zipper

Transcriptional Regulation

Analysis of the Broad-Complex (Br-C )gene suggests that it regulates myosin function during imaginal disc morphogenesis. Molecular genetic analysis shows that zinc-finger transcription factors encoded by Br-C are critical for imaginal disc morphogenesis. A screen for enhancers of a Br-C family member, broad1, has identified several loci that function during leg imaginal disc morphogenesis. Ebr, an enhancer of broad1, is a mutation in the myosin heavy chain locus. Defects in leg morphogenesis produce the malformed phenotype. The malformed phenotype reflects aberrations in cell shape changes during morphogenesis in pupal leg imaginal discs. The malformation ranges in severity from a small deformation in the femur to extreme twisting and gnarling of the femur and tibia. The genetic behavior of myosin, and the observation that myosin is subcellularly localized during leg elongation and during additional morphogenetic events, strongly support the hypothesis that myosin-based contraction drives these cell shape changes. Transcription of zip is not under ecdysone control in the imaginal discs; therefore, the gene expression directed by Br-C must affect other aspects of leg disc morphogenesis, rather than merely inducing zip expression. Genetic analysis reveals that genes other than E74 are involved with zipper as SNCCs. These studies promise to extend current understanding of the spatial and temporal control of myosin-based contractility in the cell shape changes required for metazoan development (Halsell, 1998 and references).

The Drosophila tumor suppressor gene lethal(2) giant larvae (lgl) encodes a cytoskeletal protein required for the change in shape and polarity acquisition of epithelial cells, and also for asymmetric division of neuroblasts. lgl also participates in the release of Decapentaplegic (Dpp), a member of the transforming growth factor ß (TGFß) family that functions in various developmental processes. During embryogenesis, lgl is required for the dpp-dependent transcriptional activation of zipper (zip), which encodes the non-muscle myosin heavy chain (NMHC), in the dorsalmost ectodermal cells -- the leading edge cells. The embryonic expression of known targets of the dpp signaling pathway, such as labial or tinman is abolished or strongly reduced in lgl mutants. lgl mutant cuticles exhibit phenotypes resembling those observed in mutated partners of the dpp signaling pathway. In addition, lgl is required downstream of dpp and upstream of its receptor Thickveins (Tkv) for the dorsoventral patterning of the ectoderm. During larval development, the expression of spalt, a dpp target, is abolished in mutant wing discs, while it is restored by a constitutively activated form of Tkv (Tkv^Q253D). Taking into account that the activation of dpp expression is unaffected in the mutant, this suggests that lgl function is not required downstream of the Dpp receptor. Finally, the function of lgl responsible for the activation of Spalt expression appears to be required only in the cells that produce Dpp, and lgl mutant somatic clones behave non autonomously. The activity of lgl is therefore positioned in the cells that produce Dpp, and not in those that respond to the Dpp signal. These results are consistent with the same role for lgl in exocytosis and secretion as that proposed for its yeast ortholog sro7/77: lgl might function in parallel or independently of its well-documented role in the control of epithelial cell polarity (Arquier, 2001).

Protein Interactions

Myosin II is a hexamer consisting of a pair of heavy chains (MHCs) carrying the motor domain and the tail, and pairs of the essential and regulatory light chains (EMLCs and RMLCs, respectively). The RMLC of Drosophila is coded for by the spagetti squash gene. In vertebrates the RMLC is a target of myosin light chain kinase (MLCK), a Calmodulin regulated kinase homologous to CaMKII (see Drosophila CaMKII). The EMLC bears strong homology to Calmodulin (see Drosophila Calmodulin), and is the myosin subunit responsible for sensing the level of Ca++ in the cell (see Ca2+ regulated proteins). Two other proteins are known to interact with myosin: actin and lethal (2) giant larvae. Filamentous actin is identical to muscle actin and along with myosin II is required for myosin II functions. Proteins known to interact with actin include tropomyosin, caldesmon and calponen. For information about the biological roles of the myosin interacting proteins in other organisms, with the exception of actin, see the Evolutionary Homologs section.

Spaghetti squash

Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh1, a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. sqh encodes MRLC-C. In sqh1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis failure and other defects in sqh1. Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function (Karess, 1991).

The X-linked Drosophila gene spaghetti squash (sqh) encodes the regulatory light chain of nonmuscle myosin II. To assess the requirement for myosin II in oogenesis and early embryogenesis, homozygous germline clones were induced of the hypomorphic mutation sqh1 in otherwise heterozygous mothers. Developing oocytes in such sqh1 germline clones often fail to attain full size due to a defect in 'dumping', the rapid phase of cytoplasmic transport from nurse cells. In contrast to other dumpless mutants described to date, sqh1 egg chambers showed no evidence of ring canal obstruction, and no obvious alteration in the actin network. However the distribution of myosin II is abnormal. It is concluded that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively, by nurse cell myosin II and that regulation of myosin activity is one means by which cytoplasmic transport may be controlled during oocyte development. The eggs resulting from sqh1 clones, though smaller than normal, begin development but exhibit an early defect in axial migration of cleavage nuclei towards the posterior pole of the embryo, in a similar manner to that seen in early cleavage eggs in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration require a maternally supplied myosin II (Wheatley, 1995).

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. The in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, has been asssessed by creating a series of transgenes in which these specific amino acids are altered. The transgene phenotypes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals (cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber) are abnormal, suggesting a role of myosin II in their establishment and/or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene (sqh-A20, A21), in which both serine-21 and threonine-20 have been replaced by alanines, behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20 (Jordan, 1997).

Morphogenesis is characterized by orchestrated changes in the shape and position of individual cells. Many of these movements are thought to be powered by motor proteins. However, in metazoans, it is often difficult to match specific motors with the movements they drive. The nonmuscle myosin II heavy chain (MHC encoded by zipper is required for cell sheet movements in Drosophila embryos. To determine if myosin II is required for other processes, a study was made of the phenotypes of strong and weak larval lethal mutations in spaghetti squash (sqh), which encodes the nonmuscle myosin II regulatory light chain (RLC). sqh mutants can be rescued to adulthood by daily induction of a sqh cDNA transgene driven by the hsp70 promoter. By transiently ceasing induction of the cDNA, RLC is depleated at specific times during development. When RLC is transiently depleted in larvae, the resulting adult phenotypes demonstrate that RLC is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When RLC is depleted in adult females, oogenesis is reversibly disrupted. Without RLC induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks (this involves three morphologically and mechanistically distinct types of follicle cell migration) and completion of nurse cell cytoplasm transport (dumping). Finally, in sqh mutant tissues, MHC is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail-binding protein p127. This suggests that sqh mutant phenotypes are chiefly caused by sequestration of myosin into inactive aggregates. These results show that myosin II is responsible for a surprisingly diverse array of cell shape changes throughout development (Edwards, 1996).

Studies in mammalian cells have identified several downstream substrates for Rho-kinase/ROCK. In particular, Rho-kinase regulates the phosphorylation of the nonmuscle myosin regulatory light chain (MRLC) primarily at Ser-19 and secondarily at the adjacent Thr-18. Phosphorylation of MRLC at these sites results in a conformational change that allows myosin II to form filaments and increases its actin-dependent ATPase activity (Winter, 2001 and references therein).

The amino acid sequence around the phosphorylation site of MRLC is highly conserved between mammalian MRLC and the Drosophila homolog, encoded by spaghetti squash (sqh). Therefore the phosphorylation of the Drosophila MRLC was assessed using an antibody that recognizes mammalian MRLC only when Ser-19 is phosphorylated. Immunoblot analysis shows that this antibody specifically recognizes phosphorylated Sqh in larval extracts -- the single ~20 kDa band in wild-type extract is absent both in extracts of a sqh null, and when wild-type extract is treated with phosphatase. While phosphorylated Sqh is detectable in extracts of mutants of Drosophila Rho-associated kinase (Drok²), its level is greatly reduced, whereas the Sqh protein level is not affected in Drok² mutants. Previous work with bovine Rho-kinase has established that expression of the N-terminal catalytic domain gives rise to a constitutively active kinase. Raising the level of Rok activity in vivo by transient expression of the catalytic domain of Rok (Drok-CAT) results in elevated phosphorylation of Sqh as compared to controls in which a kinase-dead form (Drok-CAT-KG) was used. Taken together, these experiments indicate that Rok is required for maintaining the proper level of MRLC phosphorylation in vivo, and that such regulation depends on its kinase activity (Winter, 2001).

The effect of loss of Rok function on MRLC phosphorylation was examined at the cellular level. In wild-type wing cells, phospho-MRLC is enriched at the cortex of the pupal wing cells, whereas in Drok² mutant cells this perimembrane staining is reduced or absent. Thus, rok is cell autonomously required for maintaining the level of cortical phospho-MRLC in the pupal wing (Winter, 2001).

The next question considered was whether MRLC/Sqh is an effector for Rok in regulating hair number in response to Fz/Dsh signaling. Use was made of a series of mutant sqh transgenes 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. These sqh transgenes are under control of the endogenous promoter and are expressed at levels similar to the native protein. Remarkably, whereas 100% of Drok² hemizygous animals die before the wandering third instar stage, introducing one copy of a sqh transgene carrying the E20E21 double mutation (mimicking phosphorylation on both sites) results in 4% hemizygous Drok² survival to adulthood. Likewise, one copy of an analogous transgene expressing SqhE21 also results in Drok² hemizygotes surviving to adulthood (albeit a lower percentage), with a large fraction surviving to late-stage pupae. No rescue was observed when transgenes expressing the alanine substituted forms (SqhA20A21 or SqhA21) were introduced into the Drok² background. These observations support the notion that MRLC is a key target (either directly or indirectly) for Rok kinase in vivo, since mimicking its phosphorylation, even in an unregulated fashion, partially rescues Drok² organismal lethality (Winter, 2001).

Moreover, the multiple hair defect resulting from rok loss of function is almost completely suppressed by the presence of the sqhE20E21 transgene in the rescued adults. Taken together with the modulation of MRLC phosphorylation by Rok, these results demonstrate that the regulation of MRLC phosphorylation is a principal function of Rok in regulating F-actin prehair number (Winter, 2001).

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference has identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. RhoGAP inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by RhoGAP. Local regulation of RhoGAP could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of RhoGAP by integrin and Src, both implicated in neural plasticity (Billuart, 2001).

Biochemical and genetic evidence indicates that a key output for Drok signaling in vivo is the regulation of phosphorylation of myosin regulatory light chain (MRLC) encoded by spaghetti squash (sqh). To test if endogenous MRLC is part of the axon retraction pathway regulated by p190, genetic interaction experiments were performed by reducing the dose of endogenous sqh in the context of the p190 dsRNA expression. Marked suppression of the phenotype was observed in flies heterozygous for a null mutation of sqh (sqh^AX3). In contrast, expression of a phosphomimetic mutant, Sqh-E20E21, markedly enhanced the p190 phenotype, whereas analogous expression of a nonphosphorylable form (Sqh-A21) had no effect. Further, truncation of the medial lobe was frequently observed when Sqh-E20E21 was expressed with the intermediate p190 RNAi line. This is evident from the FasII staining, showing that the medial ß axons (strongly FasII positive) only extend a fraction of the length of the medial lobe. This phenotype was only observed in the strongest p190 RNAi lines, never in the intermediate line alone. Taken together, these results strongly suggest that Drok and phosphorylation of Drosophila MRLC participate in mediating axon retraction as a result of p190 inactivation (Billuart, 2001).

Regulation of Zipper through phosphorylation of Spaghetti squash: Roles of myosin phosphatase during Drosophila development

Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosin II has been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, which in turn are modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit Mypt [FlyBase term: Myosin binding subunit (Mbs)]. To identify the function of these phosphorylation events, the Drosophila homolog of MYPT has been molecularly characterized, and its mutant phenotypes have been analyzed. Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. These results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes (Tan, 2003).

Myosins involved in a variety of essential processes that include muscular contraction, cytokinesis, vesicle transport, cell movement and cell shape change. Among the 17 subclasses of myosins, conventional myosins, known as myosin IIs, have been the most studied. Myosin IIs form bipolar filaments that drive contractile events by bringing together actin filaments of opposite polarity. Myosin II molecules are hexameric enzymes consisting of two heavy chains, two regulatory light chains (MRLCs - coded for by spaghetti squash in Drosophila), and two essential light chains. They can be subclassified into four groups based on their motor domain (or tail) sequences: (1) sarcomeric myosins, (2)vertebrate smooth muscle/non-muscle myosins, (3)Dictyostelium/Acanthamoeba type myosins and (4)yeast type myosins (Tan, 2003 and references therein).

The activity of smooth muscle/non-muscle myosin II is regulated by the phosphorylation of MRLC that is modulated by the antagonistic activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). MLCP is composed of three subunits: a catalytic subunit made up of protein phosphatase 1c ß (also called delta); a myosin binding or targeting subunit (MYPT), and a small subunit of unknown function. MYPT binds and confers the selectivity of PP1c for myosin (Hartshorne, 1998; Tan, 2003 and references therein).

The phosphatase activity of MLCP can be regulated in several ways (reviewed by Hartshorne, 1998; Somlyo, 2000). Rho-kinase (ROCK) phosphorylates an inhibitory phosphorylation site on MYPT and inhibits the phosphatase activity in smooth muscle. This phosphorylation may occur through ZIPK (leucine zipper interacting protein kinase)-like kinase or integrin-linked kinase. Myotonic dystrophy protein kinase phosphorylates the same inhibitory phosphorylation site, although it is not clear whether this phosphorylation event also goes through ZIPK. In addition, protein kinase C (PKC) can phosphorylate the ankyrin repeat region of MYPT, and thus attenuate the interaction of MYPT with PP1c and MRLC. Furthermore, CPI-17, a smooth muscle-specific inhibitor of MLCP, can also regulate the phosphatase activity of MLCP. Phosphorylation of CPI-17 by PKC, or ROCK, or protein kinase N, or p21-activated kinase (PAK) dramatically enhances the inhibition ability of CPI-17. Finally, MRLC can also be phosphorylated by ROCK and PAK, which itself is a substrate of Rac and Cdc42. Thus ROCK can regulate MRLC phosphorylation both through direct phosphorylation of MRLC and through inactivation of MLCP. Importantly, although the biochemistry of these phosphorylation events is well characterized, the physiological significance of these regulatory steps in vivo remains to be explored (Tan, 2003).

The in vivo function of non-muscle myosin II has been extensively analyzed in Drosophila melanogaster, Dictyostelium discoideum and Saccharomyces cerevisiae. Drosophila has a single non-muscle myosin II heavy chain encoded by zipper (zip), as well as a single non-muscle myosin II regulatory light chain encoded by spaghetti squash (sqh). Analysis of the phenotypes associated with mutations in zip and sqh have revealed that non-muscle myosin II regulates cell shape changes and cell movements in multiple processes such as cytokinesis, dorsal closure and oogenesis. In addition, mutations in both zip and sqh affect planar cell polarity during development (Tan, 2003).

The temporal requirement of zip has been studied in sqh² mutant animals that carry a sqh transgene driven by a heat shock promoter. This analysis showed that sqh activity is needed for eye and leg imaginal discs morphogenesis. Also, during oogenesis, sqh is required for morphogenesis of interfollicular stalks, border cell migration, centripetal cell ingression, dorsal appendage cell migration, and rapid transport of the nurse cell cytoplasm into the oocyte. Inhibition of this transport was also observed in animals that carry homozygous sqh¹ germline clones (GLCs) (Tan, 2003 and references therein).

The in vivo function of MRLC phosphorylation was determined by expression of sqh transgenes that contain mutated phosphorylation sites in a sqh null mutant background. Embryos carrying the null mutation sqh^AX3 die, mostly during the first larval instar, and sqh^AX3 GLCs develop extensive defects, including failure in cytokinesis, during oogenesis. SqhA20A21, in which both the primary and secondary phosphorylation sites have been changed to alanine, fails to rescue sqh^AX3, indicating that phosphorylation of Sqh is important for myosin II function. In support of this, a change of serine 21 to glutamic acid (SqhE21), that presumably mimics constitutive phosphorylation of Sqh, substantially rescues the sqh^AX3 oogenesis phenotype (Tan, 2003).

To gain further insight into the regulation of Zip and to define precisely the in vivo function of MLCP, the Drosophila homolog of the MYPT gene (DMYPT) has been cloned. DMYPT is essential for cell sheet movement during dorsal closure, morphogenesis during eye development, and ring canal growth during oogenesis. These results indicate that regulation of the phosphorylation state of MRLC, and dynamic activation and inactivation of myosin II, are essential for its various functions during many developmental processes (Tan, 2003).

A BLAST search of the Drosophila database with mammalian MYPT sequences reveals that the Drosophila genome has a single related gene, CG5891. CG5891 is predicted to encode a protein with limited homology to mammalian MYPT at the N terminus. However, sequence analysis of several cDNAs derived from CG5891 uncovered additional regions of homology between the mammalian and fly homologs, suggesting that the predicted CG5891 gene was incorrectly annotated. A representative cDNA, AT12677, encodes an ORF of 1101 amino acids (aa) that has been named Drosophila MYPT (DMYPT) to follow the nomenclature of the mammalian protein. A comparison of the compiled DMYPT cDNA and genome sequences shows that the DMYPT locus contains 18 exons and 17 introns. The start codon lies in the second exon and the stop codon in the last. Sequence alignment shows that DMYPT shares significant homology with human MYPTs in three regions: the N terminus containing several ankyrin repeats, the C terminus, and a short peptide in the middle that contains the highly conserved inhibitory phosphorylation site (Tan, 2003).

To characterize the consequences of loss of DMYPT function during development, mutations in the DMYPT gene were sought. Two P-element transposon insertions in the DMYPT locus have been defined molecularly by recovery of flanking genomic sequence. EP(3)3727, in the first intron, is homozygous viable and l(3)03802, in the tenth intron, is associated with zygotic lethality. Several deficiencies were identified that remove DMYPT sequences based on genetically defined breakpoints as well as their failure to complement l(3)03802. Df(3L)th102 deletes DMYPT entirely and thus serves as a complete loss-of-function allele for use in this study (Tan, 2003).

To determine whether the l(3)03802 P-element insertion within the DMYPT locus is responsible for the lethality, and to generate new deletion alleles, both DMYPT P-element insertions were excized using the Delta2-3 transposase. Mobilization of each element resulted in the recovery of both viable precise excisions and lethal imprecise excisions. Among the >200 excisions derived from l(3)03802, over half were viable, indicating that the lethality associated with the l(3)03802 chromosome is due to disruption of DMYPT and not another lethal hit. Thus l(3)03802 is renamed as DMYPT⁰³⁸⁰² and EP(3)3727 as DMYPT³⁷²⁷. Two of the strongest embryonic lethal excision lines, DMYPT^2-188 and DMYPT^2-199, like the original insert, DMYPT⁰³⁸⁰², fail to complement Df(3L)th102 and are described in detail below. Eleven of the 39 lethal excisions derived from DMYPT³⁷²⁷ failed to complement with DMYPT⁰³⁸⁰² and Df(3L)th102: this is consistent with the notion that they disrupt DMYPT activity (Tan, 2003).

To confirm that the DMYPT⁰³⁸⁰² insertion disrupts DMYPT function and that the cDNA derived from the DMYPT locus encodes all the functions associated with DMYPT activity, the original lethal P insertion was rescued with a transgene containing a heat shock promoter driving a DMYPT cDNA. Following 1-hour heat treatments daily from embryogenesis to eclosion, hs-DMYPT fully rescues DMYPT⁰³⁸⁰² homozygous animals to adulthood. Stopping heat treatment 1 to 2 days before eclosion led to incomplete rescue of DMYPT⁰³⁸⁰², with adults developing wing and leg defects similar to those noted for zip or sqh mutants partially rescued by a transgene. Stopping heat treatment 3 days prior to eclosion resulted in no rescue to adulthood. The complete rescue of the lethality associated with DMYPT⁰³⁸⁰² by the hs-DMYPT transgene demonstrates that loss of DMYPT activity is responsible for the lethal phenotype (Tan, 2003).

To assess the timing and cause of lethality associated with the DMYPT⁰³⁸⁰² insertion, embryos were collected and analyzed. Lethal phase analysis showed that 44% of homozygous DMYPT⁰³⁸⁰² animals died during embryogenesis, while the remaining 56% died during early first larval instar (485 total embryos counted). More than 80% of the dead mutant embryos displayed a failure of dorsal closure with a characteristic dorsal hole in their cuticles. The size of the hole in such flies is variable and is also influenced by the genetic background. Homozygous Df(3L)th102 embryos, as well as DMYPT⁰³⁸⁰²/Df(3L)th102 embryos also showed dorsal closure defects. The embryonic cuticle phenotype of DMYPT⁰³⁸⁰²/Df(3L)th102 is more severe (more embryos displayed large dorsal holes) than homozygous DMYPT⁰³⁸⁰², suggesting that DMYPT⁰³⁸⁰² is a hypomorphic allele. In addition, all of the embryonic lethal excision lines analyzed that were derived from DMYPT⁰³⁸⁰², and ten of the lethal excision lines from DMYPT³⁷²⁷, produced embryos with dorsal closure defects. Altogether, these results indicate that DMYPT is required for dorsal closure (Tan, 2003).

Dorsal closure involves a cell sheet movement where the dorsal-lateral ectoderm on both sides of the developing embryo moves toward the dorsal midline to cover a degenerative squamous epithelium, the amnioserosa. This epithelial cell sheet movement encloses the embryo in a continuous protective epidermis. Genetic loss-of-function studies have identified the Jun N-terminal kinase (JNK) signal transduction cascade as one of the key modulators of dorsal closure morphogenesis. Transcriptional targets of JNK signaling include decapentaplegic (dpp), a secreted morphogen related to the bone morphogenetic proteins (BMPs), and puckered (puc), a dual-specificity phosphatase that mediates a negative feedback loop of the JNK signal transduction pathway via dephosphorylation of JNK (Tan, 2003).

To determine whether the failure of dorsal closure in DMYPT mutants is due to an influence on JNK signaling, dpp expression was assayed in the leading cells of the ectoderm during closure. In situ hybridization revealed that the spatial and temporal expression pattern of dpp is normal in DMYPT mutant embryos, suggesting that DMYPT does not function through the JNK pathway during dorsal closure (Tan, 2003).

To further examine the cause of dorsal closure defects in the mutants, DMYPT mutant embryos were stained for markers that allowed analysis of cell polarity and shape in the dorsal ectoderm. Apically localized phosphotyrosine immunoreactivity similar to wild-type flies was observed. Moreover, there was normal basolateral fasciclin III immunostaining. Altogether, these results suggest that there are no gross defects in cell orientation or polarity. However, it was noticed that older mutant embryos begin to show abnormal cell shapes at the leading edge of the epidermis, which could account for the defects in dorsal closure observed in the DMYPT mutants (Tan, 2003).

Consistent with the late embryonic defects observed in DMYPT zygotic mutants, it was found that DMYPT is maternally contributed and ubiquitously expressed during embryogenesis. This maternal supply of DMYPT is likely the reason that the dorsal closure phenotype is variable among embryos and is influenced by genetic background. However, this question cannot be addressed directly since DMYPT is required during oogenesis (Tan, 2003).

During oogenesis, each cystoblast divides four times with incomplete cytokinesis and produces one oocyte and fifteen support nurse cells that are all connected through cleavage furrows. These cleavage furrows subsequently develop into ring canals. These open rings allow the nurse cells to transport cytoplasm into the oocyte, slowly from stage 6 to stage 10, then rapidly at stage 11. This fast phase of transport is referred to as 'dumping', and has been shown to require the activity of Sqh (MRLC). In sqh mutant germline egg chambers, dumping is blocked (Tan, 2003).

To analyze the role of DMYPT during oogenesis, homozygous mutant germline clones (GLCs) were generated of DMYPT⁰³⁸⁰² using the FLP-FRT/dominant female sterile technique. Females carrying DMYPT⁰³⁸⁰² homozygous GLCs lay few tiny eggs, about a quarter of the size of wild type eggs, that do not develop. Examination of the mutant egg chambers revealed that the dumping of nurse cell cytoplasm to the oocyte is blocked. This is similar to the dumpless phenotype observed with sqh homozygous mutant GLCs as well as for mutants in other actin binding proteins (Tan, 2003).

To investigate the basis of the dumpless phenotype associated with DMYPT⁰³⁸⁰² GLCs, actin filaments were stained using Texas Red phalloidin. The most obvious defect involves the ring canals. At stage 8, wild-type egg chambers have large bagel-shaped ring canals. In contrast, the ring canals of DMYPT⁰³⁸⁰² GLC egg chambers are very small (Tan, 2003).

To determine whether the ring canals of DMYPT⁰³⁸⁰² GLCs never enlarge, or whether they grow and then collapse, the ring canals were examined in different stage egg chambers. In wild-type egg chambers, ring canals grow from 1 µm at stage 2 to 10 µm at stage 11. In contrast, the ring canals of DMYPT⁰³⁸⁰² GLCs barely grow. Mutation of DMYPT in follicular cells have no effects on the ring canal growth, suggesting that DMYPT is required in the germline for ring canal growth. Presumably, these small ring canals cannot support the fast phase cytoplasmic transport and thus cause the dumpless phenotype resulting in tiny eggs (Tan, 2003).

In addition to actin, several other proteins, including Hu-li tai shao (Hts), Kelch, and phosphotyrosine (pY)-containing proteins, are recruited to ring canals as they form. Immunolocalization experiments have revealed that both Hts and Kelch are localized to the small DMYPT mutant ring canals. Interestingly, although pY staining is present in the mutant ring canals, an ectopic accumulation of pY staining was also observed in the nurse cells. The basis of this ectopic accumulation remains to be determined (Tan, 2003).

Next, the subcellular distribution of Zipper was examined. Mutation of Sqh causes Zip to form aggregates, thus an effect on Zip distribution in the absence of DMYPT was expected. Surprisingly, no major changes in Zip distribution were detectable between wild-type egg chambers and DMYPT GLCs. In both cases, Zip was uniformly distributed at low level with enhanced cell cortex localization. These observations are consistent with the result that DMYPT mutations have no effect on Zip localization during dorsal closure (Tan, 2003).

Previous studies have shown that the Rho family GTPases, Rac1, RhoA, and Cdc42, each play a role in dorsal closure, and may influence myosin activity through a RhoA mediated signal. Programmed overexpression of these genes by the eye-specific GMR promoter causes distinct rough eye phenotypes. To pinpoint the relationship of DMYPT with these GTPases, the effects of reducing DMYPT activity on the rough eye phenotypes was examined. Interestingly, reduction of DMYPT strongly enhances the eye phenotype caused by GMR-Rac^7A. The eyes of GMR-Rac^7A/DMYPT⁰³⁸⁰² flies are much smaller, with fewer bristles and hexagonal-shaped ommatidia, than those of GMR-Rac^7A/OreR flies. Consistent with the idea that the P-insertion and the excisions are hypomorphic alleles, Df(3L)th102 enhances the GMR-Rac^7A eye phenotype to an even greater extent than either DMYPT⁰³⁸⁰², DMYPT^2-188 or DMYPT^2-199. However, reduction of DMYPT has no effect on the size of the rough eye caused by either GMR-RhoA or GMR-Cdc42, although it does enhance the rough eye phenotype caused by GMR-RhoA since fewer bristles form. Together, these data suggest that DMYPT plays a role in eye development and functions downstream of, or in parallel with Rac and Rho (Tan, 2003).

RhoA functions downstream of Rac in determining ommatidia polarity in the eyes. Reducing the dosage of RhoA enhances the effect of sev-Rac^N17, a dominant negative form of Rac driven by the sevenless (sev) enhancer-promoter in the eye, and suppresses the activity of sev-Rac^V12, which encodes a constitutively active form of Rac. Consistently, overexpression of RhoA (sev-RhoA) rescues sev-Rac^N17, while reduction in the amount of Rac using a deficiency that uncovers Rac has no effect on the gain-of-function RhoA phenotype. Thus, similar to the Rho dependence on Rac function observed in mammalian fibroblasts, some developmental events in Drosophila also rely on a hierarchy of GTPase function (Tan, 2003).

Consistent with these observations, reducing the dosage of RhoA partially suppresses the rough eye phenotype caused by GMR-Rac. In fact, mutations of all the putative positive regulators of myosin activity (RhoA-Zip signaling pathway), including RhoA, Drok and zip itself, moderately suppress the rough eye phenotype of GMR-Rac, opposing the effect of DMYPT mutants. This suggests that the RhoA-Zip signaling pathway functions downstream of Rac, and that DMYPT is a negative regulator of the pathway (Tan, 2003).

Importantly, replacing the phosphorylation sites of Sqh with alanine remarkably suppresses the rough eye phenotype, while replacing them with glutamic acid to mimic phosphorylation slightly enhances the phenotype. This suggests that dephosphorylation of Sqh is important in eye morphogenesis and that DMYPT may be involved in regulating the dephosphorylation of myosin light chain in eye development (Tan, 2003).

To examine whether other myosins are also involved in this process, the effect of myosin VIIA, an unconventional myosin encoded by crinkled (ck), was included in the same assay. Myosin VIIA was chosen because ck and zip behave antagonistically in wing hair number determination in the Drosophila adult wing. Interestingly, ck behaves just the opposite of myosin II (Zip) during eye morphogenesis, since a reduction in ck activity enhances the GMR-Rac rough eye phenotype, nearly to the same extent as a reduction in DMYPT (Tan, 2003).

The regulation of MRLC phosphorylation is essential to modulate myosin II activity and can be controled by several distinct mechanisms. For instance, RhoA can activate its effector ROCK that in turn phosphorylates MYPT, either directly or indirectly. MYPT phosphorylation inhibits the phosphatase activity of MLCP and leads to elevation of MRLC phosphorylation. Phosphorylation of MRLC can also be increased by activation of MLCK, another downstream target of RhoA. Thus, the antagonistic activity of kinase and phosphatase is thought to engender a delicate balance of myosin II activity modulated through the phosphorylation state of its regulatory light chain (Tan, 2003).

To assess the relationship between DMYPT regulation of myosin II and signaling via the Rho GTPase family members, the Drosophila eye was examined since sensitive genetic interactions can be observed. RhoA function downstream of, or in parallel with, Rac has been implicated in regulation of orientation of ommatidia in the eye. Consistent with this, reducing the amount of RhoA, Drok and zip partially alleviates the eye defect associated with overexpression of Rac, while reducing the dosage of a putative negative regulator of myosin enhances the rough eye phenotype. Furthermore, expression of a non-phosphorylatable form of Sqh, which presumably reduces the activity of Zip, dramatically rescues the phenotype, while overexpression of a phospho-mimicking Sqh mutant, which should increase the activity of myosin, exacerbates the eye defects. Taken together, these data indicate that the regulation of myosin II activity via balancing the phosphorylation level of Sqh is critical for proper morphogenesis of the Drosophila eye. Based on these results, it is proposed that it is DMYPT that mediates myosin II downregulation in this system (Tan, 2003).

Interestingly, crinkled (myosin VIIA), an unconventional myosin, behaves antagonistically to Zip/myosin II in both eye morphogenesis and wing hair number restriction. This suggests that various myosins interact in different cell types to regulate reorganization of the actin cytoskeleton. It will be interesting to determine the specificity of functions of different myosins and their modes of regulation. Since there are many different myosins but only a single MYPT in Drosophila, it remains to be determined whether, and how, DMYPT interacts with other myosins (Tan, 2003).

In conclusion, the Drosophila homolog of mammalian MYPT, accordingly named DMYPT, has been identified. DMYPT plays multiple roles during Drosophila development. Loss of DMYPT function leads to blockage of rapid transport of nurse cell cytoplasm, inhibition of ring canal growth, failure of dorsal closure, defects of eye morphogenesis, and other unidentified processes during pupae development. Furthermore, the data indicate that dynamic regulation of myosin II activity via regulating phosphorylation level of myosin regulatory light chain by DMYPT is critical for the function of myosin II (Tan, 2003).

Nonmuscle myosin essential light chain

The essential (alkaline) light chain of nonmuscle myosin has been cloned from Drosophila. This completes the sequence of the three myosin subunits, two of which have been shown genetically to be required for morphogenesis and cytokinesis (the heavy chain encoded by zipper and the regulatory light chain encoded by spaghetti squash). The essential light chain protein is 147 amino acids in length and is 53% identical to human smooth muscle essential light chain. The sequence is consistent with the presence of four helix-loop-helix domains seen in crystallographic structures of the striated muscle myosin light chains and their close relative, calmodulin. There are several conserved contacts among the myosin subunits that may be important for the structure and regulation of the myosin motor. The gene encoding Drosophila nonmuscle essential light chain (Mlc-c) localizes to cytological position 5A6 (Edwards, 1995).

Myosin Light Chain Kinase and axon guidance

pCC/MP2 neurons pioneer the longitudinal connectives by extending axons adjacent to the midline without crossing it. These axons are drawn toward the midline by chemoattractive Netrins, which are detected by their receptor Frazzled (Fra). However, these axons are prevented from crossing by Slit, an extracellular matrix ligand expressed by glial cells and recognized by Roundabout (Robo), a receptor on the axons of most neurons. Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons (Kim, 2002).

Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues (Kim, 2002).

Several loss-of-function studies have established a critical role for conventional myosin II in growth cone motility and axon outgrowth. However, axon pathway formation requires regulated motility as the growth cone moves toward attractive cues and away from repulsive cues. This implies that myosin activity must be regulated in order to allow the growth cone to respond to attractive and repulsive cues. To provide evidence that myosin activity is regulated during pathway formation, myosin II activity was selectively elevated in Drosophila CNS neurons by using a constitutively active form of MLCK (ctMLCK). Expression of ctMLCK pan-neurally or limited to the ftzng pattern [the neurogenic enhancer element of the fushi tarazu gene (ftzng-Gal4) drives expression in a subset of neurons, including primarily neurons within the pCC/MP2 pathway] causes axons in the pCC/MP2 pathway to project across the midline incorrectly without any detectable alteration in cell differentiation. Transgenes expressing putatively active (sqhEE) or inactive (sqhAA) regulatory light chains enhance or suppress, respectively, the frequency of crossovers caused by ctMLCK expression. This suggests that ctMLCK is increasing myosin II activity via phosphorylation of the regulatory light chains and this hyperactivation of myosin II is responsible for the midline crossing errors of pCC/MP2 pathway axons. If midline repulsive signals are reduced by using heterozygous mutations of either the ligand Slit or its receptor Robo, ctMLCK expression induces many more axons to cross the midline improperly. CtMLCK expression also induces axons to cross the midline in comm mutants, where Robo-dependent repulsion remains active. Since manipulating the level of the attractive receptor Frazzled (Fra) also alters the ctMLCK phenotype, it is hypothesized that when myosin II activity is hyperactivated by ctMLCK expression, the growth cone over-responds to even transient activation of attractive mechanisms, causing it to extend across the midline. This overextension would normally be attenuated by Robo-mediated repulsive signals. Together, these results indicate that a growth cone’s response to attractive and repulsive cues requires careful regulation of MLCK and/or myosin II activity (Kim, 2002).

Growth cone steering during pathway formation is dictated by the equilibrium between attractive and repulsive cues. Attractive cues drive a growth cone forward by increasing actin polymerization and stimulating the formation of a complex of proteins that couples the actin cytoskeleton to the extracellular matrix via a membrane receptor. This complex acts as a 'clutch', allowing myosin activity to drive the growth cone forward. Repulsive cues are thought to decrease actin polymerization and inhibit membrane receptor coupling to the actin cytoskeleton. Myosin-dependent retrograde flow of actin filaments then causes filopodial and/or growth cone retraction (Kim, 2002).

The general importance of regulating myosin II activity during axon guidance decisions is confirmed by observation that pan-neural expression of ctMLCK, but not wtMLCK, in Drosophila embryos causes axons within the pCC/MP2 pathway to project across the midline incorrectly. In crossing the midline, axons in the pCC/MP2 pathway either over-respond to midline attractive cues leading them across the midline or fail to respond to repulsive signals preventing them from crossing. Indeed, it is likely that both processes are operating. Axons within the pCC/MP2 pathway move toward the midline as Fra receptors detect chemoattractive Netrins. However, they are prevented from crossing by the repulsive ligand Slit, detected by Robo, the cell surface receptor present on most growth cones. Expression of ctMLCK does not alter the onset of axon extension nor the initial pioneering events of pCC/MP2 neurons, but is sufficient to allow these axons to overcome the repellent Slit barrier and cross the midline. If midline repulsive signals are reduced by using heterozygous mutations of either slit or robo, ctMLCK expression induces many more pCC/MP2 axons to cross the midline, and decreasing myosin II activity using sqh mutations that lower the activity of the regulatory light chains suppresses some of the crossovers observed in heterozygous robo mutants. Thus, it seems that myosin II activity must be maintained below a certain threshold in order for Robo to prevent axons from crossing the midline. When myosin II activity exceeds that threshold, as in embryos expressing ctMLCK, the growth cone is unable to respond appropriately to activation of Robo (Kim, 2002).

Robo is thought to prevent axons from crossing the midline in part by reducing filopodial exploration of the midline. In cultured neurons, inhibition of MLCK using a pharmacological inhibitor is sufficient to cause filopodial collapse. This suggests that Robo-dependent decreases in MLCK activity may contribute to Robo’s ability to regulate filopodia retraction. Increasing the myosin activity associate with retrograde flow of actin would also aid in filopodia retraction. Enhanced retrograde flow of actin by ctMLCK would be expected to help Robo prevent axons from crossing the midline, a prediction clearly not born out in this study. Thus, no evidence is available to support an increase in retrograde flow as a consequence of ctMLCK expression. However, the myosin activity moving actin backwards during retrograde flow appears to propel the growth cone forward once the actin filament is coupled to a receptor complex. Thus, if ctMLCK expression enhances the response of a growth cone to attractive cues upon receptor coupling to actin, the importance of retrograde flow in retracting axons may have been masked (Kim, 2002).

Indeed, the level of midline attractive activity affects the frequency of axon crossovers observed when ctMLCK is overexpressed. Decreasing the level of the attractive receptor Fra reduces the number of axons crossing the midline in ctMLCK embryos, while coexpression of UAS-Frazzled enhances ctMLCK-induced crossovers. Activation of Fra by soluble Netrins may encourage midline crossing by enhancing MLCK and/or myosin II activity, which in turn facilitates a growth cone’s response to whatever adhesive systems are operating at the midline. The importance of these attractive systems at the midline is further supported by the ability of ctMLCK expression to rescue comm mutant phenotypes. In comm mutants, a failure to remove Robo from the membrane increases midline repulsive activity and thus commissures do not form because axons are prevented from crossing the midline. But attractive cues are also present in comm mutants and, at least in early stages, axons orient toward and explore the midline as if they are trying to respond to midline attractive cues. With ctMLCK expression, these tentative explorations appear to be converted into positive movement across the midline. This suggests that, when myosin II activity is increased by ctMLCK expression, even transient activation of midline adhesive systems, and consequent coupling to actin filaments, will provide sufficient traction to move the growth cone partially over the midline. Once over, the continued presence of Slit at the midline would actually help propel the growth cone all the way across to the contralateral connective, thus forming part of the commissure. The thickness of many of the rescued commissures suggests that fasciculation with early axons may aid later axons in continuing the formation of a commissure. Together, the data indicate that a growth cone’s response to midline attractive cues is sensitive to the overall level of myosin II activity (Kim, 2002).

The sensitivity of guidance decisions to myosin II activity levels confirms that myosin II activity must be regulated in the growth cone. One possibility is that a basal level of myosin II activity is set that permits constitutive force generation. That is, myosin II activity is permissive for outgrowth but leaves directional information to other signaling events, presumably involved in determining which actin filament myosin II exerts force upon and/or which actin filaments are coupled to the substrate. The data suggest that, if pathfinding is to remain accurate, this basal level must be carefully set so that a growth cone does not over-respond to attractive cues or fail to respond to repulsive cues along its pathway. Alternatively, myosin II activity could be regulated directly as a consequence of guidance receptor activation. In this model, activation of MLCK and/or myosin II by guidance cues is instructive; myosin II is stimulated in response to attractive receptors and inhibited by repulsive receptors. Thus, the growth cone moves toward attractive cues and is prevented from extending toward repulsive cues. At the moment, the data cannot distinguish between these two modes of myosin regulation. Indeed, since nature often compromises, a basal level of constitutive myosin II activity may stimulate axon out-growth with activation of guidance receptors fine-tuning this activity to provide directional information (Kim, 2002).

Certainly, it is striking that several signaling pathways exist that converge to regulate myosin II activity in growth cones or other motile systems and many of these signaling pathways have been implicated in the transduction of midline attractive and repulsive cues. Calcium-Calmodulin (CaM) and various downstream target proteins are required for axon extension and negotiation of choice points. This includes the ability of pCC/MP2 axons to remain on the correct side of the midline. Given the data presented here, CaM activation of MLCK may help transduce midline cues by stimulating conventional myosin II activity via phosphorylation of its regulatory light chains. CaM also binds to IQ motifs in the hinge region of unconventional myosin molecules, where it activates myosin activity in response to Ca²⁺ signals. The Rho family of GTPases are also major regulators of myosin activity. Rho and its effector Rho Kinase regulate dephosphorylation of the regulatory light chains of myosin by Myosin Phosphatase, while both Cdc42 and Rac GTPases regulate MLCK activity via p21 Activated Kinase (PAK). Given that these families of GTPases also regulate several aspects of actin polymerization and receptor coupling to actin filaments, they are expected to be key molecules in coordinating actin and myosin dynamics during growth cone motility. Various GTPases have been directly or indirectly implicated in the transduction of midline repulsive cues in Drosophila embryos. Frazzled may also signal attraction, at least in part, through activation of some Rho family GTPases. Future experiments will examine how mutations in these signaling pathways affect ctMLCK overexpression phenotypes and thus help elucidate the relative contribution of these signaling pathways to the regulation of myosin II activity during axon guidance (Kim, 2002).

In summary, by overexpressing a constitutively active form of MLCK, conventional myosin II activity in growth cones is selectively elevated independent of guidance cue information. Elevated myosin II activity causes specific axon guidance errors since axons in the pCC/MP2 pathway project across the midline abnormally. This overextension defect compliments previous loss of function data and confirms the importance of MLCK and myosin II in growth cone movement. Moreover, by determining that this phenotype is modified by mutations in midline guidance cues, it has been demonstrated that a growth cone’s response to both attractive and repulsive guidance cues requires that MLCK and/or myosin II activity be carefully regulated (Kim, 2002).

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. These data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho (Fritz, 2002).

Thus, when expressed alone, only ctDrac and ctDcdc42 cause midline crossing errors. However, the mutant GTPases interact genetically with mutations in robo, Sos, and chic and with overexpression of ctMLCK. The interactions are surprisingly specific. Midline crossing errors caused by expression of ctDrac or ctDcdc42 are suppressed by heterozygous loss of Profilin and enhanced by expression of ctMLCK. These results indicate that Drac1 and Dcdc42 encourage axons to cross the midline by regulating actin polymerization and/or myosin activity. CtRho and dnRho interact strongly with expression of ctMLCK or heterozygous loss of Robo, which suggests that regulation of myosin activity by Rho is crucial for midline repulsion. This work demonstrates that Rho, Drac1, and Dcdc42 are involved in dictating which axon may cross the midline, presumably by aiding in the transduction of attractive and/or repulsive cues operating at the midline. By using mutations in signaling molecules known to prevent pCC/MP2 axons from crossing the midline, this analysis concentrates on how Rho, Drac1, and Dcdc42 may regulate cytoskeletal dynamics in response to midline repulsive cues (Fritz, 2002).

The interactions between the Drac1 and Dcdc42 and ctMLCK indicate that misregulation of myosin activity may contribute to ctDrac- and ctDcdc42-induced axon guidance errors. Coexpression of ctMLCK with ctDrac or ctDcdc42 results in a strong enhancement of midline crossing errors, while expression of dnDrac or dnDcdc42 suppresses the defects caused by increased myosin activity. This suggests that Drac1 and/or Dcdc42 activate myosin activity in the growth cone to increase outgrowth. One mechanism may be through activation of PAK, which leads to phosphorylation of myosin regulatory light chains (MLC) to increase myosin activity. However, it has been shown that PAK also phosphorylates and inactivates MLCK, resulting in less myosin activity. In vitro, PAK phosphorylates MLCK at serine 439, which is present in ctMLCK, and serine 991, which has been removed from ctMLCK, so the impact of this pathway on the truncated ctMLCK protein is uncertain. Alternatively, it is possible that the interaction of Drac1 or Dcdc42 and ctMLCK is a secondary effect to increased actin polymerization. If increased actin polymerization is causing more filopodial exploration of the midline, increasing myosin activity through ctMLCK expression could cause axons to cross the midline before they can retract filopodia encountering repulsive signals. Separating the relative contributions of Drac1 and Dcdc42 to actin polymerization and myosin activity will require more specific experiments involving the effectors of Drac1 and Dcdc42 (Fritz, 2002). \

The role of Rho in midline repulsion is more difficult to determine since both dnRho and ctRho enhance the midline crossing phenotype of heterozygous robo mutants. This is consistent with the data in which both dnRho and ctRho enhance the ctMLCK phenotype. Similar complexities are seen in the literature; expression of a Rho GEF, which is expected to increase Rho activity, leads to increased attraction to the midline, even though activation of Rho usually leads to growth cone collapse or retraction. The complexity of the Rho interactions is understandable when the dual role of myosin activity during axon guidance is considered. The most documented connection between myosin activity and Rho is through the effector Rho Kinase (RhoK). RhoK phosphorylates MLC and also inactivates myosin phosphatase by phosphorylating its myosin binding subunit, leading to increased phosphorylation of MLC and therefore increased myosin activity. Myosin activation is needed both for the retrograde flow of actin that retracts filopodia and for the force that propels the growth cone forward. Repulsive guidance signals are expected to increase retrograde flow while preventing forward movement (Fritz, 2002).

Expression of dnRho may specifically interfere with retraction of filopodia in response to repulsive cues, leading to increased midline crossing errors. A global increase in myosin activity caused by expression of either ctRho or ctMLCK, or even a Rho GEF, may cause axon guidance errors by increasing the forward movement of the growth cone. Midline attractive activity (e.g., Netrins) probably also influences how much myosin activity is available to move a growth cone over the midline. The literature and these experiments are most consistent with a model in which Rho is activated by repulsive guidance signals. Activation of ephrinA5 receptors causes an increase in Rho activity resulting in a growth cone collapse. Plexin B, the receptor for repulsive semaphorins, binds to and seems to activate Rho. Activation of Robo by Slit recruits srGAP1, which appears to prevent it from binding to and inactivating Rho. The genetic interactions seen between Sos^e49 mutations and expression of ctRho or dnRho are consistent with Sos acting as a GEF for Rho in pCC/MP2 neurons. DnRho strongly enhances the midline crossing errors caused by loss of Sos, while ctRho almost completely suppresses them. Since Sos-dependent signaling pathways are required for response to midline repulsive cues, this is further evidence that Rho is activated downstream of repulsive guidance signals, although a role downstream of selected attractants cannot be ruled out (Fritz, 2002).

Clearly, regulation of Rho family GTPase activity is necessary to prevent axons from crossing the midline inappropriately. Midline repulsive signaling involves regulation of all three GTPases; Drac1 and Dcdc42 are likely downregulated, while Rho seems to be activated downstream of repulsive signals. The Rho family GTPases influence actin polymerization and/or myosin force generation to regulate the processes of growth cone motility that are required for proper response to axon guidance signals (Fritz, 2002).

Protein kinase C

Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, the Drosophila myosin-II tail expressed in Escherichia coli was purified and it was shown to be phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, it has been shown that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, transgenic flies were generated that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way (Su, 2001).

Lethal(2)giant larvae

Mutations in the gene, Lethal (2) giant larvae, l(2)gl, besides causing malignant tumors in the brain and imaginal discs, generate developmental defects in a number of other tissues. Much of the uncertainty regarding the function of the l(2)gl gene product, p127, results from a lack of knowledge as to the precise location of this protein in the cell. P127 is located entirely within the cell in both the cytoplasm and bound to the inner face of lateral cell membranes in regions of cell junctions. On the membrane, p127 can form large aggregates which are resistant to solubilization by nonionic detergents, indicating that p127 is participating in a cytoskeletal matrix. These findings suggest that the changes in cell shape and the loss of apical-basal polarity observed in tumorous tissues are a direct result of alterations in the cytoskeleton organization caused by l(2)gl inactivation and also suggest that p127 is involved in a cytoskeletal-based intercellular communication system directing cell differentiation (Strand, 1994a).

Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes developmental abnormalities in the germline, the ring gland and the salivary glands. The l(2)gl gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. P127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. P127 can form homo-oligomers, and p127 contains at least three distinct domains contributing to its homo-oligomerization. P127 directly interacts with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network (Strand, 1994b).

The p127 tumour suppressor protein encoded by the lethal(2)giant larvae gene is a component of a cytoskeletal network distributed in both the cytoplasm and on the inner face of the plasma membrane. P127 can be phosphorylated at serine residues. A serine kinase is associated with p127. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127, containing five serine residues that are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane (Kalmes, 1996).

Inactivation of the lethal(2)giant larvae (l(2)gl) gene results in malignant transformation of imaginal disc cells and neuroblasts of the larval brain in Drosophila. Subcellular localization of the l(2)gl gene product, P127, and its biochemical characterization have indicated that it participates in the formation of the cytoskeletal network. In experimentally overaged larvae obtained by using mutants in the production of ecdysone, the l(2)gl temperature sensitive mutation displays a tumorous potential. This temperature-sensitive allele of the l(2)gl gene has been used to describe the primary function of the gene before tumor progression. A reduced contribution of both maternal and zygotic activities in l(2)gl temperature sensitive homozygous mutant embryos blocks embryogenesis at the end of germ-band retraction. The mutant embryos are consequently affected in dorsal closure and head involution and show a hypertrophy of the midgut. These phenotypes are accompanied by an arrest of the cell shape changes normally occurring in lateral epidermis and in epithelial midgut cells. l(2)gl activity is also necessary for larval life: the critical period falls within the third instar larval stage. l(2)gl activity is also required during oogenesis: mutations in the gene disorganize egg chambers and cause abnormalities in the shape of follicle cells, which are eventually internalized within the egg chamber. These results together with the tumoral phenotype of epithelial imaginal disc cells strongly suggest that the l(2)gl product is required in vivo in different types of epithelial cells to control their shape during development (Manfruelli, 1996).

In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumor-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).

Because Lgl is a component of cortical protein complexes that include nonmuscle Myosin II, or Zipper (Zip), a test was performed for genetic interactions between lgl and zip in Miranda localization by examining embryos zygotically mutant for both lgl and zip. The zip¹ mutation does not affect Miranda localization throughout embryonic development and lgl-zip embryos show no difference in Miranda localization from zygotic lgl- embryos until late embryonic stages (stage 16) owing to the maternal contribution of zip. However, at stage 17 when maternal zip had been exhausted, lgl-zip embryos appear to restore the basal crescent of Miranda in metaphase neuroblasts, whereas zygotic lgl- embryos at the same stage do not. Thus, Lgl might act for Miranda localization in part by suppressing zip function directly or indirectly, consistent with a study on yeast that indicated negative genetic interactions between Lgl homologs and Myosin II. Alternatively, the asymmetric distribution of Pon requires myosin function in neuroblasts, as revealed by the use of 2,3-butanedione monoxime (BDM) that generally inhibits myosin function. The effect of BDM on Miranda localization was examined. Treatment of wild-type embryos with BDM phenocopies lgl mutants, resulting in a partial redistribution of Miranda from the cortex to microtubules. The effect of BDM is more marked in lglGLC embryos: as the BDM concentration increases, the relocalization of Miranda to microtubules is synergistically enhanced in most BDM-treated neuroblasts and results in the complete exclusion of Miranda from the cortex at 50 mM BDM. The phenocopy and enhancement of lgl mutations by general inhibition of myosin function are in contrast with the suppressive effects of zip mutations, suggesting that Lgl cooperates with at least one type of Myosin other than Zip to anchor Miranda at the cell cortex. It is thus inferred that Lgl regulates negatively myosin II function and also positively the function of another Myosin isotype in cortical protein targeting in neuroblasts (Ohshiro, 2000).

Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).

How does Lgl regulate basal protein targeting? Lgl binds non-muscle myosin II in all organisms tested, and sro7/77 and myo1 (encoding Lgl-related proteins and myosin II, respectively) show strong negative genetic interactions in yeast. Tests were performed for genetic interactions between lgl⁴ and two different null mutations in zipper (encoding myosin II), scoring Miranda basal localization in stage 17 neuroblasts, when maternal Lgl and Myosin II protein levels are lowest. Wild-type and zip embryos have normal basal protein localization, whereas lgl⁴ embryos show complete delocalization of basal proteins. However, lgl⁴ embryos lacking one copy of myosin II show a significant increase in basal protein targeting; and lgl⁴;zip¹ mutant embryos show virtually normal basal protein targeting. Thus, reducing myosin II levels strongly suppresses the lgl phenotype, indicating that myosin II can inhibit basal targeting when Lgl levels are low (Peng, 2000).

In addition, the general myosin inhibitor 2,3-butanedione monoxime (BDM) can suppress the lgl phenotype: stage 10 lgl⁴ embryos treated with BDM show a significant increase in basal protein localization compared with sham-treated stage 10 lgl⁴ embryos. Wild-type or lgl4 embryos treated with 50 mM BDM show delocalization of Miranda, Prospero and Pon. These data indicate that a myosin that is sensitive to 25 mM BDM inhibits basal protein localization in lgl embryos (probably myosin II), and at least one myosin that is sensitive to 50 mM BDM promotes basal protein targeting in mitotic neuroblasts (Peng, 2000).

Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).

Extradenticle

In the absence of MEIS family proteins, two mechanisms are known to restrict the PBX family of homeodomain (HD) transcription factors to the cytoplasm. (1) PBX is actively exported from the nucleus via a CRM1-dependent pathway. (2) Nuclear localization signals (NLSs) within the PBX HD are masked by intramolecular contacts. In a screen to identify additional proteins directing PBX subcellular localization, a fragment of murine nonmuscle myosin II heavy chain B (NMHCB) was identified. The interaction of NMHCB with PBX was verified by coimmunoprecipitation; immunofluorescence staining revealed colocalization of NMHCB with cytoplasmic PBX in the mouse embryo distal limb bud. The interaction domain in PBX maps to a conserved PBC-B region harboring a potential coiled-coil structure. In support of the cytoplasmic retention function, the NMHCB fragment competes with MEIS1A to redirect PBX, and the fly PBX homolog EXD, to the cytoplasm of mammalian and insect cells. Interestingly, MEIS1A also localizes to the cytoplasm in the presence of the NMHCB fragment. These activities are largely independent of nuclear export. The subcellular localization of EXD is deregulated in Drosophila zipper mutants that are depleted of nonmuscle myosin heavy chain. This study reveals a novel and evolutionarily conserved mechanism controlling the subcellular distribution of PBX and EXD proteins (Huang, 2003).

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