquitously expressed protein in Drosophila, and other mlc-c/ ELC binding proteins may be one or more orders of magnitude less abundant. Consequently, the relatively small percentage (<10%) of total mlc-c/ELC not bound to zip/MyoII heavy chain in the wild-type lysates may well be biologically important. Moreover, the proximal zip/MyoII heavy chain IQ-motif, which mlc-c/ELC binds, is very similar to IQ-motifs in other proteins (Franke, 2006).

Using candidate, mass spectrometry and bioinformatics approaches, a number of putative Drosophila mlc-c/ELC binding partners were identied. Using the candidate approach it was found that Ck/MyoVIIA, Jar/MyoVI, and Zip/MyoII bind Mlc-c/ELC using affinity purified Mlcc/ELC antibodies and a myc-tagged mlc-c/ELC construct. These interactions were verified with additional, reciprocal experiments (e.g., Mlc-c/ELC but neither Sqh/RLC nor Zip/ MyoII is present in Ck/MyoVIIA and Jar/MyoVI immunoprecipitations). Mass spectrometry on proteins associated with Myc-Mlc-c/ELC confirmed binding by Zip/MyoII, and showed that two additional proteins, Asp/Abnormal Spindle and Didum/MyoV are also Mlc-c/ELC binding partners. Mass spectrometry could fail to identify all binding partners for several reasons. (1) Greater than 90% of Mlc-c/ELC is associated with Zip/MyoII in wildtype, therefore large quantities of Mlc-c/ELC must be pulled down to identify binding partners for the remaining 10%. (2) If the remaining 10% of Mlc-c/ELC associates with several proteins, pulling down sufficient quantities of each for mass spectrometry is likely to be difficult. (3) Mlc-c/ELC binding partners may either be expressed at very low levels, or expressed in a cell, tissue or developmentally specific manner. (4) Even if the binding partner is expressed ubiquitously, at high levels throughout development, Mlc-c/ELC association may be transient such that only a small amount is bound to Mlc-c/ELC at any given time. While light chains bind with high affinity to Myosin II heavy chains Calmodulin is known to bind weakly under certain solution conditions (Franke, 2006).

An in silico approach was used to identify IQ-motif containing proteins in the Drosophila genome. As expected this approach identified all proteins used to generate the profile. In addition, this approach identified one Mlc-c/ELC binding partner that was also identified by mass spectrometry, Asp/Abnormal Spindle. Seven uncharacterized candidate IQ-motif containing proteins were identified with none having published reagents to directly test for Mlc-c/ELC association (Franke, 2006).

Mutations in myosin VI cause deafness in humans and mice. Jar/MyoVI has a single IQ-motif and is expressed at low levels embryonically except for enrichment in the dorsal-most three or four rows lateral epidermal cells during dorsal closure and in neuroblasts, where it plays a role in basal protein targeting and correct spindle orientation. It was possible to co-IP Jar/MyoVI from lysates with Mlc-c/ELC antibodies, but could detect mlc-c/ELC only in jar/MyoVI pull-downs when Mlc-c/ELC was overexpressed. This suggests that Jar/MyoVI has additional light chains (possibly calmodulin). Because Jar/MyoVI has a single IQ-motif, this suggests that Mlc-c/ELC association may be cell, temporal and/or function-specific. In vitro, porcine MyoVI has been shown to bind calmodulin at two sites - at its single, conventional IQ-motif and at a structural region within a unique insert (not found in other myosins) characteristic to MyoVI’s. Therefore, it is possible that Jar/MyoVI may bind calmodulin and Mlc-c/ELC simultaneously (Franke, 2006).

Mutations in human myosin VIIA cause Usher syndrome1B, a deaf/blind disorder. Mutations in the fly homologue, Ck/MyoVIIA, cause hearing loss and lethality. Ck/MyoVIIA expression is ubiquitous at low levels throughout development, with enrichment in the fly hearing organ. This is the first report of a specific light chain for Ck/MyoVIIA which has 5 IQ-motifs that could simultaneously bind different light chains. Previous reports showed that mouse myosinVIIA binds calmodulin, Ck/MyoVIIA may also bind calmodulin as well as Mlc-c/ELC (Franke, 2006).

Myosin Vs have 6 IQ-motifs and play roles in intracellular transport, correct spindle positioning during cell division and the polarized distribution of intracellular compontents. Both ELC and calmodulin are light chains for myosin V in S. cerevisiae and S. pombe, and myosin Va in chick brain. A recent report also identified both Mlc-c/ELC and calmodulin as light chains for the fly homologue, Didum/MyoV. Animals mutant for didum/MyoV generally arrest during larval development with no obvious defects. Some adult escapers do eclose and males exhibit defects during late spermatogenesis (Franke, 2006).

Mutations in the human orthologue of Asp/Abnormal Spindle, ASPM, are the most common cause of autosomal recessive primary microcephaly-characterized by a reduction in cerebral cortex size. Mutations in Asp/Abnormal spindle cause syncitial embryos to have free centrosomes and larval neuroblasts to have a high mitotic index. Asp/Abnormal Spindle localizes to polar regions of the spindle immediately surrounding the centrosome and its removal causes centrosomes to lose their microtubule organizing center activity. Asp/Abnormal Spindle has at least 5 IQ-motifs. This is the first report of a light chain for asp/Abnormal Spindle in any organism (Asp/Abnormal spindle is conserved in human, mouse, Drosophila and C. elegans), and it is unclear what role light chains have on asp/Abnormal Spindle function (Franke, 2006).

Rho-associated kinase indirectly affects the activity of myosin II in the development of planar cell polarity

Drosophila Rho-associated 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 (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).

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 sqhAX3 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).

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).

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).


REFERENCES

Search PubMed for articles about Drosophila Crinkled

Bejsovec, A, and Chao, A. T. (2012). crinkled reveals a new role for Wingless signaling in Drosophila denticle formation. Development 139(4): 690-8. PubMed ID: 22219350

Bridges, C. B. and Brehme. K. S. (1944). The Mutants of Drosophila melanogaster. Pub. 552, Carnegie Institute, Washington, DC.

Chung, Y. D., Zhu, J., Han, Y.-G. and Kernan, M. J. (2001). nompA encodes a PNS-specific, ZP domain protein required to connect mechanosensory dendrites to sensory structures. Neuron 29: 415-428. PubMed ID: 11239432

Dror, A. A. and Avraham, K. B. (2009). Hearing loss: mechanisms revealed by genetics and cell biology. Annu. Rev. Genet. 43: 411-437. PubMed ID: 19694516.

Ernest, S., et al. (2000). Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness. Hum. Mol. Genet. 9: 2189-2196. PubMed ID: 10958658

Franke, J. D., Montague, R. A. and Kiehart, D. P. (2010). Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis. Dev. Biol. 345(2): 117-32. PubMed ID: 20599890

Franke, J. D., Boury, A. L., Gerald, N. J. and Kiehart, D. P. (2006). Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster. Cell Motil. Cytoskeleton 63(10): 604-22. PubMed ID: 16917818

Gibson, F., et al. (1995). A type VII myosin encoded by the mouse deafness gene shaker-1. Nature 374: 62-64. PubMed ID: 7870172

Hasson, T., et al. (1995). Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS. Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type 1B. Proc. Natl. Acad. Sci. 92: 9815-9819. PubMed ID: 7568224

Hasson, T., et al. (1997). Unconventional myosins in inner-ear sensory epithelia. J Cell Biol. 137: 1287-1307. PubMed ID: 9182663

Hasson, T. (1999). Sensing a function for myosin VIIa. Curr. Biol. 9: R838-R841. PubMed ID: 10574757

Kiehart, D. P, et al. (2004). Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA. Genetics 168(3): 1337-52. PubMed ID: 15579689

Kros, C. J., et al. (2002). Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations. Nature Neurosci. 5: 41-47. PubMed ID: 11753415

Liu, X.-Z., et al. (1997a). Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nature Genet. 16: 188-190. PubMed ID: 9171832

Liu, X.-Z., et al. (1997b), Autosomal dominant non-syndromic deafness caused by a mutation in the myosin VIIA gene. Nature Genet. 17: 268-269. PubMed ID: 9354784

Maniak, M. (2001). Cell adhesion: ushering in a new understanding of myosin VII. Curr. Biol. 11: R315-R317. PubMed ID: 11369222

Petit, C. (2001). Usher syndrome: from genetics to pathogenesis. Annu. Rev. Genomics Hum. Genet. 2: 271-297. PubMed ID: 11701652

Self, T., et al. (1998). Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells. Development 125: 557-566. PubMed ID: 9435277

Todi, S. V., Sharma, Y. and Eberl, D. F. (2004). Anatomical and molecular design of the Drosophila antenna as a flagellar auditory organ. Microsc. Res. Techn. 63: 388-399. PubMed ID: 15252880

Todi, S. V., Franke, J. D., Kiehart, D. P. and Eberl, D. F. (2005). Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila. Curr. Biol. 15(9): 862-8. PubMed ID: 15886106

Todi, S. V., et al. (2008). Myosin VIIA, important for human auditory function, is necessary for Drosophila auditory organ development. PLoS One. 3(5): e2115. PubMed ID: 18461180

Turner, C. M. and Adler, P. N. (1998). Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila. Mech. Dev. 70(1-2): 181-92. PubMed ID: 9510034

Watanabe, S., Ikebe, R. and Ikebe, M. (2006). Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism. J. Biol. Chem. 281(11): 7151-60. PubMed ID: 16415346

Weil, D., et al. (1995). Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 374: 60-61. PubMed ID: 7870171

Weil, D., et al. (1997). The autosomal recessive isolated deafness, DFNB2, and the Usher 1B syndrome are allelic defects of the myosin-VIIA gene. Nat. Genet. 16(2): 191-3. PubMed ID: 9171833

Winter, C. G., et al. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105: 81-91. 11301004

Yang, Y., et al. (2006). Dimerized Drosophila myosin VIIa: a processive motor. Proc. Natl. Acad. Sci.103(15): 5746-51. PubMed ID: 16585515


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date revised: 25 May 2012

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