zipper
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 (TkvQ253D). 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).
Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro. CP190 has an essential function in the nucleus as a chromatin insulator, but centrosomes and MTs appear unperturbed in Cp190 mutants. Thus, the centrosomal function of CP190, if any, is unclear. This study examined the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. It is hypothesized that coordinates CP190 myosin-driven cortical contractions with the cell-cycle state of the internal nuclei. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos (Chodagam, 2005).
CP190 and CP60 are centrosomal microtubule-associated proteins (MAPs) that form a complex and shuttle between the nucleus in interphase and the centrosome in mitosis (Oegema, 1995; Kellogg, 1995). Both proteins interact directly with MTs in vitro, but their concentration at centrosomes does not depend on MTs (Oegema, 1995; Raff, 1993). The CP190 gene is essential for viability, and homozygous mutant animals die during late stages of pupal development. Surprisingly, these mutants have no detectable defects in mitosis, or in any aspect of centrosome or MT behavior. Moreover, a form of CP190 that cannot bind to centrosomes or MTs (CP190ΔM) can rescue the lethality associated with Cp190 mutations, demonstrating that the ability of CP190 to interact with centrosomes and MTs is not essential for fly viability. Recently, CP190 has been shown to act in the nucleus as a chromatin-insulator element that sets up boundaries between different regions of chromatin. Thus, CP190 appears to have essential functions in the nucleus, but its function at the centrosome, if any, remains unclear (Chodagam, 2005).
Several Drosophila centrosomal proteins are essential for the rapid rounds of mitosis that occur in the early embryo but are dispensable for mitosis at later stages of development. Therefore, whether CP190 might have an essential role at the centrosome during early embryogenesis was tested. This was not possible previously because CP190 mutant flies are inviable as a result of the nuclear requirements for CP190, and mutant flies rescued by CP190ΔM are generally unhealthy and are sterile. Therefore the Cp1901 and Cp1902 mutations were recombined onto an FRT chromosome so that germline clone (GLC) embryos could be generated (hereafter referred to as CP190GLCs). These embryos develop from heterozygous females whose germline is homozygous for the Cp190 mutation. CP190GLCs from either mutant contained essentially undetectable levels of the CP190 protein, and similar results were obtained with both alleles. Although CP190 was no longer detectable at centrosomes, mitotic spindles appeared to function normally, and the centrosomal localization of γ-tubulin, CNN, D-TACC, and Msps was largely unperturbed (Chodagam, 2005).
Although centrosomes and MTs appeared to behave normally in CP190GLCs, it was noticed that these embryos had a defect in axial expansion. In syncytial Drosophila embryos, the first zygotic nucleus is usually positioned toward the anterior. During nuclear cycles 4-7, the process of axial expansion causes the nuclei to spread out along the anterior-to-posterior axis so that, by nuclear cycle 7-8, they are distributed evenly throughout the length of the embryo. In CP190GLCs, axial expansion failed, and the nuclei remained abnormally clustered at the anterior of the embryo (Chodagam, 2005).
Axial expansion is a highly coordinated contractile process that requires both actin and cytoplasmic myosin II. A live analysis of myosin behavior, labeled by virtue of GFP-tagged myosin regulatory light chain (RLC, an obligatory subunit of functional myosin II), has shown that during axial expansion myosin undergoes cycles of recruitment to and dispersion from the cortex, in coordination with the nuclear-division cycles of the internal nuclei (Royou, 2002). Recruitment occurs during mitotic interphase and promotes a cortical contraction that is thought to drive axial expansion. This cyclical cortical recruitment of myosin requires the phosphorylation of one of the activating residues of the RLC (Royou, 2002) but does not require either microtubules (Royou, 2002) or an intact actin network (it is not perturbed by cytochalasin or latrunculin injection) (Chodagam, 2005).
To test if these cycles of myosin accumulation occurred in CP190GLCs, RLC-GFP behavior was examined in CP190GLCs. In optical sections of wild-type (WT) embryos expressing one copy of RLC-GFP, cycles of myosin cortical accumulation and dispersion were observed prior to the arrival of the nuclei at the cortex, and these continued when the nuclei were at the cortex, with RLC-GFP being strongly recruited to the cortex in interphase and dispersing from the cortex during mitosis. By contrast, in CP190GLCs expressing one copy of RLC-GFP, only very weak cycles of myosin II accumulation at the cortex could be observed, and these were more uneven than those seen in WT embryos. Even after the nuclei had arrived at the cortex, the accumulation of RLC-GFP at the cortex in interphase was much weaker in CP190GLCs than in WT embryos. Surprisingly, however, the subsequent accumulation of RLC-GFP at the leading edge of the cellularization furrows was equally strong in CP190GLCs and WT embryos. Moreover, in cellularized embryos, the accumulation of RLC-GFP in contractile rings during cytokinesis also appeared to occur normally in CP190GLCs. Thus, the organization of myosin appears to be disrupted in CP190GLCs specifically during the syncytial phase of embryogenesis (Chodagam, 2005).
That myosin organization was disrupted in CP190GLCs was confirmed by immunostaining fixed embryos with an anti-myosin heavy chain (MHC) antibody. Although MHC staining was strong in the cortical regions surrounding the nuclei of WT embryos, in CP190GLCs, MHC staining was much reduced and more irregular. As was the case with RLC-GFP, the localization of MHC to the leading edge of the cellularization furrow appeared to be normal in CP190GLCs (Chodagam, 2005).
Although myosin II behavior was profoundly disrupted in CP190GLCs, actin organization appeared to be unperturbed. In CP190GLC blastoderm embryos, cortical actin caps form over each nucleus, just as in WT. In preblastoderm WT embryos, a network of actin fibers and granules lies below the actin-rich cortex, and an actin-rich 'central domain' is associated with the internal nuclei during axial expansion; actin is also concentrated around the centrosomes during these early syncytial divisions. All these features of actin organization were maintained in CP190GLCs (Chodagam, 2005).
These observations suggested that the failure in axial expansion in CP190GLCs is due to a failure to properly recruit cortical myosin. Western blotting confirmed that the levels of MHC were not altered in CP190GLCs. To test whether CP190 might act upstream of myosin activation, it was asked whether an 'activated' RLC could rescue the axial expansion defect in CP190GLCs. The phosphorylation of the myosin RLC (on Ser-19 and, secondarily, on Thr-18 in vertebrates; these correspond to Ser-21 and Thr-20 in Drosophila) is required for myosin II motor activity. Blocking RLC phosphorylation, either by using mutant forms of the RLC in which these residues have been replaced by alanines (RLC-A20,A21) or by inhibiting Rho Kinase, whose activity is required for phosphorylating these residues, renders myosin II non-functional, eliminates its cortical localization, and leads to a failure in axial expansion. In contrast, replacement of these sites by phospho-mimetic glutamates (RLC-E20,E21) restores activity, as defined genetically, and appears to render the myosin constitutively active. Thus, phosphorylation is essential for the function and localization of myosin (Chodagam, 2005).
It was found that expression of one copy of a transgene encoding the activated form of RLC (RLC-E20,E21) partially rescues both the axial-expansion defects and myosin cortical recruitment in CP190GLCs. Importantly, the expression of one copy of this transgene in WT flies had no effect on axial expansion, and the expression of one copy of a WT RLC-GFP transgene did not rescue the CP190GLC axial-expansion defect. Thus, an 'activated' form of RLC can recruit MHC to the cortex during interphase and can rescue the axial-expansion defect in CP190GLCs, strongly suggesting that CP190 normally acts upstream of myosin II activation to regulate axial expansion (Chodagam, 2005).
A form of CP190 lacking the centrosomal and MT binding domain of CP190 (CP190ΔM) can rescue the adult lethality associated with mutations in the CP190 gene, presumably because this form of the protein can still function as a chromatin insulator in the nucleus. Therefore whether the axial-expansion defects of the CP190GLCs could also be rescued by CP190ΔM was tested. In CP190GLCs that expressed the full-length CP190 protein driven from the polyubiquitin promoter, the axial-expansion defect was strongly suppressed, and the transgenically supplied CP190 localized to centrosomes. In CP190GLCs expressing CP190ΔM driven from the polyubiquitin promoter, the axial-expansion defect was not significantly rescued and CP190ΔM did not localize to centrosomes. Thus, it appears that CP190 requires its centrosome/MT binding domain to function properly in axial expansion (Chodagam, 2005).
How might CP190 influence myosin activity? The cycles of cortical myosin II recruitment that drive axial expansion are regulated by oscillations in the activity of Cdc2-Cyclin B, with levels of cortical myosin being high in interphase and low in mitosis (Royou, 2002). This regulation is probably indirect; Cdc2-Cyclin B activity varies only locally around the nuclei during early embryo development, and cycles of myosin recruitment are initiated at the cortex long before the nuclei arrive there. Moreover, although Cdc2-Cyclin B can directly phosphorylate RLC in vitro, the removal of the potential Cdc2 phosphorylation sites in Drosophila RLC alters neither the myosin II recruitment cycles nor the ability of myosin to drive axial expansion (Royou, 2002). How local fluctuations in Cdc2-Cyclin B activity around the nuclei direct cycles of myosin recruitment at the cortex is therefore unclear, but it is speculated that CP190 plays a role in facilitating this process (Chodagam, 2005).
Cdc2-Cyclin B, for example, could regulate myosin by regulating the activity and/or localization of Drosophila rho kinase (Drok). This kinase is required for axial expansion (Royou, 2002), it regulates myosin II activity via phosphorylation of Thr-20 and Ser-21, and it is concentrated at centrosomes in at least some cell types. Perhaps CP190 facilitates the activation of Drok at centrosomes or the targeting of Drok from centrosomes to the embryo cortex (either by diffusion or along MTs). It has been shown previously that MTs are not essential for the cycling of myosin at the cortex (Royou, 2002), but these studies were performed when the nuclei had already reached the embryo cortex. Perhaps MTs are essential for the long-range signaling that must occur between the cortex and the nuclei/centrosomes during axial expansion. Because the interaction of CP190 with centrosomes and MTs is regulated during the cell cycle (Oegema, 1995; Kellogg, 1995), the involvement of CP190 in this process could ensure that the myosin-driven cortical contractions are coordinated with the cell-cycle state of the internal nuclei (Chodagam, 2005).
These data suggest that, whatever its mechanism, CP190 serves as a crucial link between the centrosome/MT and actin/myosin cytoskeletal networks during the early stages of Drosophila embryonic development. This mechanism may be specific for organisms that have a syncytial phase of development and so require that centrosomes influence actin/myosin behavior over considerable distances. Indeed, no obvious orthologs of CP190 have been identified on the basis of sequence homology in species other than insects. On the other hand, the fertilized eggs of many species are very large, and special mechanisms that allow the long-range communication between the centrosomes and the cortical myosin network may be required in these systems (Chodagam, 2005).
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.
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 (sqhAX3). 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).
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 sqhAX3 die, mostly
during the first larval instar, and sqhAX3 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 sqhAX3, 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
sqhAX3 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, DMYPT2-188 and
DMYPT2-199, 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-Rac7A. The
eyes of GMR-Rac7A/DMYPT03802 flies are much
smaller, with fewer bristles and hexagonal-shaped ommatidia, than those of
GMR-Rac7A/OreR flies. Consistent with the idea that the
P-insertion and the excisions are hypomorphic alleles, Df(3L)th102
enhances the GMR-Rac7A eye phenotype to an even greater
extent than either DMYPT03802, DMYPT2-188 or
DMYPT2-199. 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-RacN17, a dominant negative form of Rac driven by the
sevenless (sev) enhancer-promoter in the eye, and suppresses
the activity of sev-RacV12, which encodes a constitutively
active form of Rac. Consistently, overexpression of RhoA
(sev-RhoA) rescues sev-RacN17, 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).
Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. The function of myosin light chain phosphatase, which down-regulates myosin activity, has been characterized in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. This phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and the phenotype requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. It is suggested that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration (Lee, 2004).
Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin light chains (MLC), and two myosin regulatory light chains (MRLC). Phosphorylation of key serine and threonine residues on MRLC stimulates the ATPase activity of MHC and promotes its assembly into filaments, leading to stress fiber contraction. Mutations in the Drosophila orthologs of these myosin subunits have provided insight into the developmental functions of myosin II. Mutations in zipper (zip), which encodes MHC, cause defects in cytokinesis, closure of the dorsal embryonic epidermis over the amnioserosa, axon patterning, and myofibril formation. spaghetti squash (sqh), encoding MRLC, is required for cytokinesis, oogenesis, and imaginal disc eversion (Lee, 2004 and references therein).
Actin-binding proteins of the ezrin, radixin, and moesin (ERM) family are thought to link transmembrane proteins to the actin cytoskeleton. ERM proteins are activated by phosphorylation of a conserved threonine residue, which inhibits association between the N-terminal FERM domain and C-terminal actin-binding domain of the protein, freeing them to bind to other substrates. Moesin-like (Moe) is the only representative of this family in Drosophila. Moe mutants have abnormal oocyte polarity because defects in the anchorage of actin filaments to the oocyte cortex disrupt the localization of maternal determinants. In addition, Moe mutant cells in the wing disc undergo an epithelial-to-mesenchymal transition and adopt invasive migratory behavior (Lee, 2004 and references therein).
Interestingly, genetic and biochemical studies implicate the same kinase and phosphatase in the regulation of both nonmuscle myosin II and Moesin. Rho-associated kinase (ROCK/Rok) has been shown to phosphorylate MRLC in both mammalian and Drosophila systems. Myosin light chain kinase (MLCK) also can phosphorylate and activate MRLC; MLCK seems to act at the periphery of the cell, whereas ROCK is active in more central regions. Although ERM proteins are positively regulated by Rho GTPases, it is not clear whether they are directly phosphorylated by ROCK or by phosphoinositide-regulated kinases. However, in Drosophila wing disc development Moe seems to act antagonistically to Rho1 and rok (Lee, 2004 and references therein).
A major antagonist of the Rok/myosin signaling pathway is myosin light chain phosphatase (MLCP). This serine/ threonine protein phosphatase is a heterotrimer consisting of a catalytic subunit (PP1cdelta), a 20-kDa protein of unknown function, and the myosin binding subunit (MBS) that targets MLCP to its substrates, which include both MRLC and Moesin. Phosphorylation by Rok of a specific threonine within a conserved motif in MBS has been shown to inhibit MLCP activity; this suggests that Rok can positively activate MRLC and Moesin both by direct phosphorylation of these two substrates and also by inhibition of MBS. Like zip mutants, Drosophila Myosin binding subunit (Mbs) mutants fail to complete dorsal closure, suggesting that this process requires spatially regulated myosin activation. Mbs is also required for the growth of ring canals during oogenesis, and genetic interactions suggest that it opposes the functions in imaginal disc development of zip, Rho1, and rok. Likewise, Caenorhabditis elegans mel-11, which encodes MBS, and let-502, which encodes Rok, have opposite functions in embryonic elongation (Lee, 2004 and references therein).
Photoreceptor differentiation progresses across the Drosophila eye disc from posterior to anterior and is preceded by an epithelial indentation known as the morphogenetic furrow (MF). Cells in the MF undergo a transient contraction along the apical-basal axis and constrict their apical surfaces. After emerging from the MF, some of these cells assemble into ommatidial clusters, differentiate into photoreceptors, and extend axons through the optic stalk into the brain. Mbs mutations have been identified in a screen for genes required for normal photoreceptor differentiation. Findings on the role of Mbs in photoreceptor development suggest that photoreceptor neurons require Mbs to reduce myosin activity and thus prevent their cell bodies from migrating toward their axon terminals (Lee, 2004).
Mbs exerts its effects on eye development by regulating the phosphorylation state of the Sqh MRLC subunit of nonmuscle myosin II. The level of phosphorylated Sqh is greatly increased in Mbs mutant clones in both the eye and wing discs, and nonphosphorylatable or phosphomimetic forms of Sqh strongly modulate the severity of the Mbs phenotype. In addition, the effect of zip dosage on the Mbs phenotype indicates that p-Sqh acts through Zip to control photoreceptor localization. In vivo data show that in the eye disc Mbs is not required to dephosphorylate Moe. If dephosphorylation of Moe by Mbs occurs in vivo, it may be limited to specific tissues or developmental stages (Lee, 2004).
The identity of the kinase antagonized by Mbs in the eye is less clear. Although it has been reported that Rok can phosphorylate Sqh in vitro and that p-Sqh levels are reduced in rok mutant larvae, normal levels of p-Sqh were detected in rok2 eye disc clones. In addition, overexpression of Rok-CAT in the eye disc has no visible effect on photoreceptor differentiation or localization, and does not seem to enhance the Mbs phenotype. Rok may have a more significant effect on Sqh phosphorylation in other tissues; the lethality caused by overexpression of constitutively active Mbs is partially suppressed by coexpression of the catalytic domain of Rok. Myosin seems to be a downstream effector of Rho and Rok in wing and leg development, and the MEL-11 myosin phosphatase antagonizes the LET-502 Rho kinase in C. elegans development, supporting a role for Rok in phosphorylating Sqh in some cell types (Lee, 2004 and references therein).
Another kinase that might phosphorylate Sqh in the eye disc is MLCK. It has been reported that MLCK phosphorylates MRLC at the periphery of fibroblast cells, whereas ROCK acts in the central domain of these cells. Drosophila Stretchin-MLCK is a very large compound gene that produces multiple alternatively spliced transcripts, and no mutations in this gene have been identified, preventing the analysis of its interactions with Mbs. Another possible kinase is p21-activated kinase (PAK), which has been shown to increase the level of phosphorylated MRLC in cultured cells and to phosphorylate MRLC in vitro. Interestingly, overexpression of a myristylated form of PAK in Drosophila photoreceptors causes their cell bodies to detach from the eye disc epithelium and enter the brain, strongly resembling the Mbs mutant phenotype. Pak mutant photoreceptors develop normally except for axon guidance defects, suggesting that Pak is not essential for myosin activation in these cells. However, a second Pak gene, mushroom bodies tiny, is required for late photoreceptor morphogenesis and adherens junction integrity, and a third Pak gene is present in the genome, raising the possibility that these enzymes have redundant functions and complicating any analysis of their interactions with Mbs (Lee, 2004 and references therein).
The excessive myosin activity present in Mbs mutant photoreceptors causes them to adopt a more basal location in the eye disc and sometimes to enter the optic stalk. Several possible mechanisms for this phenotype have been addressed. Myosin can affect the shape of cultured cells by promoting the assembly of stress fibers and focal adhesions, and a transient accumulation of p-Sqh accompanies the apical constriction and apical-basal contraction of cells in the morphogenetic furrow. It was therefore interesting to enquire whether loss of Mbs might induce these cell shape changes in ectopic regions of the eye disc, resulting in mutant cells that formed a constitutive furrow. However, visualization of the apical surface of mutant clones by p-Tyr or phalloidin staining did not reveal any ectopic apical constriction of cells surrounding the photoreceptor clusters, suggesting that myosin phosphorylation is not sufficient to induce the cell shape changes that occur in the morphogenetic furrow. In addition, the integrity of the epithelial surface surrounding the photoreceptor clusters indicates that loss of Mbs specifically affects the localization of photoreceptor cells (Lee, 2004).
Another possibility is that Mbs mutant cells might undergo an epithelial to mesenchymal transition and become migratory. This phenotype has been reported for wing disc cells mutant for Moe, which encodes a potential substrate of Mbs. However, Mbs mutant cells in the wing disc remain within the epithelium and show no change in their apical-basal localization, although p-Sqh is up-regulated to a similar extent in both the wing and eye discs. In addition, Mbs mutant photoreceptors seem to retain some aspects of their epithelial character; they continue to express the epithelial apical junction proteins Patj, Crumbs, and E-cadherin. These proteins are present apical to mislocalized nuclei, suggesting that the entire cell is affected rather than the position of the nucleus within the cell. In contrast, the nuclei of klarsicht or Glued mutant cells are basally located within the cell due to defective dynein function (Lee, 2004).
The model that is favored is that unregulated myosin generates a traction force that pulls photoreceptor cell bodies toward their axon terminals. This would explain why the Mbs phenotype is specific to photoreceptors rather than wing disc cells or undifferentiated cells in the eye disc. It also would explain why the movement of mutant cells is directed toward the optic stalk or, in a disco background, toward the axon terminals within the eye disc. This abnormal force also might be accompanied by changes in adhesion to other cells or the substrate. Loss of Mbs could reduce the adhesion of epithelial cells to their neighbors, preventing them from withstanding the normal forces involved in axon extension. However, Mbs clones do not show the smooth borders characteristic of changes in adhesive properties (Lee, 2004).
It is not known whether the force generated by excessive myosin activity is located at the growth cone or in the cell body, although the latter model is favored because the highest levels of p-Sqh are found in apical regions of both wild-type and Mbs mutant cells. In vertebrate growth cones, two isoforms of the heavy chain of nonmuscle myosin II seem to have different locations and functions. MHCIIB is more peripheral and is required for axon outgrowth, whereas MHCIIA is central and is required for cell adhesion. Drosophila has only a single zip gene, which may perform both functions. The importance of MHCIIB in generating the traction force that allows growth cone extension suggests that this force might be increased in the absence of MLCP activity. There is a precedent for the idea that axon outgrowth can exert a pulling force on the cell body, because it has been shown that chick motor neurons will migrate out of the spinal cord along their axons if their movement is not blocked by boundary cap cells (Lee, 2004 and references therein).
The other possibility is that the actomyosin contraction takes place within the cell body, detaching it from surrounding cells and pulling it toward the growth cone. This would resemble the normal function of myosin in retracting the rear of migrating cells. Cell detachment and shrinkage has been reported for fibroblasts treated with an inhibitor of MLCP activity. Myosin light chain phosphatase activity may be specifically required in neuronal cells to allow axon extension to occur without triggering a migratory response in the cell body (Lee, 2004).
The contractile ring protein anillin interacts directly with nonmuscle myosin II and this interaction is regulated by myosin light chain phosphorylation. Despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. A role is proposed for anillin in spatially regulating the contractile activity of myosin II during cytokinesis (Straight, 2005).
The anillin protein is a multifunctional component of the cytoskeleton that is recruited to the furrow early in cytokinesis but functions primarily late in cytokinesis to focus contractility at the furrow. Anillin is known to directly interact with actin and contribute to the organization of the septin complex along actin filaments (Field, 1995; Kinoshita, 2002). This study shows that anillin also directly interacts with nonmuscle myosin II. This interaction with myosin II depends upon phosphorylation of myosin II regulatory light chain by MLCK, suggesting that anillin only associates with active myosin II (Straight, 2005).
Whether anillin functions in cytokinesis to recruit activated myosin II to the cleavage furrow was tested. Anillin depletion data in vivo rule out this simple model because myosin II is able to localize to the division site and promote furrow contraction with normal timing in the absence of anillin. Oegema (2000) observed reduction of the initial rate of furrow contraction after inhibiting anillin by antibody injections, but the current depletion data suggest this may have been due to the presence of antibody in the furrow rather than anillin removal. This study also found that anillin targets to the furrow normally when myosin II is depleted, although in this case contraction is completely inhibited. Those data are consistent with previous pharmacological studies where it was shown that inhibition of kinases that regulate cytokinesis interfere with targeting of myosin II, but not of anillin, to the furrow (Straight, 2003). It will be interesting to test in the future where the pathways that target myosin II and anillin diverge. Both require the continual presence of microtubules (Straight, 2003) and probably also activated Rho (Somma, 2002) to target normally (Straight, 2005).
The primary defect observed in cells that lack anillin is a delocalization of contraction at the end of cytokinesis. Observation of myosin II dynamics in anillin-depleted cells revealed that myosin II is no longer constrained to the contractile ring as it is in control cells and instead is found in the cell cortex concomitant with aberrant cell contraction. This aberrant contraction often results in both mispositioning of the cleavage furrow to yield binucleate cells or to furrow regression and thus binucleation. It was not possible to deplete all of the anillin by RNAi in human cells, thus complete depletion or inhibition of the anillin protein may result in an even more severe cytokinesis phenotype. It is not clear whether the phenotype observed represents extra contraction, for example, due to hyperactivation of myosin II, or relocalization of contraction due to mislocalization of active myosin II to ectopic sites. Distinguishing these hypotheses will require measuring contractile properties of the cortex at different positions. Because anillin is restricted to the contractile ring in unperturbed cells, it is unlikely that anillin outside the furrow inhibits myosin II. Overall, these data point to a model whereby anillin binding to activated myosin II restricts its activity to the furrow until cytokinesis can complete. Anillin is retained in the fully contracted furrow much longer than myosin II, and it is also present in intracellular bridges that are no longer contracting (Field, 1995). An extension of this model proposes that loss of myosin II from the fully contracted furrow is promoted by cell cycle-dependent modification of anillin and/or myosin, such as dephosphorylation of myosin regulatory light chain (Straight, 2005).
Several lines of evidence suggest that anillin controls, or at least coordinates multiple aspects of cytokinesis. Two anillin-related proteins in yeast, Mid1 and Mid2, organize distinct steps during cytokinesis. The Mid1 protein, like anillin, relocalizes from the nucleus to the contractile ring early in cytokinesis. Mid1 mutants are defective in septum placement and formation and overexpression of Mid1 disrupts cytokinesis. Several important differences exist between Mid1 and anillin. Mid1 is not essential, does not require actin filaments or microtubules to be maintained at the division site, and does not contract with the actomyosin contractile ring However, Mid1 is important for the initial organization of myosin II at the contractile ring and can interact with myosin II. A second anillin-like protein in fission yeast, Mid2, performs other functions that depend on anillin in metazoan cells. In particular, Mid2 organizes septins in fission yeast and is necessary for proper cell separation, whereas metazoan anillin binds directly to septins and participates in targeting septins to the cortex (Oegema, 2000). Mid2 mutant cells have no defect in myosin II localization or contraction at the end of cytokinesis. Thus, metazoan anillin may encompass the activities of both Mid1 and Mid2. It is speculated that the functions of anillin may be split in fission yeast because of the different mechanical requirements for cytokinesis. In yeast, remodeling of the cell wall may be the primary requirement for cytokinesis, whereas cytokinesis in metazoan animals is dominated by the need to physically constrict the equator of the dividing cell. In budding yeast, the mechanical requirements are different again, because the cell division site is predetermined at a narrow constriction. In that system, myosin II targets very early and no anillin-like proteins have been identified (Straight, 2005).
Anillin is known to be essential for the completion of cytokinesis in vertebrate cells and in Drosophila (Somma, 2002). Anillin's interaction with both the septin complex and with filamentous actin may be required for cell abscission. Myosin II leaves the contractile ring late in cytokinesis, but anillin persists at these contracted furrows, suggesting that anillin's role in the completion of cytokinesis may only be partially explained by its interaction with myosin II. The current results suggest an early role for anillin in cytokinesis to properly organize the contractile ring and a late function for anillin in restricting myosin II contraction to the furrow. In Drosophila embryos expressing mutant anillin, actin, and myosin II are disorganized during cellularization. This may reflect an analogous role for anillin in organizing myosin II at the cellularization front as well as at the contractile ring during cytokinesis (Straight, 2005).
The events of mitosis are temporally coupled by the activities of protein kinases that drive the cell cycle and the proteasome that inactivates these kinases and degrades other proteins involved in mitosis. A role has been demonstrated for proteolysis in the disassembly of the contractile ring (Straight, 2003) in vertebrate cells. Possible substrates for this proteolysis are anillin and the cell cycle kinase Polo. In yeast, Mid2 is degraded by ubiquitin-mediated proteolysis, it will be interesting to determine whether in somatic cells anillin is degraded upon mitotic exit, although no change was observed in anillin levels during the metaphase-to-interphase transition in Xenopus egg extracts. Mid1 is controlled by the activity of Polo kinase in fission yeast. In Xenopus extracts, anillin is rapidly dephosphorylated as cells exit mitosis and is efficiently phosphorylated by Polo kinase in vitro. Regulation of anillin by phosphorylation may provide another effective means of coupling the early and late events of cytokinesis to the cell cycle (Straight, 2005).
The results demonstrate a role for anillin in localizing the contractile activity of myosin in addition to anillin's previously identified functions in binding actin and organizing the septins. Thus, anillin seems to be a central factor for coupling the filament systems that interact during cytokinesis. Understanding how proteins such as anillin dynamically organize the cytoskeletal and regulatory networks that are integrated to accomplish cytokinesis will be key to understanding the process of cell division (Straight, 2005).
Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, focus was placed on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. Using RNAi and live video microscopy in Drosophila S2 cells, Rho-kinase (Rok) and myosin II were implicated in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the Myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. It is concluded that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II in comparison to other regulators of cytokinesis; this work suggests that a distinct regulatory pathway promotes cell elongation (Hickson, 2006 full text of article).
How the complex events of mitosis and cytokinesis are seamlessly coordinated remains largely a mystery. Cell elongation is a characteristic feature linking mitosis and cytokinesis in many cell types. However, it has not been apparent how much attention this event deserves; it could be construed as a secondary consequence of spindle extension or an early manifestation of the gradual recruitment of contractile elements that form the contractile ring. The current results suggest that, although it is inextricably linked with both mitosis and cytokinesis, there are distinctive genetic contributions to its success (Hickson, 2006).
One of the most striking findings was that depletion of rok function prevented anaphase cell elongation and caused a dramatic buckling of the spindle. Taking many observations into account, it is infered that the primary defect was one where the cortex failed to respond appropriately and, as a result, the spindle suffered a mechanical disruption as it encountered the unresponsive cortex. Thus, rok is required for remodeling the cell cortex during anaphase cell elongation, and perturbation of rok function disrupts the normal temporal coordination of cortex and spindle. In this regard, Rok might be required for the spindle to communicate with the cortex to stimulate elongation, or it might simply be required to execute elongation. In either case, the anaphase spindle extension alone is clearly insufficient to push the sides of the cell out and promote cell elongation. In addition, continued spindle elongation within the restricted confines of the rigid cortex demonstrates that there is no feedback signal from the cortex to the spindle. Thus, cell elongation and spindle extension are likely coupled only in a unidirectional manner: The cortex responds to the growing spindle, but the spindle does not sense an unyielding cortex (Hickson, 2006).
The data also clearly indicate that Rok is required for normal myosin II recruitment to the equatorial cortex. Myosin II is also required for cell elongation, suggesting that it is the relevant target of Rok action. In this regard, it is noted that a similar failure of cell elongation was observed in the neuroblasts of Drosophila larvae homozygous for sqh1, a hypomorphic spaghetti squash allele. These mutants show poorly elongated anaphase and telophase cells in which the segregated DNA masses were in tight apposition with the cell cortex. The similarity between those phenotypes and the ones described in this study in S2 cells strongly suggests that a similar Rok/myosin II pathway operates in vivo in the developing fly (Hickson, 2006).
Genetic-interaction studies have demonstrated that rok functions in the Pebble pathway to influence cytokinesis in the wing disc. However, rok mutant cells can divide at least several times to produce a substantial clone in the wing disc. Although this finding may lead one to question the importance of rok in cytokinesis, apparent dispensability in this context should not be taken as a lack of importance. Continued division of S2 cells with compromised rok function occurs in the face of major perturbations, and, after prolonged rok RNAi, frequent failures appear in cytokinesis. It is suggested that rok plays an integral part in promoting and coordinating cytokinesis and that successful cytokinesis with compromised rok function is testament to the robustness of the process (Hickson, 2006).
Given that cytokinesis is so robust, a consideration of how loss of rok function alters the normal progression of the process might provide more insight than a consideration of its overall success. In addition to the extreme defect in which furrowing is blocked, rok RNAi causes a pronounced delay in the onset of furrowing and reduction in the rate of ingression of furrows. Simultaneous depletion of Mbs prevented failures in cytokinesis but did not restore the normal timing of furrowing. Thus, Rok promotes whereas Mbs suppresses furrow ingression. Additionally, the normal timing requires Rok, indicating that its activity contributes to triggering the onset of furrowing. Studies in C. elegans (Piekny, 2002) have also found that Let-502 (the Rok ortholog) and MEL-11 (the Mbs ortholog) play antagonistic roles in furrow ingression, but in contrast to the observations in Drosophila S2 cells, in C. elegans, the activity of Let-502 appeared to control the speed of ingression without influencing the timing of onset of furrowing (Hickson, 2006).
Among the cytokinesis genes that were examined, only RNAi of rok and zipper (myosin II) gave a severe block to elongation, and, in the case of rok, this often gave a strong elongation effect without blocking furrowing. It was also found that RNAi of pebble and racGAP50c slowed elongation to half its normal rate while severely suppressing furrowing. RNAi of other cytokinesis genes, such as diaphanous, citron kinase, and anillin, did not interfere with elongation. Given these findings, it is suggested that elongation and furrowing, although they share some common functions, are differentially regulated and ought to be recognized as distinct subroutines in the overall process of cytokinesis. Given that Pebble is an upstream activator of Rho and Rok in cytokinesis, the finding that pebble RNAi gave a more mild elongation defect suggests that a different Rok activator promotes elongation: For example, Rok might be activated by other Rho-GEFs, as it is in interphase (Hickson, 2006).
Two processes likely contribute to cell elongation: equatorial contraction and polar relaxation. This duality may contribute to some of the apparent overlap in the regulation of elongation and furrowing. Rok mediated equatorial recruitment and activation of myosin II might contribute to equatorial contraction and provide one input into elongation. This input is likely to depend on Pebble and RacGAP50C which also localize to the equator and are known to influence Rho function. The partial defect in cell elongation induced by pebble or racGAP50c RNAi might be explained by disruption of this contraction. Other indications suggest that polar relaxation occurs in S2 cell cytokinesis and that rok RNAi interferes with the process. During elongation, the polar cortices bleb and appear to be actively remodeled as if signaled to do so by the approaching spindle poles, whereas following rok RNAi the segregating spindle poles push right up against the cortex. It is also noted that mitotic spindle poles have been shown in other systems to harbor active Rho (as evidenced through a GFP-Rho binding domain reporter, Rho-kinase and myosin II regulatory light chains phosphorylated on the Rho-kinase phosphorylation site. In addition, mammalian ROCKI/II are responsible for membrane blebbing during apoptosis and therefore clearly able to promote such an outcome. Thus, it is not inconceivable that a Rok/myosin II pathway could operate at the spindle poles during anaphase to promote polar relaxation. It is thought that Rok and myosin II function in two pathways, one governing equatorial contraction and the other polar relaxation, either of which can support cell elongation, whereas Pebble and RacGap50C interfere only with cortical contraction and hence result in a partial elongation defect (Hickson, 2006).
It seems likely that some divisions, particularly those that are asymmetric, might be particularly dependent on polar relaxation and hence be more sensitive to rok depletion than others. For example, asymmetric divisions might rely on differential actions at the spindle poles. Indeed, the sensitivity of the initial stages of polar-body formation in mouse eggs to an inhibitor of the mammalian Rok suggests that Rok may play a role in the formation of the cortical protrusion into which the spindle migrates in this highly asymmetric division (Hickson, 2006).
In summary, this study has uncovered pivotal roles for Rok in the earliest shape change of cytokinesis: anaphase cell elongation. A model of cell elongation and the onset of cytokinesis is depicted in a Model for Anaphase Cell Elongation and Initiation of Cytokinesis in Drosophila S2 Cells . As the spindle extends in anaphase B, Rok stimulates polar relaxation, allowing the spindle to push the sides of the cell out as it extends. In addition, Rok stimulates myosin II recruitment to the equatorial cortex, where it begins to contract in a broad zone. At the center of this broad zone, the contractile actin ring then forms and the cytokinetic furrow ingresses. Distinctions in gene requirements for anaphase cell elongation versus furrowing suggest distinctions between the two processes (Hickson, 2006).
It is intriguing that Rok and/or myosin II appears to be involved in the whole gamut of cell shape changes that occur during cell division: mitotic cell rounding, anaphase cell elongation, cytokinetic furrowing, and postmitotic spreading. This implies that the same fundamental machinery mediates each of these dramatic cytoskeletal rearrangements. Understanding how these events are regulated so as to ensure the appropriate response at the appropriate time is one of the challenges ahead (Hickson, 2006).
Reorganization of the actin cytoskeleton and contraction of actomyosin play pivotal roles in controlling cell shape changes and motility in epithelial morphogenesis. Dephosphorylation of the myosin regulatory light chain (MRLC) by myosin phosphatase is one of the key events involved. Allelic combinations producing intermediate strength mutants of the Drosophila Myosin-binding subunit (Mbs) showed imaginal discs with multilayered disrupted morphologies, and extremely mislocated cells, suggesting that Mbs is required to maintain proper epithelial organization. Clonal analyses revealed that Mbs null mutant cells appear to retract basally and localization of apical junction markers such as DE-cadherin is indetectable in most cells, whereas phosphorylated MRLC and F-actin become heavily concentrated apically, indicating misconfiguration of the apical cytoskeleton. In agreement with these findings, Mbs was found to concentrate at the apical domain suggesting its function is localized. Phenotypes similar to Mbs mutants including increased migration of cells were obtained by overexpressing the constitutive active form of MRLC or Rho-associated kinase signifying that the phenotypes are indeed caused through activation of Myosin II. The requirement of Mbs for the integrity of static epithelial cells in imaginal discs suggests that the regulation of Myosin II by Mbs has a role more general than its previously demonstrated functions in morphogenetic events (Mitonaka, 2007).
Mbs is essential for maintaining the integrity of imaginal disc epithelium. Imaginal discs of Drosophila are characterized by a monolayer of tall columnar epithelial cells with an apparent apical-basal polarity and defined morphologies. However, the shapes of imaginal discs in Mbs mutants are disorganized and the cells multilayered. In addition, those imaginal discs are fused with adjacent tissues. The results suggest that Mbs is essential for maintaining the proper morphology and organization of epithelial cells. In Mbs null mutant clones, cells lose normal apical organization as indicated by a loss of localization of apical junction markers such as DE-cadherin seen in wild-type cells. However, the effects on apical markers due to loss of Mbs differed slightly with those reported with photoreceptor cells by Lee (2004). Whereas Lee reported the retainment of apical localization of DE-cadherin in basally retracting Mbs mutant photoreceptor cells, this study found that most mutant clones cells of the wing imaginal disc appeared to lose localization of DE-cadherin and Dlg when they basally retracted or changed shape. However, sice the apically exposed area in mutant clones induced in wing disc epithelia was very small, and the resolution in vertical confocal sections of epithelia was insufficient, it is impossible to conclude whether apical markers merely lose their localizations or are completely lost in mutant clone cells (Mitonaka, 2007).
Phenotypes similar to Mbs have been observed in the mutants of Moesin, and it has been suggested that Moesin facilitates epithelial morphology by antagonizing the activity of Rho GTPase/Rho1 which activates Myosin II via Rho-associated kinase/Drok. Because Moesin binds to Mbs and is a potential substrate for MLCP (Fukata, 1998) and Mbs also acts antagonistically toward the Rho/Rho-associated kinase signaling cascade (Mizuno, 2002), the possibility was considered that Mbs could be dephosphorylating Moesin, as well as dephosphorylating MRLC directly. Although this seemed rather unlikely since dephoshorylation of Moesin is reported to lead to its inactivation, it was tested by immunostaining of phosphorylated Moesin in Mbs mutant clones to make certain. As changes in the levels of phosphorylated Moesin were not detected, increased phosphorylation levels of MRLC that were observed in Mbs mutant cells are likely to be due to loss of direct dephosphorylation of MRLC by Mbs. This interpretation is also supported by findings that apical F-actin appears to increase or become more concentrated in Mbs mutant clones whereas loss of Moesin activity causes loss of apical F-actin (Mitonaka, 2007).
Immunostaining of Mbs revealed that it specifically localizes at the apical domain of the columnar epithelial cells. The results suggest that the MRLC is locally phospho-regulated in the apical region of epithelial cells and that this dynamic regulation of Myosin II is important for the organization of the actin cytoskeleton and for the maintenance of epithelial cell integrity. Overexpression of constitutive active Sqh and Drok, which up-regulates Myosin II, showed results identical to Mbs mutations supporting the conclusion that the Mbs mutant phenotype occurs via the activation of Myosin II (Mitonaka, 2007).
Hyperactivation of Myosin II by the loss of Mbs or overexpression of the constitutive active Sqh or Drok also caused the gross mislocation of marked epithelial cells. It has been reported that the mutations in Mbs cause photoreceptor cells to drop out of the eye disc epithelium and move toward and through the optic stalk (Lee, 2004). In that case also, the highest levels of phospho-MRLC have been detected in the apical region of the mutant cells suggesting dependency on Myosin II activity (Mitonaka, 2007).
The importance of Mbs in maintaining epithelial integrity has been demonstrated in cells participating in dynamic processes, such as the leading edge cells of embryonic dorsal closure, the photoreceptor cells extending axons from the retinal epithelia, and the nurse cells with growing ring canals during oogenesis. All of these cells are known for specialization of cytoskeletal actin structure corresponding to their morphological changes in normal development. This study has shown requirement for the maintenance of the integrity of undifferentiated epithelial cells of the imaginal disc at a developmental stage when no dynamic morphological events other than cell proliferation occur. This suggests that the dynamic regulation of Myosin II in the apical region by Mbs has a more general role in epithelial cells than has been previously thought (Mitonaka, 2007).
The spatiotemporal regulation of the actomyosin cytoskeleton is important for epithelial morphogenesis, and MBS/Mbs plays an essential role in this process by negatively regulating Myosin II. This study showed that defects in Mbs activity were found to cause a loss of the apical cellular architecture typical of epithelial cells, and resulted in reduced adhesiveness, in tissue overgrowth, tissue fusion, and extreme mislocation of cells. Thus, Mbs fits many of the criteria for a potential neoplastic type tumor suppressor gene, which are genes in which mutant cells are thought to become neoplastic as a secondary effect of polarity alterations (Mitonaka, 2007).
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, termed Myosin light chain cytoplasmic, 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).
Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. This study reports the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which has been designated stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms, as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a 'gene fission' event, that encode independent transcripts and proteins with distinct structural and enzymatic functions (Champagne, 2000).
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 coneís 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 Roboís 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 coneís 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 coneís 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 coneís 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 Sose49 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).
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).
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 Dros