Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. This study shows, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, Zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms, since the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for Zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex (Liu, 2008).
Myosin II undergoes dynamic filament assembly and localization during cell division and other processes. Although the molecular mechanism of these dynamics is fairly well understood in Dictyostelium, it is less clear in higher organisms that lack myosin heavy chain kinase. To contribute to understanding of myosin II filament assembly and localization in higher organisms, this study examined the filament assembly and localization properties of the Drosophila non-muscle myosin II (Liu, 2008).
Of the >1000-residue zipper tail domain, only a 90-residue segment is required to assemble into oligomers that are disrupted by high salt, similar to filament assembly domains from various organisms. The 90-residue zipper assembly domain (AD) does not include the non-helical tailpiece and is close to the end of coiled-coil rod. Based on sequence comparison, the zipper AD encompasses the assembly competence domain (AD) of sarcomeric myosin II and overlaps the second assembly competence domain of human non-muscle myosin IIb, but is distinct from the region that is critical for assembly of vertebrate smooth muscle myosin II. There is low sequence homology between zipper and Acanthamoeba or Dictyostelium myosin II heavy chain tail domain beyond the presence of a heptad repeat (Liu, 2008).
Different models have been proposed to illustrate the specific domain associated with filament assembly. Acanthamoeba myosin II heavy chain required the COOH-terminal 14-heptad repeats plus the tailpiece to initiate bipolar filament assembly through hydrophobic as well as electrostatic interactions. The AD of Dictyostelium myosin II heavy chain is a 35-residue region within an extended assembly domain flanked by two regions containing alanines in core 'a' and 'd' heptad positions, with phosphorylation of 3 threonine residues controlling filament formation by folding back the tail. The 29-residue AD of vertebrate sarcomeric myosin II has the characteristic of clustered negatively charged residues in the center flanked by positively charged residues on each side. Negatively charged and positively charged assembly competence domains from human non-muscle myosin IIB are located 100 residues apart, and antiparallel electrostatic interactions between these domains are essential for filament nucleation. The Drosophila zipper AD identified in this study possesses an evenly distributed alternating charge repeat and is necessary and sufficient to oligomerize in vitro (Liu, 2008).
The lack of a consensus filament assembly model among different myosin IIs may be due to the considerable diversity in filament properties essential for the wide range of cellular activities within various cell types. However, investigation of the interaction between identified assembly domains and other interacting sites should reveal the general principles of filament assembly. The zipper tail domain possesses an alternating charge repeat that is thought to be important for filament assembly. This charge repeat is more evenly distributed in the AD than the rest of the tail, which fails to assemble into filaments. Currently a mutational analysis is being undertaken to test the importance of the charge repeat in filament stability (Liu, 2008).
This study found that interphase and mitotic cortical localization in S2 cells requires distinct tail domain elements. In interphase S2 cells, myosin II partitions between cortical and cytoplasmic pools, but the targeting domain that was identified, an interphase cortical targeting domain (iCTD), is biased toward cortical localization. This suggests that cortical targeting is regulated during interphase. The iCTD does not contain the filament AD so it is expected that it does not form filaments in the cell. Thus, at least one mode of interphase cortical targeting utilizes a mechanism that does not require filament assembly. However, when the iCTD is combined with the filament AD, the localization becomes punctate rather than the even distribution observed with the iCTD alone. It has been observed that deletion of the AD of alpha-cardiac MYH expressed in COS cells abolished the formation of a needle-shaped structure. Surprisingly, inclusion of the non-helical tailpiece causes this punctate localization to be lost, although no effect of the tailpiece was observed on the in vitro filament assembly characteristics. If punctate localization represents filament assembly, which is supported by the correlation of punctate localization with the presence of the AD, then the tailpiece may be involved in regulating filament assembly (Liu, 2008).
Myosin II localization becomes very dynamic during mitosis, and these dynamics appear to be important for function. For example, photobleaching experiments in Dictyostelium have shown that myosin II dynamically cycles between the cytoplasm and furrow and that this cycling requires filament assembly and disassembly. In S2 cells, myosin II is highly regulated and is not detected on the cortex early in mitosis but forms dynamic cortical aggregates at the metaphase-to-anaphase transition and ultimately forms a concentrated band around the central spindle that becomes the cleavage furrow. The initial recruitment of myosin II to the F-actin-rich equatorial cortex requires myosin regulatory light chain phosphorylation through Rho1 signaling. Because the recruitment of myosin to the equatorial cortex during mitosis is independent of F-actin, it is likely that the role of regulatory light chain is to regulate myosin II filament assembly rather than its actin-dependent ATPase activity. These data show that non-muscle myosin II filament formation is essential for cleavage furrow localization and requires the zipper AD but localization to the interphase actin cortex is independent of filament assembly. Consistent with this, the AD of vertebrate smooth muscle and human non-muscle myosin IIa is required for furrow localization in COS cells (Liu, 2008).
Surprisingly, the minimum filament AD (1849-1940) that forms filaments in vitro fails to localize to the cortex at any stage of the cell cycle. However, a slightly larger region (1744-1969), the furrow-targeting domain, efficiently localizes to the cleavage furrow. It is unlikely that these tail fragments localize to the cleavage furrow through mixed coiled-coils with endogenous myosin since several long tail domain fragments, including the iCTD, were not recruited to the cleavage furrow. However, based on the data, the possibility that these tail fragments are recruited to the cleavage furrow through oligomerization with endogenous myosin II cannot be ruled out (Liu, 2008).
What is the cortical anchor that recruits myosin II? Few proteins have been identified that bind the heavy chain tail domain. Possible cortical anchors include membrane phospholipids; the tail domains of mammalian non-muscle myosin IIs have been shown to bind phosphatidylserine-containing liposomes. Another candidate is the tumor suppressor Lethal (2) giant larvae (Lgl), which binds the myosin II tail domain and is cortically associated with the cytoskeleton. Given that different requirements were observed for interphase and mitotic cortical localization, it is likely that cortical recruitment in these two contexts utilizes distinct anchoring mechanisms. Future work will be directed at the identification of the cortical anchoring factors and possible filament regulatory mechanisms. The reagents described in this study should be useful in this regard (Liu, 2008).
E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. This study shows that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis results in cell intercalation defects. A pathway is delineated that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, evidence is provided that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin (Levayer, 2011).
Epithelial tissues have a robust architecture that is essential for their barrier function. This barrier function depends on their ability to build adhesive contacts at adherens junctions through the recruitment and stabilization of E-cadherin (E-cad), β-catenin (β-cat) and α-catenin (α-cat) by actin filaments (F-actin). During development, epithelia are also extensively reshaped by remodelling of cell contacts. This plasticity is essential for morphogenesis during embryogenesis and organogenesis. Work in the past decade showed that this requires force generation by actomyosin networks and their anchoring at cell junctions by E-cad/β-cat/α-cat complexes. Thus, E-cad plays a pivotal role in junction robustness and plasticity by mediating both adhesion (cohesion) and tension transmission (remodelling). Understanding what controls the distribution and dynamics of E-cad/β-cat/α-cat complexes is therefore key to understanding cell packing and the mechanics of tissue morphogenesis. Disruption of this balance marks key steps in the progression of solid tumours. The loss of epithelial organization during the epithelial to mesenchymal transition is an extreme example in which E-cad endocytosis causes the loss of adhesion and tension transmission at the cell cortex (Levayer, 2011).
The early development of the Drosophila embryo is a powerful system to study epithelial morphogenesis. Spatial regulation of force generation by actomyosin networks and force transmission to adhesion by E-cad both contribute to apical cell constriction in the invaginating mesoderm1, and cell intercalation in the elongating ectoderm called the germ band. Germ-band extension (GBE) is driven by cell intercalation in the ventrolateral region, whereby cells exchange neighbours through planar polarized junction remodelling, namely shrinkage of 'vertical' junctions (that is, junctions oriented along the dorsoventral axis). Intercalation is powered by non-muscle Myosin-II (Myo-II): anisotropic actomyosin contractile flows from the medial apical region to 'vertical' junctions drive junction shrinkage. The shortening of vertical junctions is stabilized by Myo-II at the cortex. Actomyosin contractility is transmitted at the cortex by E-cad complexes through β-cat. Interestingly, E-cad/β-cat/α-cat complexes also exhibit a planar polarized distribution complementary to that of Myo-II: E-cad is less abundant in shrinking 'vertical' junctions. This E-cad polarity is also required to orient actomyosin flows to 'vertical' junctions. It is unknown what controls the planar polarized distribution of E-cad. This may depend on Rho kinase (ROCK), which is required for the polarized distribution of Par3 (Levayer, 2011).
This study shows that the planar polarized distribution of E-cad is also controlled by an upregulation of clathrin- and dynamin-mediated endocytosis at adherens junctions, in particular in 'vertical' junctions of intercalating cells. Blocking endocytosis causes the loss of E-cad planar polarization and a block of intercalation. This led to an investigation of the mechanisms that control planar polarized upregulation of clathrin-mediated endocytosis (CME) of E-cad at adherens junctions. Activation of WASP (Wiscott-Aldrich Syndrome Protein) and the Arp2/3 (Actin-Related Protein 2/3) complex by Cdc42 controls the branched actin polymerization that is required for vesicular scission. This study identified an additional pathway controlling the initiation of E-cad endocytosis through the recruitment of the AP2 (Adaptor Protein 2) complex and clathrin. This recruitment is driven by lateral clustering of E-cad that relies on unbranched actin polymerization induced by Dia, and the presence of Myo-II. Dia and Myo-II are both activated by the guanine exchange factor RhoGEF2 (Levayer, 2011).
This study has delineated two distinct roles for actin in E-cad endocytosis. Dia and Myo-II control the initiation of E-cad endocytosis by enrichment of clathrin and AP2 in an E-cad-dependent manner. This is tightly spatially regulated in the ventrolateral region and in 'vertical' junctions during cell intercalation by cortical RhoGEF2 localization, an activator of Dia and Myo-II in Drosophila embryos. This is distinct from the role of branched actin polymerization by Arp2/3, which promotes vesicular scission similarly to dynamin. At later stages of development, this depends on WASP and is controlled by Cdc42, aPKC (atypical protein kinase C) and Cip4 (Cdc42-interacting protein 4. In early embryos, as WASP is inhibited by the JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription), Scar instead plays a critical role in vesicular scission. Inhibition of Arp2/3 in scar mutants and its constitutive activation (artificially induced by myrWASP) did not affect clathrin and AP2 concentration at adherens junctions, unlike Dia. The different tiers of regulation of E-cad endocytosis by Arp2/3 and Dia may reflect different roles for actin in constitutive versus regulated E-cad endocytosis. Certain situations require a rapid change in the rate of endocytosis, and may do so by tuning the rate of initiation by clathrin and AP2. It will be interesting to see whether rapid collapse of adherens junctions during epithelial to mesenchymal transition relies on a similar process (Levayer, 2011).
Crosslinking E-cad with an IgG is sufficient to promote dorsal endocytosis of E-cad by upregulating the concentration of clathrin, similarly to Dia activation, even following inhibition of Dia, Myo-II or RhoGEF2. Considering the highly correlated localizations of E-cad complexes with AP2 and RhoGEF2, it is proposed that Dia and Myo-II control the initiation of E-cad endocytosis by inducing lateral clustering of E-cad, similar to Fc receptor clustering during phagocytosis or nanoclusters of GPI (glycosylphosphatidylinositol)-anchored proteins. This may have been co-opted by the pathogen Listeria, whose entry into epithelial cells requires E-cad endocytosis. This mechanism may also require specific 'priming' of E-cad, by ubiquitylation as in mammals, although these tyrosines are not conserved in flies. Importantly, the mechanism of AP2 recruitment by E-cad remains unknown in all systems (Levayer, 2011).
Inhibition of E-cad endocytosis increased E-cad levels and disrupted its planar polarized distribution. Myo-II also accumulated in the medial apical region of cells. The GBE defects in shi-ts mutants or following clathrin inhibition are the result of the altered distribution of actomyosin tensile forces. E-cad/β-cat/α-cat complexes affect the lateral flow of medial actomyosin pulses and Myo-II polarized junctional accumulation, presumably through the regulation of tension transmission within the medial network and/or at the junctions. The medial accumulation of Myo-II when E-cad endocytosis is inhibited may thus reflect an inhibition of actomyosin flow towards the cortex. These results emphasize the interplay between actomyosin contractile dynamics and E-cad adhesive complexes during epithelial morphogenesis (Levayer, 2011).
Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle
myosin II are redistributed in the cells adjacent to the wound,
forming a supracellular purse string around the lesion. Purse string
contraction coordinates cell movements and drives rapid wound closure. By
using fluorescence recovery after photobleaching in Drosophila
embryos, this study found that myosin turns over as the purse string
contracts. Myosin turnover at the purse string is slower than in other actomyosin
networks that have a lower level of contractility. Mathematical
modelling suggested that myosin assembly and disassembly rates are both
reduced by tension at the wound edge. Laser ablation was used to show that
tension at the purse string increases as wound closure progresses, and
that the increase in tension is associated with reduced myosin turnover.
Reducing purse string tension by laser-mediated severing results in
increased turnover and loss of myosin. Finally, myosin motor activity is
necessary for its stabilization around the wound and for rapid wound
closure. These results indicate that mechanical forces regulate myosin
dynamics during embryonic wound repair (Kobb, 2017).
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).
Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. This study provides evidence for endocytic inhibition of actomyosin networks. Specifically, it was found that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, it was also found that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. It is proposed that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm (Lee, 2013).
Coupling actomyosin networks to the plasma membrane (PM)
is essential for cells to migrate, interact, change shape,
and divide. As examples, actin networks form and function
at the leading edge of migratory cells, at cell-substrate
adhesion complexes, and at cell-cell adhesion complexes in
multicellular tissues. To assemble these complexes, receptors
can physically engage the actin cytoskeleton and also induce
cytoskeletal assembly via Rho- family guanosine triphosphate
(GTP)ases and phosphoinositide signaling. Inversely,
endocytosis can remove receptors from the PM promoting
the turnover of adhesion and signaling complexes.
More generally, the close links of both actin networks and
endocytic machinery with the PM suggest possible crosstalk
between these subsystems. Indeed, endocytic signaling
nucleates local actin networks to help drive membrane invagination
and scission (Mooren, 2012; Anitei, 2012). In contrast, more widespread
membrane cytoskeleton activity can create tension that
inhibits membrane invagination. Conceivably, endocytosis
could also inhibit the membrane cytoskeleton, but such
activity is undocumented (Lee, 2013).
The syncytial Drosophila embryo is a well-established
model for studying actomyosin networks and membrane trafficking
during PM furrow ingression. In the early syncytial
embryo, nuclei divide synchronously just beneath the PM.
At each division cycle, the activities of Rho-family GTPases, the Arp2/3 complex, and the formin Diaphanous
(Dia) organize actomyosin-based PM ingressions
(pseudocleavage furrows) that surround each nucleus to prevent
nuclear collision and loss. Once ~6,000 nuclei form,
similar mechanisms induce a final round of PM ingressions.
These furrows persist and elongate through membrane trafficking
to apical and lateral sites, and with support of
actomyosin networks at their basal tips (the furrow canals). This massive PM growth cellularizes the first embryonic
epithelium, a process completed with constriction of
actomyosin rings formed at the base of each cell.
Recently, endocytic events were detected at the tips of
pseudocleavage furrows and early cellularization furrow canals
by the presence of Amphiphysin (Amph)-positive tubules
and the internalization of labeled PM (Sokac, 2008). These events have
provided a model for studying how the actin cytoskeleton
can both promote and inhibit endocytosis (Sokac, 2008; Yan, 2013). However,
the role of this endocytosis is unclear, and paradoxically, it
would appear to counteract membrane growth. This study examined
how Arf G protein (Arf) activation might be involved. In other
contexts, Arfs promote endocytosis by recruiting coat proteins,
activating lipid signaling, and triggering actin polymerization. Like other G proteins, Arfs are activated
by guanine nucleotide exchange factors (GEFs). Cytohesins
are a major class of PM Arf-GEFs (Donaldson, 2011), and roles for cytohesin
Arf-GEFs have been documented at migratory leading edges,
focal adhesions, and adherens junctions in mammalian cell
culture (Santy, 2005; Torii, 2010; Ikenouchi, 2010). Drosophila contains one cytohesin, called
Steppke (Step). Step is known to function in postembryonic
insulin and EGF signaling, which mammalian
cytohesins do as well, but its contributions to
the Drosophila embryo and to other cellular processes are
unknown. This study shows that Step promotes endocytosis
at pseudocleavage furrows and furrow canals to restrain actomyosin
networks at these sites (Lee, 2013).
These data provide the first description of cytohesin function in
a developing embryo. Drosophila Step promotes
a subset of endocytic events at the tips of ingressing PM
furrows during embryo cellularization. Endocytosis has been
documented previously at these sites (Sokac, 2008), but its role has
been unclear. By manipulating a conserved upstream activator
of endocytosis, this study has identified an important role of endocytosis
in controlling the membrane cytoskeleton. The data argue
that Step acts at furrow tips to induce local Arf-dependent
endocytosis, which in turn antagonizes Rho1-dependent
actomyosin network assembly at these sites.
It was also found that the cytoskeleton can inhibit endocytosis at
the furrow tips, as has been previously shown in this system
(Sokac, 2008; Yan, 2013) and in other contexts. An overall
model is proposed in which this reciprocal relationship is one-sided at specific
developmental stages. At newly forming PM furrows, Step
dominates, promoting endocytosis that keeps cytoskeleton
activity in check for proper pseudocleavage and cellularization
furrow architecture and growth. During later cellularization,
the cytoskeleton dominates. Zygotic expression of actin regulators
such as Nullo normally increases actomyosin activity
as cellularization proceeds and appears to work in
conjunction with Dia to block endocytic events at the furrow
tips (Sokac, 2008; Yan, 2013)]. By counteracting the inhibitory endocytosis,
cytoskeletal activity would elevate further but at these later
stages is locally restrained by a distinct mechanism requiring
Bottleneck (Schejter, 1993). To form the blastoderm, this second restraint
mechanism is removed, and contractile rings close off the
base of each cell. In the absence of the initial step-mediated
restraint mechanism, it is proposed that the cytoskeleton abnormally
dominates the relationship at all early PM furrows.
Without Step-based endocytic inhibition, it is speculated that
actomyosin networks abnormally expand and inhibit other
endocytic events leading to coexpansion of cytoskeletal polymers
and PM from the furrow tips (Lee, 2013).
An important element of the model is the local induction
of endocytic events. The data localize Step to the tips of
ingressing PM furrows, and both the loss and overexpression
of Step alter membrane organization specifically at these
sites. This localized Step-regulated activity occurs in a dynamic
global membrane trafficking system within each forming
cell. During the peripheral nuclear divisions, each nucleus
acquires its own endoplasmic reticulum and Golgi apparatus
that function with recycling endosomes to direct exocytosis
to growing PM furrows at cellularization. Simultaneously,
endocytic events occur over the apical PM and at
the furrow tips, with endocytosed material recycled
to the growing furrows. Thus, the overall membrane system
is in continual flux, and coordination by local regulation
would be expected. The data identify a polarized endocytic
activator required for the process. Step Arf-GEF activity is
critical for restraining the membrane cytoskeleton at furrow
tips, and a subset of AP-2 activities is involved as well (Lee, 2013).
How could endocytosis and actomyosin networks impact
each other at the tips of PM furrows or elsewhere? This question
can be considered from several levels of organization.
First, a simple and direct connection could be endocytic
removal of one or more PM actomyosin regulators. This work
identifies Rho1 or an upstream regulator as a candidate.
Intriguingly, membrane trafficking has been previously
linked to the Rho1 pathway in this context. Specifically, recycling
endosomes have been implicated in the trafficking of
RhoGEF2 to the PM (Cao, 2008). It was hypothesized that RhoGEF2
might also be a target of Step for its removal from the PM
but no difference was found in RhoGEF2 levels at furrow canals
in step loss-of-function embryos. Thus, Rho1 may be a more
specific target of Step, although a direct connection to the
Rho1 pathway remains to be determined. Of note, a number
of septins have also recently been observed on the Amph-positive tubules (Lee, 2013).
Second, interplay between different pools of actin is
possible. For example, actin contributes to the invagination
and scission of endocytic vesicles, and thus, endocytic
actin and other PM actin networks could compete for regulators
or components. Additionally, there could be signaling
crosstalk between regulators of the different networks. For
example, Arf signaling often elicits local Rac or Cdc42 activity, and this might trigger crosstalk affecting Rho activity. Interestingly, overexpression of Cdc42-interacting protein
4 (Cip4) appears to antagonize Dia at furrow canals,
although Cip4 mutants have no cellularization phenotype on
their own. Significantly, however, this study found that Step
acts with AP-2 to control the membrane cytoskeleton. This
Step-AP-2 cooperation suggests that clathrin-coated pits are
involved in the antagonism, although it does not exclude the
possibility of separate cytoskeletal crosstalk (Lee, 2013).
Third, larger scale interactions should be considered. Endocytosis
could remove membrane in bulk that would otherwise
support the membrane cytoskeleton, although observations
of residual furrow canal endocytic activity with step
loss of function suggest a more specific mechanism. Inversely,
the membrane cytoskeleton could block endocytosis by
elevating PM tensio or possibly by sterically blocking
endocytic machinery from accessing the PM (Lee, 2013).
Endocytic-cytoskeletal crosstalk is relevant to many cellular
processes. For example, receptor endocytosis occurs in proximity
to actomyosin networks in various contexts, including
migratory leading edges, focal adhesions, and adherens junctions. However, these endocytic events and actomyosin
networks have mainly been studied independently,
and thus their functional integration is not understood. This study highlights the possibility that endocytic activity at such assemblies could simultaneously remove receptors and
antagonize local cytoskeletal networks, with both effects promoting
complex turnover and cellular dynamics (Lee, 2013).
Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. This study shows that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. A similar role in cell segregation was shown for the Abdominal-B Hox gene. The results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells (Curt, 2013).
The sorting out of cells with distinct Hox activity in Drosophila has been reported before and in the case of the Hox gene Deformed a possible function in cell segregation has been assigned to such activity. This study has observed some cases that show that Ubx is needed to maintain segregation of cells from different segments during pupation. It is possible that Drosophila Hox genes may have a function in cell segregation during this pupal stage, where cells from different discs and histoblast nests fuse to develop the adult cuticle. The mechanism of segregation seems to rely on the confrontation of cells with different Hox function and not on the absolute levels of Hox expression. This implies that Hox activity in neighboring cells may be checked through proteins at the cell membrane whose expression or levels must be controlled by Hox genes. In the embryo, the Hox gene Abd-B has been shown to regulate molecules like cadherins, and such proteins may mediate segregation between adjacent cells with distinct Hox input (Curt, 2013).
In vertebrates, cells from different rhombomeres are also almost completely prevented from freely mixing. As was shown in this study for Drosophila, it has been proposed that the tension provided by the activity of actomyosin molecules, controlled by Hox genes, could prevent mixing of cells in the vertebrate's rhombomeres. Hox-directed cell segregation, therefore, prevents cells with different Hox code to intermingle, and therefore the appearance of homeotic transformations. This function of Hox genes may be an old one in evolution, required in animals in which development of different body regions is not coupled to the mechanisms of segmentation. In Drosophila, this role of Hox genes may not be needed in cells that are physically separated during most of development (as in imaginal discs and histoblasts from different segments) or superseded by the activity of proteins like Engrailed and Hedgehog, but the maintenance of different affinities by Hox genes and signaling pathways through myosin accumulation may be a general mechanism to segregate cell populations in different species (Curt, 2013).
The Drosophila body axes are established in the oocyte during oogenesis. Oocyte polarization is initiated by Gurken, which signals from the germline through the epidermal growth factor receptor (Egfr) to the posterior follicle cells (PFCs). In response the PFCs generate an unidentified polarizing signal that regulates oocyte polarity. A loss-of-function mutation of flapwing, which encodes the catalytic subunit of protein phosphatase 1β (PP1β) was identified that disrupts oocyte polarization. PP1β, by regulating myosin activity, controls the generation of the polarizing signal. Excessive myosin activity in the PFCs causes oocyte mispolarization and defective Notch signaling and endocytosis in the PFCs. The integrated activation of JAK/STAT and Egfr signaling results in the sensitivity of PFCs to defective Notch. Interestingly, the results also demonstrate a role of PP1β in generating the polarizing signal independently of Notch, indicating a direct involvement of somatic myosin activity in axis formation (Sun, 2011).
The AP body axis of Drosophila is established during oogenesis through intracellular communication between the oocyte and the somatic follicle cells. Correct oocyte polarity requires a polarizing signal generated by the PFCs, in response to an earlier signal (Gurken) that is secreted from the oocyte and received by the PFCs via Egfr. Previous studies have shown that genes regulating PFC proliferation, differentiation and epithelial polarity must function normally to render the PFC competent to signal back to the oocyte; however, the nature of this polarizing signal is still unknown, neither is it clear how the signal is produced or transmitted from the PFCs to the germline. This study reports a direct role of Drosophila PP1β in the production of the polarizing signal. Loss of PP1β in the PFCs due to the flwFP41 mutation causes a disruption of the oocyte MT polarity and the mislocalization of determinants of embryonic AP polarity indicative of a defect in the polarizing signal. This oocyte polarity defect was not observed with anterior or lateral follicle cell clones mutant for flwFP41, demonstrating that the activity of PP1β is required in the PFCs to repolarize the oocyte. It was also shown that heterozygous mutants of positive regulators of myosin activity suppress the oocyte polarity defect, whereas constitutive activation of Rok or expression of a mutant myosin targeting subunit in the PFCs induces a similar oocyte polarity phenotype. This supports the conclusion that myosin activity controls the polarizing signal in the PFCs (Sun, 2011).
The fact that elevated myosin activity in the PFCs interferes with the production of the polarizing signal raises the question of the specific function of myosin in this process. There are two separable effects of elevated myosin activity in the PFCs: an effect on Notch signaling and a Notch-independent effect. Loss of Notch signaling in the follicle cells inhibits the developmental progress of the PFCs and results in the disruption of the formation of the AP polarity in the oocyte. In flwFP41 PFC clones, the cells are still responsive to the patterning signals of Egfr and the JAK/STAT pathway and the mutant PFCs are able to adopt the posterior fate as indicated by the expression of pnt-lacZ. Therefore, the major problem in the generation of the polarizing event by loss of PP1β is not cell specification or cell survival. Instead, it is proposed that loss of Notch signaling directly affects the production of the polarizing signal, and that myosin activity is further required for the proper generation of this signal independently of its effects on Notch signaling, as discussed below (Sun, 2011).
It was shown that defective Notch signaling in flwFP41 mutant PFCs can be rescued by expression of NICD, but not by full-length Notch or Notch extracellular truncation (NEXT). This indicates that myosin hyperactivation through loss of PP1β disrupts Notch signaling probably at the level of the final Notch cleavage. This cleavage, which is γ-secretase dependent and generates the functional NICD, is subject to regulation at the level of endosomal trafficking. In mutants that disrupt entry of the receptor into early endosomes, Notch accumulates at the cell surface or below the plasma membrane with significantly reduced signaling activity. In mutants affecting the function of the Vacuolar ATPase, Notch signaling is also blocked at the step of the third cleavage, indicating that this cleavage requires an endosomal environment. An elevated level of Notch protein at the cell surface and in early and late endosomal compartments in the subapical cell cortex is observed in the flwFP41 mutant PFCs. It is therefore likely that the defective Notch activity in flwFP41 is caused by a failure of the receptor to efficiently enter early endosomes and subsequent sorting compartments. Such a defect in endosomal trafficking might be a direct consequence of abnormal myosin activity. The regulation of the actin cytoskeleton and of actin motor proteins plays an important role in the endocytic pathway in yeast and mammalian cells. In Drosophila embryos, cortical actin regulates endocytic dynamics at early cellularization. In addition, studies in mammalian cell culture have shown that Rho, Rok and myosin II directly regulate phagocytosis, revealing important roles of myosin II in the process of endocytosis. However, loss of PP1β does not cause a significant block in endocytosis in all cell types. It was found that flwFP41 clones in the eye discs allow apparently normal Notch signaling to occur and do not show ectopic Notch accumulation. Also no an overt endocytic defect in mutant eye disc cells was detected by performing a trafficking assay. In addition, mutant clones in anterior and lateral follicle cells did not show a defect in Notch signaling. This indicates a particular sensitivity of the PFCs to problems in Notch endocytosis and Notch activation, which is due to the coordinated activities of JAK/STAT and Egfr signaling (Sun, 2011).
The data strongly suggest that PP1β has an independent role in axis formation apart from its effects on regulating Notch cleavage and activation. Excessive myosin activity resulting from constitutive Rok activity, or from expression of a mutant myosin targeting subunit in the PFCs, disrupts Stau localization without inducing a measurable Notch phenotype. Additionally, expression of NICD only marginally suppresses Stau mislocalization caused in the flwFP41 mutant cells, whereas it strongly rescues the Notch signaling defect. Therefore, oocyte polarity defects were observed by myosin misregulation even in the presence of normal Notch signaling (Sun, 2011).
The effects of excessive myosin activity are also different from those of the Hippo pathway, which is also specifically required in the PFCs for axis formation. Similar to flwFP41, hippo mutant PFCs are defective in Notch signaling and result in oocyte mispolarization, and these defects are restricted to PFCs. However, previous studies demonstrate that the effects of the Hippo pathway are mediated solely by its effects on Notch. Hippo signaling itself appears to occur normally in the flwFP41 mutant follicle cells (Sun, 2011).
The abnormal accumulation of membrane proteins suggests a general membrane trafficking problem associated with myosin hyperactivation. It raises the possibility that PP1β regulates the polarizing signal, which might be a membrane associated protein, by controlling its intracellular trafficking as it is trafficked to the cell surface. However, hyperactive myosin caused by loss of PP1β function might also directly impede the interaction between the PFCs and the oocyte, possibly by affecting the function of cellular structures, such as microvilli, required for the presentation of the polarizing signal on the apical surface of the PFCs to the oocyte. Higher levels of components of apical membrane complexes as well as of the adherens junction proteins were observed on the apical surface, which might result from changes in the underlying actin cytoskeleton caused by excessive myosin activity. Consequently, changes in the membrane properties, especially on the apical side that contacts the germline, might also change cell surface xtions between the PFCs and the oocyte, which might then affect the transmission of the polarizing signal. A very local effect on oocyte polarity is observed when a subset of PFCs are mutant for flwFP41, where Staufen protein is still localized correctly in the oocyte underneath the wild-type cells, but is absent from the region underneath the mutant cells. This strongly suggests that the polarizing signal is not freely diffusing over longer distances, and points to local interactions between the PFCs and the oocyte (Sun, 2011).
One very puzzling aspect of the flwFP41 phenotype is the fact that the phenotypes of defective Notch signaling and cell overproliferation are restricted to the PFCs. Position-dependent phenotypes have been observed in mutants disrupting the epithelial integrity of the follicle cells, such as dlg1 and crb mutants. There, defects of the epithelial architecture, such as multilayering, are mostly observed at the poles of the egg chamber. In mutants of the Hippo pathway, dramatic Notch defects are observed in PFC clones but only modest ones in clones at other sites of the epithelium. Such position-dependent responses might be due to the special terminal positions of the cells at the poles where they could experience more mechanical stress than the lateral cells. Excessive myosin activity caused by loss of PP1β function might exacerbate the mechanical forces experienced by the PFCs, leading to posterior-restricted phenotypes. Alternatively, signaling events specific to subpopulations of follicle cells might cause the cells to react differentially to the loss of common gene products. Strikingly, it was found that the hyperactive myosin can lead to loss of Notch signaling and overproliferation when the Egfr pathway is activated in anterior follicle cells where JAK/STAT activity is normally present. Even the lateral cells produced these phenotypes when subject to the combined activity of JAK/STAT and Egfr signaling. Therefore, whereas loss of PP1β function elevates myosin activity in all the mutant cells independent of cell position, the coordinated activation of JAK/STAT and Egfr signaling creates a sensitized intracellular environment in the PFCs and renders them particularly susceptible to phenotypes such as defects in protein trafficking due to myosin misregulation. It is likely that particular targets of the combined activity of Egfr and JAK/STAT enhance the defects generated by the elevated myosin activity; however, it is presently unknown what these target proteins might be (Sun, 2011).
Overall this study has shown that the regulation of myosin activity by PP1β is crucial in the posterior follicle cells where overactive myosin interferes with intracellular trafficking and with the generation of the posterior polarizing signal. This demonstrates the importance of the general cellular physiology in both signal transduction as well as signal generation, and adds a layer of complexity to the analysis of developmental signals important for cell specification (Sun, 2011).
Dorsal closure of the Drosophila embryo is an epithelial fusion in which the epidermal flanks migrate to close a hole in the epidermis occupied by the amnioserosa, a process driven in part by myosin-dependent cell shape change. Dpp signaling is required for the morphogenesis of both tissues, where it promotes transcription of myosin from the zipper (zip) gene. Drosophila has two members of the Activated Cdc42-associated Kinase (ACK) family: DACK and PR2. Overexpression of DACK (Ack) in embryos deficient in Dpp signaling can restore zip expression and suppress dorsal closure defects, while reducing the levels of DACK and PR2 simultaneously using mutations or amnioserosa-specific knock down by RNAi results in loss of zip expression. ACK function in the amnioserosa may generate a signal cooperating with Dpp secreted from the epidermis in driving zip expression in these two tissues, ensuring that cell shape changes in dorsal closure occur in a coordinated manner (Zahedi, 2008).
The results on the regulation of zip expression by Dpp are consistent with the model of Fernandez and colleagues (2007) in which two rounds of dpp expression in the DME cells regulate dorsal closure. In the first round of dpp expression, before completion of germband retraction, Dpp signals from the DME cells to the amnioserosa. This signaling can be visualized by pMad staining in the amnioserosa, which is obvious during germband retraction but fades away by the beginning of dorsal closure, a pattern paralleled by zip expression. In the second round of signaling, occurring during dorsal closure, Dpp signals to the dorsal epidermis, as demonstrated by robust pMad in this tissue and high zip levels in the DME cells. Dpp signaling and ACK function are both necessary but not sufficient for zip expression in the embryo during dorsal closure. Activation of either Dpp signaling or ACK function in prd stripes does not lead to ectopic zip expression, indicating that in each case additional inputs are required. DACK is able to elevate zip expression only on the dorsal side of the embryo in regions where zip is normally expressed, indicating that required additional inputs are present; it is proposed that one such input is Dpp signaling. When DACK is overexpressed in the amnioserosa, either in prd stripes or throughout the whole tissue, its effects on zip expression are non–cell-autonomous, leading to up-regulation of zip expression throughout the dorsal side of the embryo. With regard to the zip expression pattern, seen with prd-Gal4-driven DACK transgenes, a diffusible signal emitted from prd stripes in the amnioserosa could attain a fairly uniform distribution over the dorsal side of the embryo. It is proposed that Dpp secreted from the DME cells cooperates with a diffusible signal from the amnioserosa (regulated by ACK in a kinase-independent manner) to drive coordinated zip expression in these two tissues. ACK appears to make the larger input into zip expression as DACK overexpression results in a clear elevation in zip levels on the dorsal side of the embryo, but this is not seen with excessive Dpp signaling. Simultaneously activating Dpp signaling and overexpressing DACK with prd-Gal4 is not sufficient to promote ectopic zip expression (for example in the ventral epidermis), indicating that other components are required for zip expression, consistent with DACK operating through downstream signaling events (Zahedi, 2008).
It is well established that communication between the amnioserosa and the epidermis is critical for embryonic morphogenesis, and this study has identified the zip locus as one target of such crosstalk, with zip transcription in both tissues dependent on signals secreted by both tissues. A diffusible signal from the amnioserosa to the epidermis has been proposed in the regulation of germband retraction by Hindsight, a transcription factor that is member of the U-shaped group of genes expressed in the amnioserosa, and preliminary results indicate that the U-shaped group is involved in the regulation of zip expression in both the amnioserosa and the epidermis. How could ACK tie into transcriptional regulation of a diffusible signal from the amnioserosa to the epidermis? There is evidence that ACK functions in clathrin-mediated receptor endocytosis in a kinase-independent manner, and is possible that ACK regulates by receptor endocytosis a pathway in the amnioserosa that leads to a transcriptional response. These data suggest that the kinase activity of DACK may actually impair its ability to drive zip expression, as KD-DACK promoted higher zip levels than wild-type DACK. Interestingly the binding of mammalian ACK1 to SNX9/SH3PX1, a member of the sorting nexin family of proteins involved in the sorting of proteins in the endosomal pathway, is inhibited by ACK1 kinase activity . The interaction of ACK with a sorting nexin is conserved in flies, where Drosophila SH3PX1 binds to DACK (Zahedi, 2008).
In addition to the transcription of myosin in the DME cells being dependent on input from the amnioserosa, assembly of the actomyosin contractile apparatus in the DME cells requires an expression border for the adhesion molecule Echinoid between the amnioserosa and the epidermis. The juxtaposition in epithelia of cells expressing Ed with those not expressing Ed triggers actomyosin cable assembly. Ed is expressed in the epidermis but not the amnioserosa and this provides a means, in addition to ACK-mediated signaling, by which the amnioserosa 'communicates' with the epidermis in regulating the actomyosin contractile apparatus (Zahedi, 2008).
Previous studies have found that global activation of Cdc42 signaling in tkv mutant embryos could suppress the dorsal closure defects caused by a reduction in Dpp signaling, and the present results indicate a major route of action for this suppression is ACK. The activation of Cdc42 throughout the embryo leads to increased expression of DACK specifically in the amnioserosa, and this study has shown that overexpressing DACK in this tissue can suppress tkv dorsal closure defects. The results indicate a tissue-specific regulation of DACK levels by Cdc42 that may be part of a sophisticated signaling network enabling the coordinated morphogenesis of tissues in the embryo. DACK does not bind Cdc42 but PR2 does and Cdc42 may also regulate ACK function during dorsal closure through direct interaction with PR2. The serine/threonine kinase dPak, an effector for Rac/Cdc42, may also be a component of Cdc42-mediated communication between the amnioserosa and the epidermis during dorsal closure. It has been shown that dPak expression in the amnioserosa is regulated by Cdcd42, but in the opposite direction from DACK in that impairment of Cdc42 signaling leads to elevated dpak transcription in this tissue. Impairing dPak kinase activity through amnioserosa-specific expression of the dPak autoinhibitory domain leads to defects in head involution and dorsal closure (Zahedi, 2008).
Head involution defects and germband retraction failures are seen in ACK-deficient embryos and loss of DACK can suppress the head involution defects and germband retraction failure caused by overexpression of Dpp. These results suggest that Dpp signaling and ACK cooperate in the regulation of these morphogenetic events in addition to dorsal closure. A recent review has highlighted the parallels between dorsal closure and head involution in terms of morphogenetic events and the genes required, with both involving epithelial sheet migration, zip expression and Dpp signaling. DACK overexpression leads to excessive zip levels in the head and it is likely that ACK and Dpp signaling work together to provide myosin for head involution. That signaling from the amnioserosa is involved in regulating head involution is supported by an earlier finding that impairing dPak function in the amnioserosa causes failures in this process (Zahedi, 2008).
Does ACK impact Dpp signaling other than at the level of zip transcription? Homozygosity for a DACK allele suppresses the ectopic wing vein phenotype caused by excessive Dpp signaling. It is likely that this phenotype is caused by something other than misregulation of zip expression, and ACK could be regulating the expression of a subset of Dpp target genes (other than dad or salm) or may be interacting with the Dpp pathway at another level (Zahedi, 2008).
TGF-β family signaling is a central regulator of dorsal closure and other epithelial fusions, but how Dpp controls dorsal closure has not been well-defined. We have shown that regulation of zip expression in cooperation with the Drosophila ACKs constitutes a major route of action of Dpp during dorsal closure. These findings may be relevant to vertebrate wound healing, in which closure of the wound involves both epithelial movement and TGF-β–dependent contraction of connective tissue in the wound (Zahedi, 2008).
The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. This study identified a missense allele of Drosophila Survivin (FlyBase name: Deterin) that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this 'separation-of-function' allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems (Szafer-Glusman, 2011).
The chromosomal passenger complex (CPC), composed of the Ser-Thr kinase Aurora B and three partner proteins, plays several key roles in mitosis and meiosis, including regulation of attachment of kinetochores to microtubules, the spindle checkpoint that delays anaphase onset until all chromosomes are under tension on the spindle, regulation of sister chromatid cohesion, and cytokinesis. To accomplish these different tasks, the Aurora B kinase must be exquisitely localized in space and regulated in time. During mitosis, Aurora B associates with the microtubule-binding protein Inner Centromere Protein (INCENP), Borealin/DASRA/CSC-1, and the small, multifunctional BIR-motif protein Survivin to form the CPC (Szafer-Glusman, 2011).
Dissecting the role of individual CPC components has been hampered by the extraordinary interdependence of the four subunits; depletion of any single CPC protein by RNA interference knockdown in human cells affected the structural unit, localization, and function of the CPC. The structural basis of this interdependence is evident in the crystal structure of the Survivin-Borealin-INCENP core of the CPC complex, in which Borealin and INCENP associate with the C-terminal helical domain of Survivin to form a tight three-helix bundle (Szafer-Glusman, 2011 and references therein).
Strict localization of Aurora B by the CPC ensures that this kinase, which has multiple substrates, phosphorylates the correct targets at the proper points in cell cycle progression. Concentrated on chromosomes from G2, then at inner centromeres from prometaphase until the metaphase-to-anaphase transition, the CPC is required to regulate chromosome condensation, spindle formation and dynamics, kinetochore maturation, kinetochore-microtubule interaction, correct chromosome alignment, and control of the spindle checkpoint. At anaphase onset the CPC then translocates to the central spindle (CS) midzone and equatorial cortex and is involved in CS formation. At the CS Aurora B phosphorylates the centralspindlin components, Pav/mitotic kinesin-like protein 1 (MKLP1)/Zen-4 and the RacGAP50/MgcRacGAP/Cyc-4. However, the mechanisms that target the CPC to the spindle midzone and equatorial cortex after onset of anaphase and the mechanisms by which the CPC regulates central spindle formation and cytokinesis are not understood. In addition, the requirements for CPC function for critical events in metaphase and at the metaphase-anaphase transition have complicated analysis of how the CPC is localized and functions at later stages for cytokinesis (Szafer-Glusman, 2011).
This study characterized the role of Drosophila Survivin (dSurvivin), previously termed deterin and analyzed in its antiapoptotic activity, as a regulator of cell division, identifying a missense mutation (scapolo) in the Drosophila Survivin BIR domain that allows recruitment and function of the CPC in metaphase but disrupts CPC localization and function in anaphase and telophase. The findings reveal that Survivin plays a role in targeting the CPC and centralspindlin to the central spindle and the equatorial cell cortex during anaphase. In spermatocytes, Survivin function is also required to localize Polo and localize the small GTPase RhoA to set up the contractile ring machinery at the onset of cytokinesis. In larval neuroblasts undergoing mitotic division, however, the scapolo mutant did not block initial accumulation of Rho to a band at the equatorial cortex, although it did cause failure of cytokinesis. The different requirements for Survivin function for equatorial accumulation of Rho in spermatocytes versus neuroblasts may reflect a fundamental difference in the series of steps that lead to formation of the contractile ring in these two cell types (Szafer-Glusman, 2011).
A missense mutation leading to substitution of serine for the wild-type Pro-86 of Drosophila Survivin uncouples the function of Survivin in metaphase from function during anaphase and telophase, indicating a direct requirement for Survivin and the chromosomal passenger complex in orchestrating the profound reorganization of the cortical cytoskeleton at the cell equator at the onset of cytokinesis. This 'separation-of-function' allele allowed analysis of Survivin and CPC function during cytokinesis, which is normally obscured by the better-known roles of the CPC at centromeres during metaphase, when it facilitates alignment of chromosomes to the spindle equator and mediates the spindle checkpoint. The finding that a point mutation in the BIR domain disrupts activity of Survivin during cytokinesis challenges the model that the C-terminal domain of Survivin is sufficient for cytokinesis function (Lens, 2006) and indicates that residues in the BIR domain are important for localization and activity of Survivin at the central spindle (Szafer-Glusman, 2011).
Survivin associates with kinetochores and the central spindle with different dynamics, being highly mobile in prometaphase and metaphase and strongly immobile at the anaphase central spindle. This change in dynamics may underlie the largely normal localization and function of scapolo (scpo) (a missense allele of the Drosophila Survivin) at metaphase but the fully penetrant effect on assembly of the F-actin contractile ring and cytokinesis observed in scpo mutants (Giansanti, 2004; Szafer-Glusman, 2011).
Cytokinesis depends on the assembly of an equatorial actomyosin ring regulated by local activation of the small GTPase RhoA at the cortex, in turn catalyzed by the RhoGEF Ect2/Pebble. It has been proposed that association of RhoGEF/Pebble with centralspindlin promotes local RhoA activation at the cortex. In addition, the kinase polo (PLK1) has been implicated in RhoGEF localization and Rho activation, at least in part by phosphorylation of the centralspindlin component MgcRacGAP. The current observations that the Drosophila RhoA homologue, Rho1, failed to accumulate at the equatorial cortex in scpo mutant spermatocytes implicate Survivin and the CPC in the mechanism(s) that localize and activate RhoA at the equatorial cortex in these cells. This requirement may in part act through effects on Polo kinase. Failure to localize Polo to the central spindle in scpo mutant spermatocytes could prevent localization of RhoGEF by the centralspindlin complex and the consequent activation of Rho at the cortex. In this model, failure to localize Polo may contribute to the failure to form an equatorial ring of localized Rho1 and, in consequence, the inability to form a localized ring of myosin regulatory light chain and F-actin in scpo mutant male germ cells undergoing meiotic division. This mechanism may also explain the failure to maintain pole-to-equatorial microtubules observed in scpo mutant spermatocytes. It is likely that Rho-mediated activation of the Formin Dia helps stabilize microtubule arrays at the equatorial cortex of dividing cells, as active Rho and Formin (mDia) have been shown to regulate stabilization of microtubule arrays at the cortex of migrating fibroblasts. Consistent with this model, this study found that microtubules reached the plasma membrane at the equator of scpo dividing spermatocytes, but the bundles are transient and fail to form stable arrays at the cortex (Szafer-Glusman, 2011).
A striking finding of this work is the difference in requirement for Survivin function for localization of the Polo kinase and RhoA in anaphase neuroblasts versus spermatocytes. This difference raises two possibilities: either Survivin is not part of a universal signaling mechanism that directs cytokinesis, or different semiredundant mechanisms can drive cytokinesis, similar to redundancy between astral pulling and sliding of central spindle microtubules for anaphase B, and different cell types rely more strongly on one mechanism or the other. Indeed, consistent with the latter possibility, spermatocytes and neuroblasts display different cytoskeletal architectures during cytokinesis (Giansanti, 2006). In neuroblasts, actomyosin initially accumulates in a broad cortical band, presumably because this is the region of the cell cortex that escapes repression of Rho associated with the plus ends of astral microtubule. This initial wide band gradually narrows into a tight equatorial ring as the cell progresses into telophase. Thus assembly of the contractile apparatus in neuroblasts proceeds, as proposed for Caenorhabditis elegans embryos, in 'two genetically separable steps' in which localization of contractile machinery is initially independent of the central spindle. In support of this model, this study found that Rho1 accumulates in a broad cortical band in scpo mutant neuroblasts, suggesting that the first stage can occur independent of Survivin and CPC localization to the central spindle (Szafer-Glusman, 2011).
Spermatocytes, in contrast, do not form an initial wide equatorial band of contractile ring components. Instead, from their first appearance in early anaphase, the actomyosin rings in spermatocytes are tightly focused at the cell equator). It is speculated that this restricted initial localization of contractile ring components and the apparent lack of a preceding wide equatorial band may be a consequence of a more stringent global block to Rho1 activation at the cortex in spermatocytes than in neuroblasts. It is proposed that this global block is eventually overridden by positive regulation of Rho1 by local concentration of RhoGEF, in turn facilitated by CPC-dependent events associated with and/or localized by central spindle microtubules. Rho1 activation would then occur within a narrow peak exactly at the site where pole-to-equator microtubules interact to maximize RhoGEF deposition/concentration at the cortex. Indeed, F-actin ring assembly occurs locally and cytokinesis initiates immediately after the pole-to-equator microtubules contact the cortex in Drosophila spermatocytes. It is proposed that, according to this model, the defects in Survivin lead to lack of CPC activity and abnormal centralspindlin, resulting in absence of Rho1 and Polo kinase from the equator of scpo mutant spermatocytes (Szafer-Glusman, 2011).
In neuroblasts, where a more permissive cortex allows a broad belt of Rho1 activation at the cell equator, Survivin and CPC appear to promote gradual convergence of the initial broad band into a narrow ring centered at the maximum of RhoGEF activity at the cortex. In scpo mutants, which display irregular anaphase central spindles devoid of Pav, the broad Rho1 cortical band fails to narrow, the cells fail to form a focused, narrow ring of myosin, and cell division proceeds with inefficient and incomplete constriction (Szafer-Glusman, 2011).
A key difference between neuroblasts and spermatocytes that may account, at least in part, for the differences in behavior of Rho1 and myosin complex proteins is in the relationship between Polo kinase and the CPC observed in scpo mutant mitotic versus male meiotic cells. In spermatocytes, Polo and the CPC are interdependent and Polo colocalizes with the CPC along its full journey from metaphase through anaphase and telophase. In neuroblasts, in contrast, Polo localization during cytokinesis appears to be independent of the CPC and centralspindlin, at least at early stages of cell division, but Polo appears to colocalize with Feo, the Drosophila homolog of PRC1, that required for central-spindle formation and cytokinesis. A second difference between neuroblasts and spermatocytes may be the recently described, spindle-independent backup system that can localize myosin to a broad band at the cell cortex near the future cleavage plane under control of the neuroblast cell polarity system. The broad localization of myosin to the cell cortex observed in ana/telophase neuroblasts in scpo mutants may be in part due to these redundant mechanisms (Szafer-Glusman, 2011).
The formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, this study found that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. This study shows that Crumbs directs aPKC anisotropy which negatively regulate myosin II, probably through Rok. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized (Roper, 2012).
Myosin II has emerged as a key player in morphogenesis because of its ability to form contractile structures together with F-actin that can directly alter the shapes of cells. Different pools of myosin II within epithelial cells undergoing morphogenesis have been observed, namely apical junctional myosin, apical medial myosin, and in addition myosin organized into supracellular structures termed myosin cables or purse-strings. All three myosin II pools have been shown to be important for epithelial morphogenesis, but how much the activities of the pools depend on each other and how their specific assembly is regulated is much less clear (Roper, 2012).
Using the formation of the invagination of the salivary glands in the fly embryo as a model allowed me to analyze a morphogenetic process in which all three different pools of myosin are present. Upon specification of the gland placode, myosin II levels are drastically upregulated in the secretory cells of the placode, and myosin accumulates at cortical regions and medially within the apical 'dome' of each cell. In addition, a supracellular myosin cable surrounding the placode is formed in a process by which parts of existing structures (remnants of parasegmental cables) are joined together with a newly specified dorsal section of the cable (Roper, 2012).
Compared to mesoderm invagination in the fly, a well-studied process that depends on both apical medial and cortical myosin assemblies, the invagination of the tubes of the salivary gland topologically rather resembles wound healing or dorsal closure processes, as the surrounding epidermis is drawn in from around the placode to cover the patch where cells are invaginating into the embryo (Roper, 2012).
All three processes have in common that the patch of cells 'disappearing' from the plane of the epithelium is surrounded by a contractile actomyosin cable. In contrast to wound healing and dorsal closure, the cable in the case of salivary gland tubulogenesis is assembled within the cells on the inside, whereas it is assembled in the surrounding epithelial cells in the former two instances. Thus, the signal for cable assembly is provided by the 'inside' cells in the salivary gland placode (Roper, 2012).
The laser ablation data presented in this study clearly demonstrate that the cable around the placode is under increased tension, even when compared to other myosin enriched edges. The tension is in magnitude comparable to the tension determined for shorter supracellular myosin cables observed during germband extension in the fly embryo. This increased tension indicates active involvement in the invagination process. Previous modeling studies on sea urchin invagination have shown that a contractile apical ring surrounding a placode could be a driving force for invagination. Interestingly, upon laser ablation the cable around the salivary gland placode was very quickly repaired, suggesting a continuous signal to assemble myosin at the outermost surface of the placode. This fast repair precluded laser ablation as a means of probing function of the cable in the invagination in contrast to medial and junctional myosin (Roper, 2012).
Crumbs, the transmembrane component of the apical protein complex, shows a very striking anisotropic localization at the border of the placode, that is complementary to the accumulation of myosin II forming the cable. The data strongly support a model whereby Crumbs intracellular tails at cell edges facing toward the inside of the placode recruit aPKC, which can act as a negative regulatory factor impinging on Rok, thus preventing cable assembly at edges containing high levels of Crumbs tails. This leaves active Rok at the cell edges forming the placode boundary, where it acts to recruit myosin into the cable.
Interestingly, only the presence or artificial introduction of cortical anisotropy of Crumbs and downstream aPKC has this effect. The central cells of the placode that are not forming the boundary all show strongly upregulated levels of Crumbs, aPKC, Rok, and myosin II, but in these cells a high density of Crumbs tails does not preclude accumulation of junctional membrane-proximal myosin. Thus, the change in density of Crumbs tails, not the overall concentration, is instructive in this system (Roper, 2012).
Crumbs has previously been shown to have an effect on salivary gland morphogenesis through a proposed regulation of the apical membrane domain and has been implicated in tracheal pit invagination through regulation of phospho-Moesin). Also, members of the Crumbs polarity complex have been shown to be able to interact with the Par3 (Bazooka)/Par-6/aPKC complex (e.g., Par-6 can bind to Crumbsintra; aPKC can phosphorylate Crumbsintra. Also, an anticorrelation between localization of Crb and aPKC compared to Lgl and myosin has been described in the denticle belts of the fly epidermis (Kaplan, 2010). This work now describes a potential link from Crumbs through aPKC to Rok and myosin II, which would link the interaction of two different polarity factors directly with the coordination of morphogenesis through myosin II at a molecular level (Roper, 2012).
The large extracellular domain of Crumbs has long posed an enigma with regard to its role. Crumbs' function in epithelial polarity can mostly be mediated by its intracellular domain (Klebes, 2000). Only for photoreceptor morphogenesis, the extracellular domain appears required within the fly, though its molecular role is unclear. The protein domains present in the extracellular domain, namely EGF repeats and lamG domains, are both found in many classical and nonclassical cadherins. Data presented in this study suggest that Crumbs could be organized in the plasma membrane through homophilic interactions of the extracellular domains between molecules on neighboring cells: Crumbs shows highly anisotropic localization within the wild-type placode but also within wild-type cells bordering a crumbs mutant clone (Chen, 2010) or in clusters of Crumbs expressing cells in a null mutant embryo and within cells at the edge of an ectopic step change in Crumbs expression levels. Also in vitro, Crumbs accumulates at contact zones between expressing cells. The extracellular domain appears the ideal candidate to mediate this anisotropy, which is supported by the following findings: (1) the CrbTMextra-GFP shows anisotropic localization at borders with cells not expressing the construct; (2) endogenous Crumbs in wild-type cells is induced to localize in an anisotropic fashion when neighboring cells are depleted of endogenous Crumbs and only express the intracellular domain; and (3) the Crbintra-FLAG shows uniform expression in cells. These observations exclude that another transmembrane protein that interacts with the intracellular domain of Crumbs in equal stoichiometry could mediate the anisotropy, though it cannot formally exclude that another extracellular factor might act as an intermediary between two Crumbs extracellular domains. These data are strongly supported by recent evidence from zebrafish, where vertebrate Crumbs isoforms appear to mediate homophilic interactions to promote orderly arrays of photoreceptors. Also, recent data analyzing the establishment of polarity in the Drosophila follicular epithelium suggest a role for cis-interaction of Crumbs molecules within a single cell (Fletcher, 2012). Thus, a clear role for the Crumbs extracellular domain in organizing plasma membrane domains through homophilic interactions in cis and in trans is prominently emerging (Roper, 2012).
Data presented in this study describe a link between the transmembrane protein Crumbs and myosin II structures actively engaged in controlling morphogenesis. Crumbs' ability to interact in trans allows the step change in Crumbs levels between placode and surrounding cells to be translated into a subcellular asymmetry, the anisotropic localization of Crumbs. This mechanism provides the cells at the border of the salivary gland placode with the means of sensing this positional information and allows them to turn the positional information into a morphogenetic readout: myosin cable formation. In the future it will be interesting to determine if the arrangement of Crumbs and myosin II described in this study is conserved during topologically similar processes of tube invagination, such as, for instance, the side budding of branches during lung or mammary gland morphogenesis (Roper, 2012).
During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. This study shows that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. Mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. It is proposed that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding (Kanesaki, 2013).
The coordinated movement of cells is one of the foundations of tissue morphogenesis. The forces driving the cellular movements are generated by surface dynamics, such as rearrangements of cell adhesions and changes of the contractility of cortical acto-myosin networks. However, the surface mechanics resisting deformation forces and maintaining cortical integrity are not well understood (Kanesaki, 2013).
The shape of the cell surface can change dynamically. One notable surface feature is the bleb, a spherical protrusion of the plasma membrane observed in diverse cellular processes such as locomotion, division, and apoptosis. Formation of blebs is driven by hydrostatic pressure in the cytoplasm. According to the current model, blebbing starts with local compression of the cytoskeletal network and proceeds according to a subsequent increase of the pressure. The compression of the cytoskeleton is mediated by the contractile force of non-muscle myosin II (MyoII). Though it has been shown that various cells, such as germ line and cancer cells, utilise blebs for their motility, the role of blebs and the mechanism of blebbing in tissue morphogenesis are still largely unclear (Kanesaki, 2013).
Invagination of a cellular layer is one of the common events in tissue morphogenesis. In gastrulation in Drosophila, ventral cells of the blastoderm embryo invaginate and then differentiate to mesoderm. The process of mesoderm invagination can be grossly divided into two sequential steps: apical constriction and furrow internalisation. During apical constriction, ventral cells collectively contract their apices and consequently form a shallow furrow on the embryo. During furrow internalisation, the ventral furrow becomes deeper and the layer of cells becomes engulfed in the embryonic body. The molecular and cellular mechanisms underlying apical constriction have been studied extensively. The change of cellular shape is mediated by integrated functioning of the cortical acto-myosin network and cellular adherens junction complex. The force driving the constriction is generated by pulsed contractility of MyoII. The tensile force from individual cells is transmitted to epithelial tissue through the adherens junction, and the tissue generates feedback force that leads to anisotropic constriction of ventral cells (Kanesaki, 2013).
Heterotrimeric G-protein (HGP) has an important role in apical constriction in Drosophila gastrulation. Signaling triggered by the extracellular ligand folded gastrulation (fog) promotes surface accumulation of MyoII in ventral cells, and the Fog signaling is mediated through an HGP α subunit encoded by concertina (cta). HGP belongs to the GTPase family, and its activity is regulated by multiple factors, including guanine nucleotide exchange factor (GEF). A previous study showed that ric-8 mutation results in a twisted germ-band due to abnormal mesoderm invagination. ric-8 was first identified as a gene responsible for synaptic transmission in Caenorhabditis elegans, and was shown to interact genetically with EGL-30 (C. elegans Gαq). Nematoda and vertebrate Ric-8 has GEF activity and positively regulates HGP signalingin vivo and in vitro. In Drosophila, Ric-8 is essential for targeting of HGPs toward the plasma membrane and participates in HGP-dependent processes such as asymmetric division of neuroblasts (Kanesaki, 2013 and references therein).
In this study, the precise role of ric-8 in mesoderm invagination was investigated. It was found that cortical stability of ventral cells is impaired in a ric-8 mutant. By a combination of genetic and pharmacological analyses, blebbing of ventral cells was found to be induced by either disruption of cortical actin or mutation of ric-8. It is suggested that HGP signaling constitutively organises cortical actin, thereby reinforcing the resistance of cells against deformation (Kanesaki, 2013).
Ventral cells intrinsically exhibit a few small blebs during mesoderm invagination. This indicates that surface contraction during apical constriction induces blebbing even in normal invagination. This study found that Ric-8 and HGP signaling are required for suppression of abnormally large blebs, and for the stabilisation of the cortex in invaginating cells. The physical mechanism underlying blebbing has been studied extensively in cultured cells. The contractile force of the acto-myosin network causes an increase of hydrostatic pressure in the cytoplasm, which leads to detachment of the plasma membrane from the cortical actin layer. The dynamics of blebs observed in ric-8 ventral cells were similar to those reported in cultured cells in terms of time and size, suggesting that the mechanisms underlying blebbing in these two systems are conserved (Kanesaki, 2013).
The average size of blebs changes as development proceeds: blebs become larger during furrow internalisation. Immuno-fluorescence imaging revealed that MyoII is abnormally accumulated beneath enlarged blebs in the ric-8 mutant. This correlation suggests that MyoII acts to induce an increase of hydrostatic pressure. Although MyoII is an indispensable factor for apical constriction, its activity can also cause malformation of the cells. How MyoII accumulates abnormally in the ric-8 mutant remains unclear. It cannot be ruled out that other processes of mesoderm invagination, such as mechanical stress from surrounding cells, also contributes to the enlargement of blebs. During apical constriction, epithelial tissue generates tension along the anterior-posterior axis, and ventral cells undergo constriction in an anisotropic manner. Similar force may also be generated at the tissue level during furrow internalisation, causing the cells there to be squeezed, and consequently increasing the intracellular pressure. Blebbing in the ric-8 mutant may be a consequence of abnormal cytoskeletal networks and physical stress acting cell to cell. In normal situations, cells would resist such physical stress and maintain the surface integrity, thereby supporting correct morphogenetic movements (Kanesaki, 2013).
This study demonstrates that HGP signaling has two functions in mesoderm invagination: induction of the apical constriction via MyoII accumulation and maintenance of the cellular surface via organisation of cortical actin. Although Fog is required for apical constriction, F-actin is organised in a Fog-independent manner, suggesting that these two functions are regulated in different ways. cta mutants and G-iα65A mutants showed similar phenotypes regarding cortical actin, suggesting that these Gα paralogs have overlapping functions. Because the Drosophila genome encodes 6 Gα subunits and 5 of them are expressed in early embryos, the contribution of G α paralogs other than Cta and G-iα65A to the suppression of blebbing cannot be rule out. The finding that ric-8, Gβ13F, and Gγ1 mutants showed blebbing, a hallmark of severely disturbed cortical actin, supports the idea that multiple HGP pathways control cortical actin redundantly. However, currently it is not known whether those signaling pathways act on the same downstream effectors. Considering that most blastoderm cells showed a dispersed signal of GFP-Moesin in the mutants, HGPs appear to be rather constitutive regulators of cortical actin organisation. Nevertheless, the abnormality of the cortex does not affect the morphology of the 'standstill' cells that do not carry out the inward movement. Thus, HGPs are required to reinforce the cortex so that the cells can endure the stress generated during tissue folding (Kanesaki, 2013).
It was previously reported that ric-8 is required for Drosophila gastrulation. This study extensively investigated mesoderm invagination and found that apical constriction is indeed compromised in the ric-8 mutant. Based on the observation of Fog-dependent MyoII accumulation, it is concluded that ric-8 is required for Fog-Cta signaling. It is unlikely that this phenotype is a secondary consequence of the disorganised F-actin in the ric-8 mutant, because actin was organised normally in the fog mutant embryo and ectopic Fog expression induced cell flattening even in late B-treated embryos. These findings instead suggested that Fog-Cta signaling and actin organisation are separate pathways and Ric-8 is involved in both pathways (Kanesaki, 2013).
Given that HGPs constitutively regulate F-actin, the signaling seems to be active in most blastoderm cells. Some unknown extracellular ligand and its receptor thus appear to be expressed to activate HGPs. It is also possible that cytoplasmic HGP regulators such as Pins, Loco, or other RGS proteins are involved in the activation. In the formation of the blood-brain barrier in Drosophila, Pins and Loco positively regulate HGP signaling. Embryos mutant for Pins also show abnormal cellular movements during mesoderm invagination. It is also intriguing to hypothesise that Ric-8 participates in the activation of HGPs through its GEF activity, which has been characterised both in vivo and in vitro. This hypothesis suggests the possibility that HGPs are endogenously activated. Future analysis of the responsible cytoplasmic regulators may clarify the mechanism of HGP regulation, and may give new insights regarding the intricate network of HGP signaling in animal development (Kanesaki, 2013).
How might HGP be functionally linked to actin polymerisation? Since G α12/13 participates in the activation of Formin family proteins in mammalian fibroblasts and a human Formin inhibits the formation of blebs in a prostate cancer cell line, a candidate factor regulating actin filaments downstream of HGP could be Diaphanous (Dia), a Drosophila Formin. Although it has been shown that organisation of actin via Dia is required for ventral furrow invagination, it is unclear whether Dia is also required for cortical stability during morphogenesis. Considering that Dia is an actin nucleator, it is speculated that Dia might act in the assembly of the actin meshwork and thereby reinforce the cortex. Indeed, it was observed that the dia mutant embryos showed cellular deformation during gastrulation, suggesting the functional relevance of the actin nucleator in the suppression of blebs. Further analysis will be required to clarify the functions of Dia (Kanesaki, 2013).
Previous studies demonstrated that ventral cells form a particular type of F-actin meshwork that makes a basic frame for apical constriction. RhoA- and Abelson-mediated signaling is required for organisation of the apical F-actin meshwork, while the Fog-Cta pathway is not. Thus, it is surprising that the mutants for HGPs, including Cta, showed a defect of cortical actin. HGP signaling may organise only a moiety of F-actin which is distinct from the one specifically accumulated at apices. HGP signaling regulates the organisation of cortical actin and mediates the establishment of the blood-brain barrier in Drosophila , suggesting that this function of HGPs is rather common in fly embryogenesis (Kanesaki, 2013).
Cell and tissue morphogenesis depends on the correct regulation of non-muscle Myosin II, but how this motor protein is spatiotemporally controlled is incompletely understood. This study shows that in asymmetrically dividing Drosophila neural stem cells, cell intrinsic polarity cues provide spatial and temporal information to regulate biased Myosin activity. Using live cell imaging and a genetically encoded Myosin activity sensor, Drosophila Rho kinase (Rok) was found to enrich for activated Myosin on the neuroblast cortex prior to nuclear envelope breakdown (NEB). After NEB, the conserved polarity protein Partner of Inscuteable (Pins) sequentially enriches Rok and Protein Kinase N (Pkn) on the apical neuroblast cortex. These data suggest that apical Rok first increases phospho-Myosin, followed by Pkn-mediated Myosin downregulation, possibly through Rok inhibition. It is proposed that polarity-induced spatiotemporal control of Rok and Pkn is important for unequal cortical expansion, ensuring correct cleavage furrow positioning and the establishment of physical asymmetry (Tsankova, 2017).
In asymmetrically dividing fly neural stem cells the protein kinases Rok and Pkn respond to cell polarity cues to regulate Myosin activity and dynamics in a stereotypical, spatiotemporal manner. It is proposed that the sequential regulation mediated by these two kinases is necessary to control Myosin activity and actomyosin dynamics, triggering stereotypic cell shape changes at various steps in the neuroblast cell cycle; first to induce cell rounding as neuroblasts enter mitosis, to permit cell elongation and unequal cortical expansion during anaphase, and finally to complete cytokinesis and the establishment of physical asymmetry (Tsankova, 2017).
Myosin recruitment before NEB is mediated by Rok. This kinase, implicated in Myosin phosphorylation, is already localized at the neuroblast cortex before NEB and in rok mutants, Myosin remains cytoplasmic. At NEB both Rok and Myosin enrich on the apical neuroblast cortex. This apical enrichment, but not cortical localization, depends on the polarity protein Pins since only apical Rok and Myosin enrichment is lost if Pins localization is compromised. Based on these data it is proposed that Rok responds to cell cycle cues, presumably through the small GTPase Rho1, to phosphorylate Myosin's regulatory subunit, enabling activated Myosin to engage with F-actin at the cell cortex prior to NEB. Subsequently, polarity cues enhance Rok on the apical cortex, resulting in the elevation of phosphorylated and, thus, activated Myosin on the apical neuroblast cortex at NEB (Tsankova, 2017).
With Pkn a second kinase has been identified, responding to polarity cues since its apical localization, starting at NEB and peaking by the end of metaphase, is dependent on Pins. Pkn is not absolutely necessary for cortical Myosin enrichment; pkn mutant neuroblasts still retain apical Myosin, although elsewhere on the cortex its localization is dramatically reduced. However, Pkn is required for Myosin's timely relocalization from the apical cortex. Wild-type neuroblasts clear Myosin from the apical cortex in early anaphase, creating an asymmetric distribution that is necessary for the unequal cortical expansion. In pkn mutants, however, both Rok and Myosin dynamics are changed, retaining both on the apical neuroblast cortex, causing aberrant cortical constrictions and concomitantly inverted polar expansion (Tsankova, 2017).
Based on these results, the following model is proposed. (1) Rok triggers cortical Myosin accumulation before NEB. (2) At NEB, apically localized Pins enriches Rok on the apical neuroblast cortex and concomitantly increases phospho-Myosin apically. (3) Pins also induces the apical enrichment of Pkn, which is necessary for the timely relocalization of Myosin from the apical neuroblast during metaphase. It is further proposed that Pkn is downregulating Myosin activity through inhibiting or downregulating apical Rok activity. Whether Pkn downregulates Rok activity by direct phosphorylation remains an attractive hypothesis, since vertebrate Rock2 has recently been identified as a Pkn target. Alternatively, Rok activity could be regulated independently of phosphorylation but governed by the length of its coiled-coil tether, linking the kinase domain with the membrane binding domain (Tsankova, 2017).
This sequential regulation of Myosin dynamics seems to be a key regulatory mechanism underlying physical asymmetric cell divisions. For instance, apical Myosin relocalization always precedes basal Myosin clearing in wild-type neuroblasts. Similarly, biasing the localization of activated Myosin affects cleavage furrow positioning and physical asymmetry (Tsankova, 2017).
It is hypothesized that polarity cues provide a cell-intrinsic timer, priming Myosin relocalization on the apical cortex, thereby ensuring the generation of physical asymmetry through unequal cortical extension. Polarity-induced enrichment of activated Myosin on the apical cortex could thus provide a symmetry breaking event, necessary for the subsequent induction of apical Myosin clearing. Consistent with this model is the finding that pins mutants, or uniform cortical localization of Pins, cause Myosin to clear from both poles at the same time and divide symmetrically by size (Tsankova, 2017).
Tissue and organ growth critically depends on the correct spatiotemporal regulation of cell division. This study provides a conceptual framework of how Rok and Pkn respond to both cell cycle and polarity cues. These cues, in conjunction with spindle-dependent signals, ensure correct physical asymmetric cell division that is necessary for stem cell homeostasis and cell differentiation. Spatial and temporal regulation of Myosin activity has also been shown to be important for pulsatile cell shape changes in the Drosophila embryo. Rok and Pkn play important roles during vertebrate development and morphogenesis , and it will be interesting to see how spatiotemporal cues, affecting local cell shape changes, are coordinated with overall tissue morphogenesis in flies and beyond (Tsankova, 2017).
To identify novel regulators of nonmuscle myosin II (NMII) an image-based RNA interference screen was performed using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. The Rab-specific GTPase-activating protein (GAP) RN-tre was identified as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, these results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility (Platenkamp, 2020).
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).
Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. This study shows that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis (Kasza, 2014).
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 Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. This study examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (1) Dmlc2Delta2-46 (Ext), an N-terminal extension truncation; (2) Dmlc2S66A,S67A (Phos), a disruption of two MLC kinase phosphorylation sites; and (3) Dmlc2Delta2-46;S66A,S67A (Dual), expressing both mutations. The results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2+) rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight (Chakravorty, 2014).
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).
This study investigated the properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). This exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared to the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared to wild-type IFI, whereas for EMB-3b these values are increased compared to wild-type EMB. ATP-induced dissociation of acto-S1 (K(1)k(+2)) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicate that the exon 3 encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine-binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating acto-myosin cross-bridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms (Bloemink, 2020).
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 cones 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 Robos 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 cones 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 cones 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 cones 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).
Kinesin-1 is a major microtubule motor that drives transport of numerous cellular cargoes toward the plus ends of microtubules. In the cell, kinesin-1 exists primarily in an inactive, autoinhibited state, and motor activation is thought to occur upon binding to cargo through the C terminus. Using RNAi-mediated depletion in Drosophila S2 cells, this study demonstrated that kinesin-1 requires ensconsin (MAP7, E-MAP-115), a ubiquitous microtubule-associated protein, for its primary function of organelle transport. Ensconsin is required for organelle transport in Drosophila neurons, and Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood. An Ensconsin N-terminal truncation that cannot bind microtubules is sufficient to activate organelle transport by Kinesin-1, indicating that this activating domain functions independently of microtubule binding. Interestingly, ens mutant flies retaining expression of this truncation show normal viability. A 'hingeless' mutant of kinesin-1, which mimics the active conformation of the motor, does not require Ensconsin for transport in S2 cells, suggesting that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together with other recent work, this study suggests that ensconsin is an essential cofactor for all known functions of kinesin-1 (Barlan, 2013).
The results suggested that Ensconsin might interact with kinesin-1 to promote activation of the motor directly. Indeed mammalian kinesin-1 (Kif5b) has been shown to interact with Ensconsin in vitro. This study performed immunoprecipitation assays from S2 cell extracts in an attempt to verify the interaction with Drosophila proteins, but a complex containing both Ensconsin and kinesin-1 independent of microtubules could not be isolated. Pull-down assays were performed using purified proteins but no interaction was detected. Although Drosophila KHC and Ensconsin interact by yeast two-hybrid, there is no additional biochemical evidence demonstrating the interaction in the Drosophila system. It is concluded that any interaction between Ensconsin and kinesin-1 is likely to be indirect, transient, or highly regulated, and thus difficult to detect by biochemical assays (Barlan, 2013).
The results presented in this study are consistent with a model in which Ensconsin, through its C-terminal domain, is involved in relieving autoinhibition of kinesin-1 to stimulate in vivo activity of the motor. Although microtubule binding by Ensconsin is not strictly required for its effect on transport, it may allow for compartmentalization of its effects to the microtubule itself, where it can act as a local regulator of kinesin-1 function. Microtubule binding may also act to increase the local concentration of Ensconsin near the microtubule, which could increase Ensconsin's overall effectiveness as a regulator (Barlan, 2013).
The work adds to the growing body of evidence suggesting that microtubules not only serve as passive tracks for motor proteins but may also actively regulate transport, via posttranslational modifications of tubulin or recruitment of specific microtubule-associated proteins to inhibit or enhance organelle transport along particular stretches of the microtubule (Barlan, 2013).
The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, this study isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, 96 poorly characterized genes were screened in the Drosophila central nervous system to establish their possible role during spindle assembly. Ensconsin/MAP7 mutant neuroblasts were found to display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, these results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1 (Gallaud, 2014).
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 Drosophila, neuroblasts undergo typical asymmetric divisions to produce another
neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including
Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds
off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric
cortical localization of all basal determinants in mitotic neuroblasts, and is therefore
indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with
several types of Myosin to localize the determinants. Another tumor-suppressor protein,
Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The
localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg
act in a common process that differentially mediates cortical protein targeting in mitotic
neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).
Because Lgl is a component of cortical protein complexes that include nonmuscle Myosin II, or
Zipper (Zip), a test was performed for genetic interactions between lgl and zip in Miranda localization by
examining embryos zygotically mutant for both lgl and zip. The zip1 mutation does not affect Miranda localization throughout embryonic
development and lgl-zip embryos show no difference in Miranda localization from zygotic
lgl- embryos until late embryonic stages (stage 16) owing to the maternal contribution of zip. However, at
stage 17 when maternal zip had been exhausted, lgl-zip embryos appear to
restore the basal crescent of Miranda in metaphase neuroblasts, whereas
zygotic lgl- embryos at the same stage do not. Thus, Lgl might act
for Miranda localization in part by suppressing zip function directly or indirectly, consistent
with a study on yeast that indicated negative genetic interactions between Lgl homologs and
Myosin II. Alternatively, the asymmetric distribution of Pon requires myosin function in
neuroblasts, as revealed by the use of 2,3-butanedione monoxime (BDM) that generally
inhibits myosin function. The effect of BDM on Miranda localization was examined.
Treatment of wild-type embryos with BDM phenocopies lgl mutants, resulting in a partial
redistribution of Miranda from the cortex to microtubules. The effect of BDM is
more marked in lglGLC embryos: as the BDM concentration increases, the relocalization of
Miranda to microtubules is synergistically enhanced in most BDM-treated neuroblasts and
results in the complete exclusion of Miranda from the cortex at 50 mM BDM. The
phenocopy and enhancement of lgl mutations by general inhibition of myosin function are in
contrast with the suppressive effects of zip mutations, suggesting that Lgl cooperates with at
least one type of Myosin other than Zip to anchor Miranda at the cell cortex. It is thus inferred that
Lgl regulates negatively myosin II function and also positively the function of another Myosin
isotype in cortical protein targeting in neuroblasts (Ohshiro, 2000).
Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide
unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size,
mitotic activity and developmental potential. During neuroblast mitosis, an apical protein
complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but
the mechanisms of these two processes are unknown. The tumor-suppressor genes
lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical
complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein
is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal
protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly
suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and
myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).
How does Lgl regulate basal protein targeting? Lgl binds non-muscle myosin II in all
organisms tested, and sro7/77 and myo1 (encoding Lgl-related proteins and myosin
II, respectively) show strong negative genetic interactions in yeast. Tests were performed for genetic
interactions between lgl4 and two different null mutations in zipper (encoding myosin II),
scoring Miranda basal localization in stage 17 neuroblasts, when maternal Lgl and Myosin II
protein levels are lowest. Wild-type and zip embryos have normal basal protein localization,
whereas lgl4 embryos show complete delocalization of basal proteins. However,
lgl4 embryos lacking one copy of myosin II show a significant
increase in basal protein targeting; and lgl4;zip1 mutant embryos show virtually normal basal protein targeting. Thus, reducing
myosin II levels strongly suppresses the lgl phenotype, indicating that myosin II can inhibit
basal targeting when Lgl levels are low (Peng, 2000).
In addition, the general myosin inhibitor 2,3-butanedione monoxime (BDM) can suppress the
lgl phenotype: stage 10 lgl4 embryos treated with BDM show a significant increase in basal protein localization compared with sham-treated stage 10 lgl4 embryos. Wild-type or lgl4 embryos treated with 50 mM BDM show delocalization of
Miranda, Prospero and Pon. These data indicate that a
myosin that is sensitive to 25 mM BDM inhibits basal protein localization in lgl embryos
(probably myosin II), and at least one myosin that is sensitive to 50 mM BDM promotes basal
protein targeting in mitotic neuroblasts (Peng, 2000).
Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins
without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are
necessary to restrict proteins to the apical membrane domain. Lgl could promote protein
targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical),
similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific
membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a
secretory pathway that is independent of brefeldin A, because it has been shown that treatment with
brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein
targeting. Alternatively, Lgl may actively promote
actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels
low so that they do not interfere with myosin-dependent basal localization. A general function
of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains
of the plasma membrane (Peng, 2000).
In the absence of MEIS family proteins, two mechanisms are known to restrict the PBX family of homeodomain (HD) transcription factors to the cytoplasm. (1) PBX is actively exported from the nucleus via a CRM1-dependent pathway. (2) Nuclear localization signals (NLSs) within the PBX HD are masked by intramolecular contacts. In a screen to identify additional proteins directing PBX subcellular localization, a fragment of murine nonmuscle myosin II heavy chain B (NMHCB) was identified. The interaction of NMHCB with PBX was verified by coimmunoprecipitation; immunofluorescence staining revealed colocalization of NMHCB with cytoplasmic PBX in the mouse embryo distal limb bud. The interaction domain in PBX maps to a conserved PBC-B region harboring a potential coiled-coil structure. In support of the cytoplasmic retention function, the NMHCB fragment competes with MEIS1A to redirect PBX, and the fly PBX homolog EXD, to the cytoplasm of mammalian and insect cells. Interestingly, MEIS1A also localizes to the cytoplasm in the presence of the NMHCB fragment. These activities are largely independent of nuclear export. The subcellular localization of EXD is deregulated in Drosophila zipper mutants that are depleted of nonmuscle myosin heavy chain. This study reveals a novel and evolutionarily conserved mechanism controlling the subcellular distribution of PBX and EXD proteins (Huang, 2003).
Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. This study characterizes the properties, relationships and associations that each subunit has with one another in Drosophila. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. The stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. Heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion it was found that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are presentFour myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) are identified as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach six previously uncharacterized genes were characterized that contain IQ-motifs and may be essential light chain binding partners (Franke, 2006).
In Drosophila, the stability of the two nonmuscle myosin II light chains in the absence of their heavy chain binding partner is markedly different. zip/MyoII deficient embryos had significantly reduced levels of sqh/RLC, but wild-type levels of mlc-c/ELC. reduced due to mutations in its sqh/RLC binding partner, mlc-c/ELC also remains at wild type levels. Despite this difference in behavior, it was found that by far, the major binding protein for both light chains in soluble extracts from wild type embryos is the zip/MyoII heavy chain. The simplest explanation for these observations is that sqh/RLC protein is unstable in the absence of zip/MyoII heavy chain protein, whereas mlc-c/ELC is stable, similar to findings in D. discoideum. All three myosin II polypeptides were analyzed when either the heavy chain or RLC was overexpressed and no increase was found in polypeptide levels of those subunits not targeted for overexpression. The findings for the turnover of polypeptide subunits are similar to that of other protein complexes ranging from spectrin to cytochromes (Franke, 2006).
In sqh/RLC mutant animals, zip/MyoII levels are reduced, but not to the same extent as sqh/RLC. Unlike sqh/RLC, zip/MyoII heavy chains are partially stable and form aggregates in the absence of sqh/RLC. Staining demonstrated that mlc-c/ELC, whose levels are unaltered in sqh/RLC mutant animals, associated with aggregated heavy chains. Aggregation and/or associations may cause the partial stabilization of heavy chains in sqh/RLC mutants. Analysis of heavy chain levels in sqh/RLC and mlc-c/ELC double mutants would address this, but such analysis is currently not possible, because no mutant mlcc/ ELC alleles are reported and the interpretation may be compromised by mlc-c/ELC binding to other proteins. Overall, the data support a model in which transcriptional or translational feedback does not regulate the level of each polypeptide. Instead, the ability of each polypeptide to persist in mutant backgrounds is dependent on both the inherent stability of each polypeptide and its association with its binding partner(s) (Franke, 2006).
The experiments confirm that sqh/RLC association with zip/MyoII heavy chain is required to prevent heavy chain aggregation, demonstrating that exposure of the hydrophobic helix comprising the distal IQ-motif in zip/MyoII is sufficient to cause aggregation in Drosophila. These results are consistent with studies on slime mold (D. discoidium( and scallop (Pecten maximus) myosin IIs that show removal of the RLC promotes aggregation of myosin II heads. Interestingly, (and in contrast), in D. discoidium cells lacking ELC, myosin II heavy chain localization appears normal. sqh/RLC primarily associates with zip/MyoII heavy chain at sites where nonmuscle myosin II is believed to function. A small fraction of the sqh/RLC localizes to aggregates, but this is likely to result from the recruitment of native, nonmuscle myosin II into heavy chain aggregates. In contrast, mlc-c/ELC associates with heavy chain at sites of aggregation demonstrating that mlc-c/ELC association is not sufficient to prevent aggregation (Franke, 2006).
In lysates from wild-type embryos it was found that the majority of both mlc-c/ELC (>90%) and sqh/RLC (>95%) is bound to zip/MyoII. More quantitative experiments are necessary to determine if a substantial difference between the amount of sqh/RLC and mlc-c/ELC not associated with zip/MyoII heavy chain exists. In S. pombe, cdc4/ELC protein levels are far in excess (>7-fold) of both heavy chains and RLC, suggesting that the amount of ELC not associated with nonmuscle myosin II heavy chain varies in different organisms (Franke, 2006).
In several organisms, the nonmuscle ELC has been shown to bind proteins other than nonmuscle myosin II heavy chain. zip/MyoII is an abundant, ubiquitously expressed protein in Drosophila, and other mlc-c/ ELC binding proteins may be one or more orders of magnitude less abundant. Consequently, the relatively small percentage (<10%) of total mlc-c/ELC not bound to zip/MyoII heavy chain in the wild-type lysates may well be biologically important. Moreover, the proximal zip/MyoII heavy chain IQ-motif, which mlc-c/ELC binds, is very similar to IQ-motifs in other proteins (Franke, 2006).
Using candidate, mass spectrometry and bioinformatics approaches, a number of putative Drosophila mlc-c/ELC binding partners were identied. Using the candidate approach it was found that Ck/MyoVIIA, Jar/MyoVI, and Zip/MyoII bind Mlc-c/ELC using affinity purified Mlcc/ELC antibodies and a myc-tagged mlc-c/ELC construct. These interactions were verified with additional, reciprocal experiments (e.g., Mlc-c/ELC but neither Sqh/RLC nor Zip/ MyoII is present in Ck/MyoVIIA and Jar/MyoVI immunoprecipitations). Mass spectrometry on proteins associated with Myc-Mlc-c/ELC confirmed binding by Zip/MyoII, and showed that two additional proteins, Asp/Abnormal Spindle and Didum/MyoV are also Mlc-c/ELC binding partners. Mass spectrometry could fail to identify all binding partners for several reasons. (1) Greater than 90% of Mlc-c/ELC is associated with Zip/MyoII in wildtype, therefore large quantities of Mlc-c/ELC must be pulled down to identify binding partners for the remaining 10%. (2) If the remaining 10% of Mlc-c/ELC associates with several proteins, pulling down sufficient quantities of each for mass spectrometry is likely to be difficult. (3) Mlc-c/ELC binding partners may either be expressed at very low levels, or expressed in a cell, tissue or developmentally specific manner. (4) Even if the binding partner is expressed ubiquitously, at high levels throughout development, Mlc-c/ELC association may be transient such that only a small amount is bound to Mlc-c/ELC at any given time. While light chains bind with high affinity to Myosin II heavy chains Calmodulin is known to bind weakly under certain solution conditions (Franke, 2006).
An in silico approach was used to identify IQ-motif containing proteins in the Drosophila genome. As expected this approach identified all proteins used to generate the profile. In addition, this approach identified one Mlc-c/ELC binding partner that was also identified by mass spectrometry, Asp/Abnormal Spindle. Seven uncharacterized candidate IQ-motif containing proteins were identified with none having published reagents to directly test for Mlc-c/ELC association (Franke, 2006).
Mutations in myosin VI cause deafness in humans and mice. Jar/MyoVI has a single IQ-motif and is expressed at low levels embryonically except for enrichment in the dorsal-most three or four rows lateral epidermal cells during dorsal closure and in neuroblasts, where it plays a role in basal protein targeting and correct spindle orientation. It was possible to co-IP Jar/MyoVI from lysates with Mlc-c/ELC antibodies, but could detect mlc-c/ELC only in jar/MyoVI pull-downs when Mlc-c/ELC was overexpressed. This suggests that Jar/MyoVI has additional light chains (possibly calmodulin). Because Jar/MyoVI has a single IQ-motif, this suggests that Mlc-c/ELC association may be cell, temporal and/or function-specific. In vitro, porcine MyoVI has been shown to bind calmodulin at two sites - at its single, conventional IQ-motif and at a structural region within a unique insert (not found in other myosins) characteristic to MyoVIs. Therefore, it is possible that Jar/MyoVI may bind calmodulin and Mlc-c/ELC simultaneously (Franke, 2006).
Mutations in human myosin VIIA cause Usher syndrome1B, a deaf/blind disorder. Mutations in the fly homologue, Ck/MyoVIIA, cause hearing loss and lethality. Ck/MyoVIIA expression is ubiquitous at low levels throughout development, with enrichment in the fly hearing organ. This is the first report of a specific light chain for Ck/MyoVIIA which has 5 IQ-motifs that could simultaneously bind different light chains. Previous reports showed that mouse myosinVIIA binds calmodulin, Ck/MyoVIIA may also bind calmodulin as well as Mlc-c/ELC (Franke, 2006).
Myosin Vs have 6 IQ-motifs and play roles in intracellular transport, correct spindle positioning during cell division and the polarized distribution of intracellular compontents. Both ELC and calmodulin are light chains for myosin V in S. cerevisiae and S. pombe, and myosin Va in chick brain. A recent report also identified both Mlc-c/ELC and calmodulin as light chains for the fly homologue, Didum/MyoV. Animals mutant for didum/MyoV generally arrest during larval development with no obvious defects. Some adult escapers do eclose and males exhibit defects during late spermatogenesis (Franke, 2006).
Mutations in the human orthologue of Asp/Abnormal Spindle, ASPM, are the most common cause of autosomal recessive primary microcephaly-characterized by a reduction in cerebral cortex size. Mutations in Asp/Abnormal spindle cause syncitial embryos to have free centrosomes and larval neuroblasts to have a high mitotic index. Asp/Abnormal Spindle localizes to polar regions of the spindle immediately surrounding the centrosome and its removal causes centrosomes to lose their microtubule organizing center activity. Asp/Abnormal Spindle has at least 5 IQ-motifs. This is the first report of a light chain for asp/Abnormal Spindle in any organism (Asp/Abnormal spindle is conserved in human, mouse, Drosophila and C. elegans), and it is unclear what role light chains have on asp/Abnormal Spindle function (Franke, 2006).
Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. This study shows that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, it was found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9; see Drosophila Atg9) trafficking when cells were deprived of nutrients. These findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein (Tang, 2011).
Myosin II is a conventional two-headed myosin composed of two heavy chains, two essential light chains, and two regulatory light chains. Myosin II activation is regulated by the phosphorylation of its regulatory light chain via MLCKs. Rho GTPase and Rho kinase have been implicated in the regulation of myosin activation. However, this study found that neither RNA-mediated knockdown of dRok nor mutations in Rho1 or dRhoGEF2 could suppress the Atg1-induced wing defects. Instead, it was found that depletion of Sqa rescued Atg1-induced wing defects. This epistasis analysis showed that Sqa functioned downstream of Atg1. Moreover, it was found that Sqa but not Atg1 could directly phosphorylate Spaghetti squash (Sqh) in the in vitro kinase assay, suggesting that Atg1 stimulates myosin activity via Sqa. Importantly, Atg1 phosphorylates and interacts with Sqa, indicating that Atg1-Sqa functions in a kinase cascade to regulate myosin II activation. Moreover, Atg1 has been found to have a critical role in the regulation of autophagy induction under stress conditions in yeast, Drosophila, and mammalian cells. These results provide the first evidence that nutrient starvation stimulates myosin II activation in an Atg1-Sqa-dependent manner. Most significantly, a dramatic decrease was found in the size and number of autophagosomes in cells expressing Sqa-T279A, Sqa-RNAi, and SqhA20A21 on nutrient deprivation, indicating that Atg1-Sqa-mediated actomyosin activation has a critical role in autophagy (Tang, 2011).
The kinase domain of Sqa is also highly homologous to that of the mammalian DAPK family proteins. Recent studies have indicated that DAPK1 regulates autophagy through its association with MAP1B and Beclin1, or by modulating the Tor signalling pathway. As DAPK family proteins also regulate myosin II phosphorylation, one might speculate that Sqa may be the Drosophila counterpart of DAPK protein. Indeed, although overexpression of Sqa does not induce cell death, Sqa shares several characteristics with DAPK3/ZIPK. First, unlike MLCK family proteins, both Sqa and ZIPK contain an amino-terminal kinase domain that has 42% sequence identity and 61% similarity. Moreover, like ZIPK, recent sequence analysis from FlyBase identified a Sqa isoform that also contains a leucine-zipper domain. Second, as phosphorylation of Thr-265 in ZIPK is essential for its kinase activity, this study found that Atg1 phosphorylates Sqa at the corresponding Thr-279, and is critical for Sqa activity. Third, just as Sqa specifically associates with kinase-inactive Atg1, the results indicate a similar interaction between ZIPK and Ulk1. Importantly, depletion of Sqa and ZIPK resulted in autophagic defects in response to nutrient deprivation. These findings together suggest that ZIPK may act as a mammalian homolog of Sqa during starvation-induced autophagy. Further investigation is needed to determine whether the mammalian Atg1 (Ulk1) directly phosphorylates ZIPK at Thr-265, and the role of this regulation in autophagy (Tang, 2011).
In autophagy, the source of the autophagosomal membrane and dynamics of autophagosome formation are fundamental questions. Studies in yeast and mammalian cells have identified several intracellular compartments as potential sources for the PAS (also termed isolation membrane/phagophore). Formation of PI(3)P-enriched ER subdomains (omegasomes) has been reported during nutrient starvation and autophagy induction, and a direct connection has been observed between ER and the phagophore using the 3D electron tomography. In addition, recent studies in yeast cells have suggested Atg9 and the Golgi complex have a role in the formation of autophagosomes. It has been proposed that the integral membrane protein Atg9 may respond to the induction signal in promoting lipid transport to the forming autophagosomes. The mAtg9 has been found to localize on the TGN and the endosomes in nutrient-rich conditions and translocate to LC3-positive autophagosomes on nutrient deprivation. Although several proteins, including Ulk1, mAtg13, and p38IP, have been found to regulate starvation-induced mAtg9 trafficking, the molecular motor that controls the movement of mAtg9 between different subcellular compartments remains unknown (Tang, 2011).
The finding that myosin II redistributes from peripheral to the perinuclear region of cells on starvation suggests that myosin II has a role in membrane trafficking. In fact, it has been reported that myosin II is required for the trafficking of major histocompatibility complex (MHC) class II molecules and antigen presentation in B lymphocytes. Myosin II has also been found to be involved in the protein transport between ER and Golgi. This study has shown that there here is a molecule link between mAtg9 and the actomyosin network, indicating that myosin II may function as a motor protein for mAtg9 trafficking during early autophagosome formation. In conclusion, this work has unravelled a regulatory mechanism between Atg1 activity and the Atg9-mediated formation of autophagosomes. Further studies are needed to determine the involvement of this signalling process in other stress-induced or developmentally regulated autophagy (Tang, 2011).
Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. This study characterized the function of the Drosophila ASPM orthologue, Abnormal spindle (Asp), and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. Size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. It is proposed that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness (Rujano, 2013).
Epithelial morphogenesis involves the action of several key molecules that coordinate cell division, tissue growth, establishment and maintenance of cell polarity, the generation of mechanical forces and the organization of cytoskeleton architecture. This study shows that Asp is a key regulator of neuroepithelium morphogenesis, as it coordinates and integrates at least two essential morphogenetic events. First it regulates tissue size and layering, by contributing to accurate cell division, spindle positioning and
Interkinetic nuclear migration INM, and second, controls cell and tissue shape, by promoting apico-basal myo-II tension distribution and tissue cohesiveness during development of the optic lobe (Rujano, 2013).
In Drosophila asp mutant brain, central brain neuroblasts present defects in spindle morphology and prometaphase arrest. This study shows that in the fly neuroepithelium, cells do not arrest in prometaphase. Instead they divide with defects in chromosome segregation. These defects result in the generation of aneuploid cells that most likely die by apoptosis, as occurs in aneuploid mouse neuroepithelial cells. Furthermore, it was shown that in the fly, Asp is required for correct spindle positioning in the neuroepithelium as ASPM. Together, correct spindle assembly and positioning contribute to size determination and tissue organization in fly neuropithelium (Rujano, 2013).
As in other pseudostratified epithelia, in the fly neuroepithelium mitotic nuclei undergo INM. Myo-II cortical contractility is known to contribute to the translocation movement that allows G2 nuclei to move towards the apical cortex and the current observations support the idea that actomyosin drives apical nuclear movement during INM in the fly neuroepithelium. An even distribution of the Asp-dependent basally localized myo-II is interpreted as allowing the right amount of forces to be distributed across the basal membrane. The generation of tension and contractility then contributes to move the mitotic nucleus to the apical side of the cell. In asp, the myo-II distribution is irregular, which generates regions of high tension and contractility where the nucleus can move upwards to divide (even if too high), as opposed to regions of low tension and contractility in which low force generation impedes the upward movement of nuclei. As a consequence, the distribution of nuclei is altered and with it tissue organization (Rujano, 2013).
One of the most surprising findings in this study is the localization of Asp to interphase microtubules and to the neuroepithelium basal cortex. The fact that Asp interacts with myo-II and is required for its proper apico-basal polarization, together with the observation that myo-II is (like Asp) also enriched at the basal cortex, suggests that these proteins form part of a complex at the basal domain. Basal accumulation of myo-II most likely provides rigidity and tension to basal membranes, contributing to cell shape and tissue architecture. Thus, Asp interaction with myo-II facilitates the maintenance of a pool of myo-II at the basal side of the neuroepithelium, thereby creating a polarized distribution of myo-II within these cells, which could serve several purposes: to provide the necessary forces to push mitotic nuclei apically during mitosis; to organize a basal structural constraint that ensures tissue integrity and shape; and to prevent myo-II-mediated apical constriction. Several studies have shown the importance of apical membrane and adherens junctions in the organization of an epithelium. This work shows that basal membrane organization and the balance of forces between these two domains are of the utmost importance for the maintenance of epithelium integrity (Rujano, 2013).
Mutations in ASPM are the most common cause of MCPH. Intriguingly, ASPM knockout mouse present only mild brain size reduction. Detailed sequence analysis has shown that this knockout still contains an intact CH1 domain, which in the fly was found to be essential for Asp function. This work shows that in flies Asp is a major regulator of brain size, but in addition also of neuroepithelium morphogenesis. Microcephaly has been mainly attributed to mutations that exclusively influence the balance between neural stem cell division and differentiation by regulating mitotic spindle positioning. This study identifies however, a non-mitotic function of a microcephaly gene in brain development and neuroepithelium morphogenesis and reveals that Asp plays a role beyond spindle organization. Future work should address whether these finding can be extended to other microcephaly proteins (Rujano, 2013).
Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/beta-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. This study shows that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, it is proposed that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm (Vuong, 2014).
Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 +/- 4.1 microM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity (Heissler, 2015).
The Hippo pathway controls tissue growth through a core kinase cascade that impinges on the transcription of growth-regulatory genes. Understanding how this pathway is regulated in development remains a major challenge. Recent studies suggested that Hippo signaling can be modulated by cytoskeletal tension through a Rok-myosin II pathway. How cytoskeletal tension is regulated or its relationship to the other known upstream regulators of the Hippo pathway remains poorly defined. This study identifies the spectrins, α-spec, β-spec, or βH-spec contractile proteins at the cytoskeleton-membrane interface, as an upstream regulator of the Hippo signaling pathway. In contrast to canonical upstream regulators such as Crumbs, Kibra, Expanded, and Merlin, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These results uncover an essential mediator of Hippo signaling by cytoskeleton tension, providing a new entry point to dissecting how mechanical signals regulate Hippo signaling in living tissues (Deng, 2016).
In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. This study demonstrates that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. These results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. It is proposed that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly (Sechi, 2020).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.