InteractiveFly: GeneBrief

flapwing: Biological Overview | References


Gene name - flapwing

Synonyms - PP1β9C

Cytological map position - 9B14-9C1

Function - signaling

Keywords - regulation of non-muscle myosin

Symbol - flw

FlyBase ID: FBgn0000711

Genetic map position - X: 10,279,966..10,302,119 [+]

Classification - PP2Ac superfamily

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Rodrigues, N. T., Lekomtsev, S., Jananji, S., Kriston-Vizi, J., Hickson, G. R. and Baum, B. (2015). Kinetochore-localized PP1-Sds22 couples chromosome segregation to polar relaxation. Nature [Epub ahead of print]. PubMed ID: 26168397
Summary:
Cell division requires the precise coordination of chromosome segregation and cytokinesis. This coordination is achieved by the recruitment of an actomyosin regulator, Ect2, to overlapping microtubules at the centre of the elongating anaphase spindle. Ect2 then signals to the overlying cortex to promote the assembly and constriction of an actomyosin ring between segregating chromosomes. By studying division in proliferating Drosophila and human cells this study demonstrates the existence of a second, parallel signalling pathway, which triggers the relaxation of the polar cell cortex at mid anaphase. This is independent of furrow formation, centrosomes and microtubules and, instead, depends on PP1 phosphatase and its regulatory subunit Sds22. As separating chromosomes move towards the polar cortex at mid anaphase, kinetochore-localized PP1-Sds22 helps to break cortical symmetry by inducing the dephosphorylation and inactivation of ezrin/radixin/moesin proteins at cell poles. This promotes local softening of the cortex, facilitating anaphase elongation and orderly cell division. In summary, this identifies a conserved kinetochore-based phosphatase signal and substrate, which function together to link anaphase chromosome movements to cortical polarization, thereby coupling chromosome segregation to cell division.

Valencia-Exposito, A., Grosheva, I., Miguez, D. G., Gonzalez-Reyes, A. and Martin-Bermudo, M. D. (2016). Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis. Nat Commun 7: 10746. PubMed ID: 26888436
Summary:
Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. This study used live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit Flapwing (Flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, a quantitative model is proposed in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

BIOLOGICAL OVERVIEW

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. Drosophila PP1β, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1β. Loss of PP1β leads to increased levels of phosphorylated nonmuscle MRLC (Squash) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1β, and activates PP1β's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1β and MYPT genes suggests this role may be conserved (Vereshchagina, 2004).

Nonmuscle myosin II, a molecular motor closely related to vertebrate smooth muscle myosin, powers the actomyosin cytoskeleton. It is required for the coordinated changes in the shape and position of individual cells during morphogenesis as well as for cytokinesis and other cell movements. Nonmuscle myosin II activity is also modulated by metastasis-related and tumor suppressor genes (reviewed Bresnick, 1999: Vereshchagina, 2004).

The regulation of nonmuscle myosin is thought to be broadly similar to that of vertebrate smooth muscle myosin (Bresnick, 1999). Contraction and relaxation of vertebrate smooth muscle are regulated by the reversible phosphorylation of myosin regulatory light chain (MRLC), principally on Ser-19. The motor activity of smooth muscle myosin is regulated by the balance of activatory phosphorylation, leading to muscle contraction, and inhibitory dephosphorylation, leading to relaxation. The spectrum of stimulating kinases includes myosin light-chain kinase (MLCK), Rho-associated protein kinase (ROK), p21-associated kinase (PAK), integrin-linked kinase (ILK) and leucine zipper-interacting protein kinase (Dlk/ZIP kinase). The antagonistic protein phosphatase is the catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) in association with its myosin phosphatase targeting subunit MYPT1 or MYPT2, and a small subunit of unknown function (reviewed by Hartshorne, 1998). These kinases and phosphatases are themselves subject to regulation by reversible phosphorylation, for example ROK not only phosphorylates and activates MRLC, but also phosphorylates MYPT1 and inhibits MRLC dephosphorylation (reviewed Kaibuchi, 1999; Somlyo, 2000). The nonmuscle roles of these myosin-regulating kinases are less clear, though at least one (ROK) also regulates non-muscle myosin II in both mammals and Drosophila. Similarly, though PP1 is often assumed to be the major non-muscle MRLC phosphatase, PP2A has also been implicated. The various phosphorylation events have been investigated biochemically, but little is known about their physiological significance, particularly in nonmuscle cells (Vereshchagina, 2004).

Drosophila nonmuscle myosin II heavy chain zipper (zip) and regulatory light chain spaghetti squash (sqh) are essential for the normal development of a very wide range of cells and tissues. Drosophila Rho-kinase (Drok) phosphorylates both Sqh and DMBS (the single Drosophila homolog of MYPT1/2; Mizuno, 2002; Tan, 2003). By analogy to the vertebrate smooth muscle system it was proposed that this phosphorylation activates myosin and inhibits myosin phosphatase (Vereshchagina, 2004).

PP1 is involved in the regulation of many cellular functions including glycogen metabolism, muscle contraction, and mitosis (reviewed Bollen, 2001; Cohen, 2002). In Drosophila, the four genes encoding isoforms of PP1c are named by their chromosome location and subtype: PP1β9C, PP1α13C, PP1α87B, and PP1α96A (Dombrádi, 1990b, Dombrádi, 1993). Of these, PP1α87B contributes 80% of the total PP1 activity, therefore the phenotypes of PP1α87B loss of function mutants (Axton, 1990; Dombrádi, 1990a; Baksa, 1993) may be due to a loss of overall PP1 activity, rather than identifying specific functions unique to the PP1α87B protein. Mice and humans have three PP1 genes: PP1α and PP1γ are related to the fly PP1α genes, although PP1δ (also known as PP1β) corresponds to fly PP1β. Of the mammalian genes, functional analysis by gene knockout in mice has so far only been performed for PP1γ (Varmuza, 1999). This knockout eliminates both the widely expressed PP1γ 1 and the testis-specific PP1γ 2. Homozygous mutant female mice are viable and fertile; homozygous mutant males are viable but sterile, with defects in spermatogenesis. Presumably the somatic and female germline functions of PP1γ are redundant with PP1α and/or PP1δ (Vereshchagina, 2004).

The in vitro biochemical activities of the PP1c isoforms are very similar. However, genetic analysis provides a powerful approach to analyze the specific, nonredundant functions of each isoform. The Drosophila PP1β catalytic subunit gene PP1β9C corresponds to flapwing (flw), weak alleles of which are viable but flightless (Raghavan, 2000). The semilethality of a strong allele, flw6, demonstrated that PP1β is essential in flies. flw6 larval body wall muscles appeared to form normally, but then detached and degenerated, leading to a semiparalyzed larva that could not feed properly (Raghavan, 2000). In addition to muscle defects, the occasional male flw6 survivors were sterile and had blistered wings, indicating a nonredundant role for PP1β9C in nonmuscle cells as well as in muscles (Vereshchagina, 2004).

This study shows that the essential role of PP1β in flies is to regulate nonmuscle actomyosin. The lethality of strong flw (PP1β) mutants is suppressed by reducing the level of phospho-Sqh (MRLC), either using nonphosphorylatable point mutants of sqh or by reducing the gene dosage of key regulators such as Rho1 or RhoGEF2. flw mutants are also suppressed by reducing the gene dosage of nonmuscle myosin heavy chain (zipper). Clones of ovarian follicle cells mutant for flw6 have increased levels of phospho-Sqh, leading to disorganized or absent F-actin and to increased levels of myosin. Therefore, although PP1 isoforms collectively have many known roles, the essential, nonredundant role for PP1β in Drosophila is in the regulation of nonmuscle myosin activity and actin organization (Vereshchagina, 2004).

Drosophila has been reported to have only one MYPT homolog, named DMBS (Mizuno, 2002; also known as DMYPT, Tan, 2003). This study demonstrates that DMBS binds both α and β isoforms of PP1 and is therefore unlikely to mediate a PP1β-specific function. However a Drosophila PP1β-specific regulatory subunit, MYPT-75D, has been identified that is similar to mammalian MYPT3, a prenylated MYPT1/2 paralog (Skinner, 2001). MYPT-75D binds specifically to PP1β in vitro and the two proteins coimmunoprecipitate from fly extracts. MYPT-75D can stimulate PP1β's Sqh phosphatase activity in vitro and MYPT-75D, PP1β and Sqh proteins coimmunoprecipitate. Expression of a nonPP1 binding form of MYPT-75D in flies results in elevation of phospho-Sqh and phenotypic consequences that can be suppressed by reducing the level of Sqh phosphorylation. It is concluded that PP1β is targeted to Sqh by MYPT-75D, where it performs an essential role in the regulation of Sqh phosphorylation, and hence myosin activity, for which other PP1c isoforms cannot substitute. The conservation of all of these components, including the PP1α and β isoforms, suggests that regulation of nonmuscle myosin in mammals may also involve the activity of PP1β and an isoform-specific myosin targeting subunit (Vereshchagina, 2004).

This study shows that two semilethal mutant alleles of PP1β can be dominantly suppressed by loss-of-function extragenic mutations. The existence of single-gene extragenic suppressors indicates that PP1β has a single essential role, the identity of the suppressors indicates that this role is in the regulation of actin and/or myosin. Though the main defect observed in flw mutants is muscle detachment and degeneration, it is clear from these data that it is nonmuscle myosin, rather than muscle myosin, that is affected. Zipper and Sqh are components of nonmuscle myosin; the muscle version of Sqh, Mlc2, does not interact with flw (Raghavan, 2000). Similarly, Tm1, but not the muscle-specific Tm2, suppresses flw. Disruption of nonmuscle myosin in flw mutants may lead to disruption of the actin cytoskeleton and affect cell adhesion in many cell types, but seems to be most readily apparent in contractile muscle, particularly the highly specialized indirect flight muscles (Raghavan, 2000). Though not directly involved in generation of contractile force, nonmuscle myosin seems to be necessary for the correct development of striated myofibrils (Vereshchagina, 2004).

The dominant suppression of the lethality of flw6 and flw7 mutants by SqhA20A21, coupled with the enhancement of flw1 by SqhE20E21, implies that the essential role of PP1β 9C is related to the regulation of the phosphorylation state of Sqh. To address whether this interaction is direct or indirect, it has been shown that PP1β can directly dephosphorylate phospho-Sqh in vitro and that the two proteins coimmunoprecipitate from Drosophila extracts. Furthermore, a new PP1β-specific MYPT has been identified, and this study shows that binding of MYPT-75D to PP1β stimulates dephosphorylation of nonmuscle MRLC both in vitro and in vivo. It is therefore concluded that the major or only essential role of PP1β in Drosophila is to dephosphorylate Sqh and that this role is mediated, at least in part, by association with a β-specific MYPT protein. Although flw6 behaves as a null allele by genetic tests (Raghavan, 2000), the possibility cannot be ruled out that it has some residual activity and that this is sufficient to perform one or more additional essential functions of PP1β, which for some reason require only a very low level of PP1β activity. PP1β, MRLC and MYPT proteins are highly conserved between flies and mammals, so it seems likely that dephosphorylation of MRLC is also an essential role of PP1β in humans (Vereshchagina, 2004).

Though PP1β can dephosphorylate Sqh directly and manipulating the phosphorylation state of Sqh is sufficient to suppress strong mutants of flw, the possibility that flw has other substrates in the same pathway cannot be excluded. For example, nonmuscle myosin heavy chain, which in mammals can be phosphorylated by PKC and CKII, could also be a substrate for PP1β (Vereshchagina, 2004).

What is the molecular basis of the suppression of flw? It is believed that the key defect, both in flw mutants and in flies expressing MYPT-75DF117A, is the hyperphosphorylation of Sqh, particularly on Ser-21; this is directly suppressed by the nonphosphorylatable Sqh mutants. In these experiments a pool of normal Sqh remains, so essentially the ratio of phosphorylated and nonphosphorylated Sqh is being manipulated. Phosphorylation of Sqh leads to activation of the myosin motor; reduction in the amount of myosin heavy chain in zipper+/- presumably reduces the amount of active motor. Sqh is known to be a substrate for Rho-kinase, itself activated by a pathway that includes two more suppressors: Rho1 and RhoGEF2. Rho-kinase itself is located on the X chromosome and was therefore not accessible to the genetic screen (Vereshchagina, 2004).

Tm1, a strong suppressor of flw6, is not a member of Rho-kinase pathway but a cytoskeletal actin-binding protein. Several functions have been ascribed to nonmuscle tropomyosin in mammals: modulation of myosin function, actin polymerization , regulating microfilament branching, and suppression of neoplastic transformation. Reduction in the amount of Tm1 appears to mitigate the consequences of hyper-phosphorylated Sqh; the obvious mechanism is by reducing the binding of active myosin to actin, though Tm1 could have its effect through regulation of actin structure and polymerization (Vereshchagina, 2004).

The phenotypes described for flw somewhat resemble those of DMBS, particularly in the female germ line (Tan, 2003) and in that they both lead to the accumulation of phospho-Sqh (Mizuno, 2002), though DMBS mutants do not show the accumulation of myosin aggregates (Tan, 2003). The differences in lethal phase (embryonic for DMBS, predominantly larval for flw) might be accounted for by maternal contribution and differences in protein stability; it was not possible to investigate this further since both DMBS and flw are required for oogenesis. Furthermore, the flw suppressors sqhA20A21, Rho1 and zipper have been shown or deduced to modify at least some of the DMBS phenotypes. This might indicate that the critical role of flw is mediated by DMBS. However, DMBS is not specific for PP1β. PP1α87B is much more abundant than PP1β, so flw mutants should have little effect on the DMBS: PP1c complex. It is possible that DMBS:PP1β has a unique role not shared by DMBS:PP1α; it is also possible that DMBS, which is phosphorylated by Rho-kinase, is itself directly or indirectly activated by a PP1β-specific phosphatase complex. However, because an additional, PP1β-specific MYPT has been identified, it seems much more likely that this is the key targeting subunit that mediates the essential role of PP1β in vivo and that the suppression of flw by Rho and RhoGEF is through a decrease in phosphorylation of nonmuscle MRLC by Rho-kinase (Vereshchagina, 2004).

Why do flies have two MYPTs apparently doing the same job, one PP1β-specific and the other not? Clearly DMBS is not completely redundant with MYPT-75D, since DMBS mutants are lethal; mutants for MYPT-75D are not available to test the converse. One possible explanation for the presence of multiple myosin targeting subunits in mammals, flies and nematodes lies at the C-termini: MYPT-75D/MYPT3 have a CaaX prenylation motif, whereas DMBS/MYPT1/2 do not. MYPT-75D localizes to the cell periphery; this implies the existence of two different nonmuscle myosin phosphatases in different compartments of the cell: DMBS:PP1c (PP1α or PP1β) in the cytoplasm and MYPT-75D:PP1β at the plasma membrane. These myosin phosphatases have different roles and may be subject to different regulation. However, gross perturbation, such as complete removal of one complex in either DMBS or flw mutants, may lead to hyperphosphorylation of Sqh throughout the cell and hence to similar phenotypic consequences. Similarly, overexpression of the cytoplasmic form at a sufficiently high level may compensate for loss of the membrane-associated form: it was found that overexpression of a DMBS cDNA can suppress flw6, indicating that greatly increased levels of DMBS:PP1α87B can partially compensate for loss of functional MYPT-75D: PP1β9C complexes. A reduction in DMBS gene dose did not enhance flw1, indicating that DMBS is not itself the key targeting subunit for PP1β. Overexpression of MYPT-75D did not suppress flw6, presumably because MYPT-75D is not limiting or because increased levels of a defective MYPT-75D:PP1β9C complex are not helpful. High-level overexpression of MYPT-75D is lethal to wild-type flies, and modest overexpression somewhat reduces the viability of flw1 flies. Both of these results are interpreted as being due to excess MYPT-75D diverting some PP1β from its normal role or location. flw1 flies, in which the MYPT-75D:PP1β9C myosin phosphatase is already somewhat defective, would be predicted to be more sensitive to this effect, as was observed (Vereshchagina, 2004).

In conclusion, PP1β has an essential role, which is in the regulation of nonmuscle myosin, and this can be entirely explained by its role as an MRLC phosphatase. It associates with two different myosin-targeting subunits, one of which is specific for PP1β. These two myosin phosphatases have different roles, though sufficiently high-level expression of the putative cytoplasmic form can partially compensate for loss of the putative membrane-associated form. Loss of PP1β, and hence the PP1β-specific myosin phosphatase, leads to cytoskeletal defects and death, as does loss of the other myosin phosphatase, indicating that each has an important, nonredundant role. All of the components of the system analyzed are well conserved between flies and humans, suggesting that the PP1β-specific myosin phosphatase may also be conserved (Vereshchagina, 2004).

Regulation of somatic myosin activity by protein phosphatase 1β controls Drosophila oocyte polarization

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 protein interactions 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).

Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration

Localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing edges of migrating cells. Although many cells migrate in collective groups during tissue morphogenesis, mechanisms that coordinate actomyosin dynamics in collective cell migration are poorly understood. Migration of Drosophila border cells, a genetically tractable model for collective cell migration, requires nonmuscle myosin-II (Myo-II). How Myo-II specifically controls border cell migration and how Myo-II is itself regulated is largely unknown. This study shows that Myo-II regulates two essential features of border cell migration: (1) initial detachment of the border cell cluster from the follicular epithelium and (2) the dynamics of cellular protrusions. It was further demonstrated that the cell polarity protein Par-1 (MARK), a serine-threonine kinase, regulates the localization and activation of Myo-II in border cells. Par-1 binds to myosin phosphatase (Flapwing) and phosphorylates it at a known inactivating site. Par-1 thus promotes phosphorylated myosin regulatory light chain, thereby increasing Myo-II activity. Furthermore, Par-1 localizes to and increases active Myo-II at the cluster rear to promote detachment; in the absence of Par-1, spatially distinct active Myo-II is lost. This study has identified a critical new role for Par-1 kinase: spatiotemporal regulation of Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the actomyosin dynamics required for border cell detachment and migration. Such a link between polarity proteins and cytoskeletal dynamics may also occur in other collective cell migrations (Majunder, 2012).

Myo-II plays a fundamental role in establishing the front-rear axis of migrating cells to promote directional migration. Myo-II localizes to the cell rear and stimulates motility at the front, likely by local stabilization of adhesions and actomyosin bundles at the cell rear but not at the front. In contrast to single cells, the mechanisms that set up or maintain polarized actomyosin contraction during collective migration are still poorly understood. This study identified a new role for Par-1 kinase, namely that Par-1 regulates myosin phosphatase to control Myo-II activation. A model is proposed in which Myo-II is activated in a polarized manner). Myosin phosphatase, which is distributed uniformly in the cluster, is locally inactivated by Par-1 at the basolateral side (back) of the cluster. The consequent polarization of active Myo-II induces contraction and cell morphological changes critical for detachment and motility. The question of how Par-1 becomes localized to the basolateral side of border cells is largely unknown. Phosphorylation by the apical polarity protein aPKC restricts Par-1 to basolateral membranes in epithelial cells and is also critical for Par-1 function in border cells. This mechanism may thus restrict basolateral localization of Par-1 in border cells, although a role for border cell-specific factors cannot be ruled out (Majunder, 2012).

The observation that there is an increase in Sqh:GFP at the rear of the border cell cluster during detachment is consistent with a specific role for Myo-II in promoting epithelial detachment of border cells. Loss-of-function sqh mosaic clone experiments demonstrated a requirement for Myo-II in border cells and possibly adjacent epithelial follicle cells. Indeed, live imaging analyses revealed that disruption of Myo-II function inhibited the ability of border cells to detach. This raises the question of how Myo-II contributes to detachment. Activation of Rok by Rho GTPase can destabilize cell-cell junctions by inducing actomyosin contraction in normal and tumor-derived epithelial cells. In other contexts, however, Rho-dependent Myo-II stabilizes cell junctions through regulation of the junctional protein E-cadherin. The overall levels of E-cadherin were unchanged when Rok was knocked down in border cells, suggesting that activated Myo-II more likely contributes directly to detachment. It is suggested that the localized increase in active Myo-II at the rear specifically contracts the border cell cluster and helps it pull away from the epithelium. In the absence of Par-1, overall levels of activated Myo-II were decreased and Sqh:GFP foci, which correlate with active Myo-II, exhibited altered dynamics; this potentially leads to uncoordinated or decreased contractile forces and thus to defects in detachment (Majunder, 2012).

Par-1 promotes increased p-MRLC/Sqh levels and higher levels of activated myosin by phosphorylation of myosin phosphatase at a known inactivating threonine. Regulated myosin phosphatase activity is essential for many cellular processes, including cell motility and epithelial morphogenesis. Despite identification of kinases that inactivate myosin phosphatase in vitro, few have been shown to do so in vivo during migration. Notably, vertebrate MARK2 (Par-1) phosphorylates the Mbs homolog MYPT1 in vitro at several sites, including the conserved threonine examined in the current study, although this has not been confirmed in vivo. This raises the intriguing possibility that vertebrate Par-1 homologs regulate myosin phosphatase, and thus Myo-II, during cell migration. However, Par-1-mediated regulation of Myo-II phosphorylation via myosin phosphatase may be cell or context specific. In contrast to the situation in border cells, Par-1 does not colocalize with myosin phosphatase in Drosophila ovarian epithelial follicle cells; active p-MRLC/Sqh and Mbs localized to apical domains in follicle cells whereas Par-1 localizes to basolateral membrane (Majunder, 2012).

Active Myo-II accumulates at the apical side/front of the border cell cluster in addition to its localization at the rear. Myo-II that is localized near the leading edge of single cells has been proposed to promote retraction by coordinating cell-substrate adhesions with the actin cytoskeleton. Likely roles for Myo-II at apical (front) side of the border cell cluster include retraction of protrusions, as well as resolving protrusion dynamics from the pre- to post-detachment phases of migration. The data do not explicitly support a role for Par-1 at the apical side of the cluster. Moreover, in the absence of Par-1, a low level of phosphorylated MRLC/Sqh was detected that was still partially localized. Thus, Myo-II is activated by at least one other kinase in addition to Par-1 (Majunder, 2012).

It was hypothesize that Par-1 promotes higher levels of Myo-II activity at the basolateral side (back), whereas another kinase activates Myo-II (and/or inactivates the phosphatase) specifically at the front. Rok can phosphorylate both MRLC and myosin phosphatase. Knockdown of Rok by RNAi significantly reduced p-MRLC/Sqh levels and disrupted border migration. The combined depletion of both Par-1 and Rok almost completely abolished detachment, suggesting that the two kinases converge (directly or indirectly) on the same target (Myo-II). Epithelial morphogenesis during C. elegans embryonic elongation and Drosophila larval tissue development require multiple kinases to optimally activate Myo-II. Furthermore, different kinases have been shown to regulate MRLC activation at discrete locations within single migrating cells. For example, MLCK phosphorylates MRLC at the front or leading edge, whereas Rok targets MRLC in the cell body and at the trailing edge of fibroblasts. However, it remains to be determined whether Rok and/or additional kinases have a polarized or more general role in Myo-II activation in border cells (Majunder, 2012).

This study demonstrates that maintaining localized Myo-II activity is a critical feature of collective cell detachment and motility and identifies the conserved polarity kinase Par-1 as a key new regulator of this pathway. Active Myo-II is polarized within the border cell cluster, rather than in individual border cells, emphasizing that asymmetrically activated Myo-II contributes to collective behavior. Notably, in a model of collective cancer cell invasion, high actomyosin activity at cell-matrix contacts combined with low activity at contacts between cells within the group, produced optimal contractile force around the outside and thus promoted collective cell movement. It will be important to determine whether vertebrate Par-1 homologs also regulate actomyosin contraction during processes that depend on collective cell motility, such as wound healing or tumor invasion and metastasis. Given that many metastasizing tumors detach from epithelia both as single cells and collective groups, it will be important to further probe the mechanisms of myosin-mediated contraction in this process (Majunder, 2012).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. This study shows that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting (Loubery, 2017).

Asymmetric cell division plays many roles in development. In particular, stem cells divide asymmetrically to self-renew while also forming differentiated cells. Asymmetric cell division involves the specific partitioning of cell fate determinants (RNA, proteins or organelles) in one of the two sibling daughter cells. The Sensory Organ Precursor cells (SOPs) of the Drosophila notum are a model system of choice to unravel the molecular mechanisms of asymmetric cell division (Loubery, 2017).

The division of each SOP gives rise to a pIIa and a pIIb daughter cell and, after two more rounds of asymmetric cell divisions, to the four cells of the sensory organ: the outer cells (shaft and socket) are progeny of the pIIa, while the pIIb forms the inner cells (sheath and neuron) and a glial cell that rapidly undergoes apoptosis. The Notch signalling pathway controls cell fate determination in this system: a signalling bias between the pIIa-pIIb sibling cells is essential to obtain a correct lineage (Loubery, 2017).

The asymmetric dispatch of cell fate determinants during SOP division is governed by the polarity of the dividing cell. The Par complex (composed by the aPKC, Par-3 and Par-6 proteins) is the master regulator of the establishment of this polarity. Downstream the Par complex, Notch signalling is regulated by endocytosis and endosomal trafficking through four independent mechanisms: (1) The E3 Ubiquitin ligase Neuralized is segregated to the pIIb cell, where it induces the endocytosis and thereby the activation of the Notch ligand Delta; (2) Recycling endosomes accumulate in the perinuclear region of the pIIb cell, in which they enhance the recycling and activation of Delta; (3) The endocytic proteins α-adaptin and Numb are segregated to the pIIb cell, where they inhibit the Notch activator Sanpodo; (4) During SOP mitosis, Sara endosomes transport a signalling pool of Notch and Delta to the pIIa cell, where Notch can be activated. Asymmetric Sara endosomes have also been shown to operate in the larval neural stem cells (Coumailleau, 2009) as well as in the adult intestinal stem cells in flies, where they also play a role during asymmetric Notch signalling. In fish, Sara endosomes mediate asymmetric cell fate assignation mediated by Notch during the mitosis of neural precursor of the spinal cord (Loubery, 2017).

Sara endosomes are a subpopulation of Rab5-positive early endosomes characterised by the presence of the endocytic protein Sara. Sara directly binds the lipid phosphatidyl-inositol-3-phosphate and both molecules are found at the surface of these endosomes. A pulse-chase antibody uptake assay has been established to monitor the trafficking of endogenous internalised Notch and Delta and showed that both Notch and Delta traffic through Sara endosomes. Furthermore, it was shown that Sara endosomes are specifically targeted to the pIIa cell during SOP division, mediating thus the transport of a pool of Notch and Delta that contribute to the activation of Notch in the pIIa. The Notch cargo and its Uninflatable binding partner are required for this asymmetric dispatch. Targeting of Sara endosomes to the central spindle is mediated by a plus-end-directed kinesin, Klp98A. The asymmetric distribution of endosomes at the central spindle results from a higher density of microtubules in pIIb with their plus ends pointed towards pIIa15 (Loubery, 2017).

This study shows that the Sara protein itself controls both the targeting and the final dispatch of Sara endosomes to the pIIa daughter cell. Sara binds and is a target of the PP1 phosphatase complex. The phosphorylation state of Sara functions as a switch that enables the targeting of Sara endosomes to the central spindle of the dividing SOP, and their subsequent detachment from the central spindle, which is necessary to allow their movement to the pIIa daughter cell (Loubery, 2017).

Previous work has shown that a subpopulation of Rab5 early endosomes positive for Sara are asymmetrically dispatched into the pIIa daughter cell during cytokinesis of the SOP. This was monitored by following in vivo either GFP-Sara or internalized Delta or Notch, which reach the Sara endosomes 20 min after their endocytosis in the mother cell. These vesicles were termed iDelta20' endosomes. In contrast, the pools of Notch in endosomal populations upstream or downstream of the Sara endosomes (that is, the Rab5 early endosomes with low Sara levels and the Rab7 late endosomes, respectively) were segregated symmetrically. Rab5 endosomes show different levels of Sara signal: by a progressive targeting of Sara to the Rab5 endosomes, Rab5 early endosomes mature into Sara endosomes. This prompts the question whether the levels of Sara in endosomes correlate indeed with their asymmetric behaviour (Loubery, 2017).

To study the relationship between the levels of Sara in endosomes and their targeting to the spindle, Matlab codes were written to perform automatic 3D-tracking of the Sara endosomes. Sara endosomes were detected by monitoring a GFP-Sara fusion, which was overexpressed through the UAS/Gal4 system. This way, the position of the endosomes, their displacement towards and away from the central spindle was monitored as well as the levels of Sara. In addition, the position was detected automatically of the Pon cortical crescent, which forecasts the side of the cell that will become the pIIb cell (Loubery, 2017).

The localization of endosomes was studied with respect to a 2 μm-wide box centered in the central spindle during SOP mitosis. The enrichment was measured of endosomes in this central spindle as a function of time. Two phases were observed in the movement of the endosomes during mitosis: (1) targeting to the central spindle and (2) departure into the pIIa cell. The endosomes are progressively accumulating in the central spindle area from the end of metaphase (~450 s before abscission) through anaphase and during cytokinesis until they are enriched at the central spindle by about 10-fold at 250 s before abscission (Loubery, 2017).

Subsequently, the endosomes depart from the central spindle area into the pIIa cell. By fitting an exponential decay to the profile of abundance of the endosomes at the central spindle, the characteristic residence time of the endosomes at the central spindle was measured after the recruitment phase: after recruitment, endosomes remain at the central spindle 98±9.8 s before they depart into one of the daughter cells, preferentially the pIIa cell (Loubery, 2017).

To address a potential role of Sara on central spindle targeting and asymmetric segregation, the behaviour was tracked and quantified of the endosomes in a Sara loss of function mutant (Sara12) and in conditions of Sara overexpression in the SOP (Neur-Gal4; UAS-GFP-Sara). In Sara12 SOPs, targeting of iDelta20' endosomes to the cleavage plane is severely impaired. Consistent with the fact that the asymmetric dispatch of endosomes to pIIa requires first their targeting to the central spindle as previously shown, in Sara12 SOPs the dispatch to the pIIa daughter is strongly affected. A slight bias (60% pIIa targeting) is, however, retained in the mutant, consistent with a previous report (Loubery, 2017).

Conversely, overexpression of Sara increases targeting to the central spindle. In these conditions, Sara is found not only in Rab5 endosomes, but also in Rab7 late endosomes as well as in the Rab4 recycling endosomes. Correlating with this, Rab4, Rab5 and Rab7 endosomes, which are not all recruited to the central spindle in wild-type conditions, are now targeted to the central spindle upon Sara overexpression and are asymmetrically targeted (Loubery, 2017).

Furthermore, consistent with the correlation that is observed between the levels of Sara at the endosomes and their displacement towards the cleavage plane, quantification of central spindle targeting of the Sara endosomes upon its overexpression shows that targeting of the endosomes to the cleavage plane is increased by a factor of 2.5 in these conditions. These observations indicate that Sara plays a crucial role on the targeting of the endosomes to the spindle and the subsequent dispatch of the Notch/Delta containing endosomes to pIIa. Does this play a role during Notch-dependent asymmetric cell fate assignation? (Loubery, 2017).

Sara function contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized. Neuralized E3 Ubiquitin ligase does play an essential role during the endocytosis and activation of the Notch ligand Delta. Therefore, during larval development, Neuralized is essential for Notch-mediated lateral inhibition in the proneural clusters, which leads to the singling-out of SOP cells from the proneural clusters. Later, during pupal development, Neuralized appears as a cortical crescent in the pIIb side of the dividing SOPs, thereby biasing Delta activation in the pIIb cell and asymmetric activation of Notch in pIIa6 (Loubery, 2017).

Consistently, a partial loss of function of Neuralized by RNAi interference in the centre of the notum (Pnr>NeurRNAi Control) showed lateral inhibition defects in the proneural clusters, causing the appearance of supernumerary SOPs as well as asymmetric Notch signalling defects in the SOP lineage, leading to supernumerary neurons and loss of the external shaft/socket cells in the lineage. The remaining Neuralized activity in this partial loss of function condition allows many sensory organs (more than forty in the centre of the notum) to perform asymmetric cell fate assignation and to develop, as in wild type, into structures containing at least the two external cells (Loubery, 2017).

In Pnr>NeurRNAi, Sara12/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>NeurRNAi controls (668±38 versus 498±52). This indicates that during lateral inhibition, Sara endosomes contributes to Notch signalling. This general role of Sara is uncovered when the Neuralized activity during Notch signalling is compromised (Loubery, 2017).

In the case of Neuralized, its localization to the anterior cortex biases Notch signalling to be elicited in the pIIa cell. This is the same in the case of Sara endosomes: asymmetric dispatch of Sara endosomes also biases Notch signalling to pIIa10. Indeed, in Pnr>NeurRNAi, Sara12/Df(2R)48 transheterozygote mutants, the number of bristles (external shaft/socket cells) in the notum is strongly reduced at the expense of supernumerary neurons compared to the Pnr>NeurRNAi controls. This indicates that Notch-dependent asymmetric cell fate assignation in the SOP lineage is synergistically affected in the Sara/Neuralized mutant. This implies that the SOP lineages which still could generate bristles with lower levels of Neuralized function in Pnr>NeurRNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>NeurRNAi, Sara12/Df(2R)48 and Pnr>NeurRNAi, Sara12/Sara1 transheterozygote mutants, these lineages failed to perform asymmetric signalling, causing the notum to be largely bald. Therefore, Sara contributes to Notch signalling and asymmetric cell fate assignation, as observed in conditions in which other redundant systems for asymmetric Notch signalling are compromised (Loubery, 2017).

Both Neuralized and Sara play general roles in Notch signalling: they are both involved in lateral inhibition at early stages and, at later stages, in asymmetric cell fate assignation. Indeed, both Neuralized and Sara mutants show early defects in lateral inhibition and, accordingly, they show supernumerary SOPs. In addition, Neuralized and Sara mutant conditions also show defective Notch signalling during cell fate assignation in the SOP lineage and therefore cause the transformation of the cells in the lineage into neurons. In this later step, Notch signalling is asymmetric. The possibility that both Sara and Neuralized play key roles in ensuring the asymmetric nature of this signalling event is only correlative: in the case of Neuralized, it is enriched in the anterior cortex of the cell, which will give rise to pIIb; in the case of Sara, (1) both Delta and Notch are cargo of these endosomes, (2) cleaved Notch is seen in the pIIa endosomes and (3) Sara endosomes are dispatched asymmetrically to pIIa10. It is tantalizing to conclude that the asymmetric localization of these two proteins mediate the asymmetric nature of Notch signalling in the SOP lineage, but further assays will be necessary to unambiguously address this issue. Clonal analysis is unfortunately a too slow assay to sort out the specific requirement of these cytosolic factors (Sara and Neuralized) in the pIIa versus the pIIb cell (Loubery, 2017).

Sara mediates the targeting of Notch/Delta containing endosomes to the central spindle and could contributes to Notch-mediated asymmetric signalling in the SOP lineage. What machinery controls in turn the Sara-dependent targeting of endosomes to the central spindle? Previous proteomic studies uncovered bona fide Sara-binding factors, including the Activin pathway R-Smad, Smox17 and the beta subunit of the PP1c serine-threonine phosphatase (PP1β(9C)). In an IP/Mass Spectrometry approach, those interactions were confirmed and in addition to PP1β(9C), two of the other three Drosophila isoforms of PP1c: PP1α(87B) and PP1α(96A) were found. Furthermore, the PP1c regulatory subunit Sds22 was found, suggesting that Sara binds the full serine-threonine PP1 phosphatase complex. The interaction with Sds22 was confirmed by immunoprecipitation of overexpressed Sds22-GFP and western blot detection of endogenous Sara in the immunoprecipitate (Loubery, 2017).

Prompted by these results, whether the PP1 complex plays a role in the asymmetric targeting of the Sara endosomes was explored by manipulating the activity of Sds22, the common regulatory unit in all the complexes containing the different PP1 isoforms. Sds22 was overexpressed specifically during SOP mitosis, by driving Sds22-GFP under the Neur-Gal4 driver with temporal control by the Gal80ts system. In SOPs where PP1-dependent dephosphorylation is enhanced by overexpressing Sds22, the Sara endosomes fail to be dispatched asymmetrically toward the pIIa daughter cell (Loubery, 2017).

The role of PP1-dependent dephosphorylation in the SOP was examined by knocking down Sds22 (through a validated Sds22-RNAi). Loss of function Sds22 did also affect the asymmetric targeting of endosomes. These data uncover a key role for phosphorylation and PP1-dependent dephosphorylation as a switch that contributes to the asymmetric targeting of Sara during asymmetric cell division (Loubery, 2017).

The observations raise the question of which is the step in the asymmetric dispatch of the endosomes that is controlled by the levels of phosphorylation: central spindle targeting, central spindle detachment or targeting to the pIIa cell? PP1/Sds22-dependent dephosphorylation controls a plethora of mitotic events, including mitotic spindle morphogenesis, cortical relaxation in anaphase, epithelial polarity and cell shape, Aurora B activity and kinetochore-microtubule interactions as well as metabolism, protein synthesis, ion pumps and channels. Therefore, to establish the specific event during the asymmetric dispatch of Sara endosomes that is controlled by PP1/Sds22 dephosphorylation, focus was placed on the phosphorylation state of Sara itself and its previously identified phosphorylation sites. This allowed specific interference with this phosphorylation event and thereby untangle it from other cellular events also affected by dephosphorylation (Loubery, 2017).

PP1/Sds22 was shown to bind Sara. It has previously been shown that mammalian Sara itself is phosphorylated at multiple sites and that the level of this Sara phosphorylation is independent on the level of TGF-beta signalling. Three phosphorylation sites have been identified at position S636, at position S709, and at position S774 in Sara protein and these sites were confirmed by Mass Spectrometry of larval tissue expressing GFP-Sara. Phosphorylation of Sara had been previously reported to be implicated in BMP signalling during wing development. However, the role of these three phosphorylation sites during asymmetric division are to date unknown (Loubery, 2017).

ProQ-Diamond phospho-staining of immunoprecipitated GFP-Sara confirmed that Sara is phosphorylated. To test whether PP1/Sds22 controls the phosphorylation state of Sara, ProQ-Diamond stainings of GFP-Sara were performed with and without down-regulation of Sds22. Downregulating Sds22 induced a 40%-increase in the normalized quantity of phosphorylated Sara, showing that PP1/Sds22 does control the phosphorylation state of Sara (Loubery, 2017).

To study the role of Sara phosphorylation during asymmetric targeting of the endosomes, the mitotic behaviour of the endosomes was analyzed in conditions of overexpression of mutant versions of Sara where (1) the three phosphorylated Serines (at position S636, S709, and S774) were substituted by Alanine (phosphorylation defective: GFP-Sara3A) or (2) the PP1 interaction was abolished by an F678A missense mutation in the PP1 binding domain (hyper-phosphorylated: GFP-SaraF678A). Neither mutation affects the general levels of abundance of the Sara protein in SOPs, the targeting of Sara itself to the endosomes, nor the residence time of Sara in endosomes as determined by FRAP experiments. Also, the targeting dynamics of internalized Delta to endosomes are not affected in these mutants (Loubery, 2017).

Upon overexpression of GFP-Sara3A in SOPs, the rate of targeting of the endosomes to the central spindle is greatly increased. In addition, GFP-Sara3A shows impaired departure from the spindle: while the residence time of Sara endosomes at the central spindle after their recruitment is around 100 s in wild type, GFP-Sara3A endosomes stay at the spindle significantly longer (151±21 s). In GFP-Sara3A endosomes, impaired departure leads to defective asymmetric targeting to the pIIa cell while, in wild type, departure from the central spindle occurs well before abscission, in the GFP-Sara3A condition, endosomes that did not depart are caught at the spindle while abscission occurs. These data indicate that the endosomal targeting to the central spindle is greatly favoured when these three sites in Sara are dephosphorylated and suggest that the departure from the microtubules of the central spindle requires that the endosomes are disengaged by phosphorylation of Sara (Loubery, 2017).

Loss of Sara phosphorylation in these sites impairs disengagement from the central spindle. Conversely, impairing Sara binding to the PP1 phosphatase results in defective targeting to the central spindle. Indeed, when binding of Sara to the PP1/Sds22 phosphatase is impaired in the GFP-SaraF678A overexpressing SOP mutants, Sara endosomes fail to be targeted to the spindle. Mistargeted away from the central spindle, the GFP-SaraF678A endosomes fail thereby to be asymmetrically targeted to the pIIa cell. Loss and gain of function phenotypes of the Phosphatase regulator Sds22 during endosomal spindle targeting support the role of Sara phosphorylation during targeting to the central spindle microtubules suggested by the GFP-Sara3A and GFP-SaraF678A experiments (Loubery, 2017).

What are the functional consequences on signalling of impaired phosphorylation/dephosphorylation in Sara mutants? The presence of Sara in endosomes is itself essential for Notch signalling. Sara loss of function mutants show a phenotype in SOP specification (supernumerary SOPs) as well as during fate determination within the SOP lineage (all cells in the lineage acquire a neural fate). In addition, this study showed that Sara is also essential for the targeting of endosomes to the spindle: in the absence of Sara, endosomes fail to move to the spindle in the SOP. They are therefore dispatched symmetrically, but those endosomes do not mediate Notch signalling. As a consequence, both daughters fail to perform Notch signalling in sensitized conditions in which Neuralized is compromised. The result is a Notch loss of function phenotype: the whole lineage differentiates into neurons (Loubery, 2017).

In both Sara3A and SaraF678A mutants, because of reasons that are different in the two cases (either they do not go to the spindle or their departure from the spindle is impaired), functional Sara endosomes are dispatched symmetrically (Fig. 6a,b,e). In contrast to the situation in the Sara loss of function mutant, those endosomes are functional Sara signalling endosomes, which can mediate Notch signalling in both cells. Therefore, these mutations are consistently shown to cause a gain of function Sara signalling phenotype: supernumerary sockets are seen in the lineages (88% of the lineages for Sara3A and 82% of the lineages for SaraF678A). A milder version of this phenotype can be also seen by overexpressing wild-type Sara (34% of the lineages) consistent again with some gain of function Notch signalling phenotype when Sara concentrations are elevated. In summary, this implies that the 3A and F678A mutations impair the phosphorylation state of Sara (with consequences in targeting), but not its function in Notch signalling (Loubery, 2017).

These results indicate that Sara itself plays a key, rate limiting role on the asymmetric targeting of the endosomes by controlling the targeting to the spindle and its departure. Maturation of the early endosomes by accumulating PI(3)P leads to accumulation of the PI(3)P-binding protein Sara to this vesicular compartment. At the endosome, the phosphorylation state of Sara indeed determines central spindle targeting and departure: in its default, dephosphorylated state, Sara is essential to engage the endosomes with the mitotic spindle. Phosphorylation of Sara disengages the endosomes from the central spindle allowing the asymmetric departure into the pIIa cell (Loubery, 2017).

PP1β9C interacts with Trithorax in Drosophila wing development

Type 1 Ser/Thr protein phosphatase (PP1) has many roles in Drosophila: regulating diverse processes from chromatin condensation to transforming growth factor-beta signaling. The presence of four PP1 genes, PP1alpha87B, PP1beta9C (Flapwing), PP1alpha96A, and PP1alpha13C, encoding very similar proteins complicates analysis of their particular functions. This study reports that the minor PP1 isoform PP1beta9C binds in vitro and in vivo and genetically interacts with Trithorax (TRX), the archetypal member of the Trx-G family of epigenetic regulators in Drosophila. Direct binding was demonstrated by GST pull-down experiments and PP1β9C/TRX interaction in vivo was confirmed by coimmune precipitation from Drosophila embryonic extracts. PP1β9C was found to be present at all TRX sites on the polytene chromosomes. Flies homo- and hemizygous for loss-of-function alleles of PP1beta9C exhibited specific wing defects when combined with various trx mutants, which indicates that PP1beta9C and TRX cooperate in Drosophila wing development (Rudenko, 2004).

The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1ß (PP1ß). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. This study shows genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw1, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. puc expression is upregulated in wing imaginal discs mutant for flw1 and pucA251, and this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw1 enhanced by puc. Together, this study shows that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs (Kirchner, 2007; full text of article).

This study shows that the nonmuscle myosin phosphatase flw interacts genetically with components of the JNK signaling pathway. The proteins JNK and PP1ß (Flw), as well as the mechanisms of JNK signal transduction and nonmuscle myosin activation, are highly conserved between Drosophila and humans. This suggests that findings of Drosophila PP1ß regulating JNK through nonmuscle myosin may be relevant for similar processes in human cells and tissues (Kirchner, 2007).

bsk (JNK) mutants suppressed the semilethality of the strong allele flw6, while puc and constitutively active hepCA enhanced the weak viable allele flw1, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw1/Y ; pucA251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw1/Y ; pucA251/+, implies that flw can act as a negative regulator of JNK. This was further supported by the fact that puc expression was not upregulated in flw1/Y ; bsk1/+ ; pucA251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw1/Y ; pucA251/+. A possible difficulty with using pucA251 (or pucE69, another frequently used puc enhancer trap) as a reporter of puc expression is that both lines are also puc mutants, and puc probably regulates its own expression through a negative feedback loop involving JNK. However, it was confirmed in an independent assay with an anti-P-JNK antibody that JNK was indeed ectopically activated in flw1/Y ; pucA251/+. Wing imaginal disc extracts from both flw1 and pucA251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK. In flw1/Y ; pucA251/+, it is suggested that dephosphorylation of JNK fails to such an extent that JNK is substantially diphosphorylated and thereby activated. Since it has been shown that activation of JNK in wing imaginal discs induces apoptosis, it is likely that the flw1/Y ; puc/+ wing phenotype is due to increased death of cells with aberrantly activated JNK (Kirchner, 2007).

A zip mutant suppresses the upregulation of puc in wing imaginal discs as well as the adult wing phenotype of flw1/Y ; pucA251/+. This shows that nonmuscle myosin acts upstream of JNK and mediates an activating signal on JNK in a flw1/Y ; puc/+ mutant background; this is also consistent with the finding that a mutant in the myosin phosphatase-targeting subunit Mbs enhances flw1/Y ; pucA251/+ to lethality. Drosophila encodes two myosin phosphatase-targeting subunits, Mbs and MYPT-75D. Mbs binds both Flw and Pp1-87B, while MYPT-75D binds Flw specifically Vereshchagina, 2004). Unfortunately, it was not possible to test for genetic interaction between flw1/Y ; pucA251/+ and MYPT-75D because no MYPT-75D mutants have been described. It was also found that a Rho1 mutant abolishes ectopic lacZ staining in flw1/Y ; pucA251 wing imaginal discs. Rho1 is an activator of Rho-dependent kinase, which phosphorylates and activates myosin light chain, as well as phosphorylating and inhibiting Mbs. Furthermore, both zip and Rho1, in combination with other genetic interactors, have been reported to show a malformed third-leg phenotype that resembles that of flw1/Y ; puc/+ (Kirchner, 2007).

Dorsal closure and wound healing depend on both nonmuscle myosin and JNK activity, but a clear genetic or molecular interaction between these pathways has not been previously demonstrated. This is the first study that shows that disruption of nonmuscle myosin can induce activation of JNK, although the mechanism of signal transduction is not clear. The single essential and nonredundant function of flw is the inhibition of nonmuscle myosin activity, presumably by dephosphorylating Sqh at T21 and S22. However, the results suggest that hyperphosphorylation of Sqh at these residues may not explain the elevated expression of puc in flw1/Y ; pucA251/+ flies. Additional mechanisms may exist to regulate nonmuscle myosin assembly and activity through phosphorylation, and the complex Flw/Mbs may have other targets in the actomyosin network in addition to Sqh. For example, moesin and focal adhesion kinase are potential targets for mammalian myosin phosphatase. Interestingly, overexpression of the focal adhesion protein tensin (blistery) in Drosophila wing imaginal discs activates JNK and induces apoptosis (Kirchner, 2007).

Two important questions remain unanswered regarding the results on the activation of JNK through nonmuscle myosin. (1) What is the molecular pathway from nonmuscle myosin to JNK? Several mechanisms have been identified that activate JNK and induce apoptosis in wing imaginal discs. For example, mutations in the caspase inhibitor DTraf1 (Drosophila tumor necrosis factor receptor-associated factor 1), as well as inhibition of DTraf1 by overexpression of Hid (head involution defective), Rpr (reaper), or Grim, induces JNK-mediated apoptosis, possibly through Msn (misshapen) or Ask1 (apoptosis signal-regulating kinase 1). Other factors involved in inducing apoptosis and activating JNK are Eiger (Drosophila tumor-necrosis factor superfamily ligand), the serine C-palmitoyltransferase Lace, Blistery (Drosophila Tensin), and Decapentaplegic and Wingless. It is not clear how these factors signal to JNK or whether they act in a single pathway, let alone whether any of them interact with nonmuscle myosin. It is likely that there are several independent ways of activating JNK, and it has been suggested that induction of apoptosis through JNK activation is a regulatory mechanism to eliminate abnormally developing cells in wing imaginal discs. (2)This leads directly to the second question: are the results of nonmuscle myosin signaling to JNK significant in a developmental context? The fact that bsk1 suppresses the semilethality of flw6 indicates that the interactions that uncovered are not confined to the main experimental model of ectopic JNK activation in wing imaginal discs. The obvious system for studying possible interactions between nonmuscle myosin and JNK would be dorsal closure, which depends on both the coordinated assembly and the contraction of the actomyosin cytoskeleton and on activation of JNK. So far, there is no evidence that dorsal closure is affected in flw mutant embryos; however, there is a maternal contribution of flw that may conceal embryonic phenotypes. Actomyosin and JNK do not promote dorsal closure completely independently from each other; for example, the expression of many components of the actomyosin cytoskeleton is upregulated in response to JNK. Also, actin and myosin failed to accumulate along the leading edge of the epidermis in the puc mutant background. Both findings would place the actomyosin cytoskeleton downstream of JNK, whereas genetic data place flw and zip upstream of JNK and puc. It is possible, however, that actomyosin acts both upstream and downstream of JNK during dorsal closure. Because of the conserved nature of the components involved, it is likely that the finding that nonmuscle myosin can signal to and activate JNK is relevant to furthering the understanding of processes like dorsal closure and wound healing in Drosophila and humans (Kirchner, 2007).

The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila

The signalling activities of Merlin and Moesin, two closely related members of the protein 4.1 Ezrin /Radixin/Moesin family, are regulated by conformational changes. These changes are regulated in turn by phosphorylation. The same sterile 20 kinase-Slik co-regulates Merlin or Moesin activity whereby phosphorylation inactivates Merlin, but activates Moesin. Thus, the corresponding coordinate activation of Merlin and inactivation of Moesin would require coordinated phosphatase activity. Drosophila protein phosphatase type 1 β (Flapwing) fulfils this role, co-regulating dephosphorylation and altered activity of both Merlin and Moesin. Merlin or Moesin are detected in a complex with Flapwing both in-vitro and in-vivo. Directed changes in flapwing expression result in altered phosphorylation of both Merlin and Moesin. These changes in the levels of Merlin and Moesin phosphorylation following reduction of flapwing expression are associated with concomitant defects in epithelial integrity and increase in apoptosis in developing tissues such as wing imaginal discs. Functionally, the defects can be partially recapitulated by overexpression of proteins that mimic constitutively phosphorylated or unphosphorylated Merlin or Moesin. These results suggest that changes in the phosphorylation levels of Merlin and Moesin lead to changes in epithelial organization (Yang, 2012).

The results suggest that Flw would act antagonistically to the kinase Slik during the coordinate regulation of Mer, acting as a tumour suppressor protein, and Moe, required to maintain epithelial integrity. If Flw acts as a coordinate regulatory phosphatase for Mer and/or Moe, it would be expected that Flw is in a protein complex with both Mer and Moe, and this was found to be true. A reproducible increase was found in the ratio of dephosphorylated to phosphorylated Mer isoforms when flw is overexpressed, and a decrease in this ratio was found when flw expression is reduced. In addition, four distinct Mer phosphorylation isoforms were detected. Supporting these observations, the over-expression of flw increases the amount of dephosphorylated Mer signal present as compared to the wild type tissue. Flw also affects the phosphorylation of Moe. The amount of phosphorylated Moesin protein is reduced when flw is over-expressed as compared to when flw expression is reduced. Thus, Flw appears to be a phosphatase specific for both Mer and Moe (Yang, 2012).

Most importantly, using functional assays in whole animals, Flw mediated regulation of Mer and Moe has clear effects on both Mer and Moe protein localization to the plasma membrane and on epithelial organization. There is a higher intensity of staining of both Mer and phosphorylated Moe associated with the plasma membrane upon reduction of flw expression. When the levels of other typical apical domain markers as well as basolateral markers were examined by maximum intensity projection analysis, it was found that maximum projections from larval wing discs show increased brightness of p-ERM, F-actin and anti-Armadillo, within the cells in which flw expression is reduced, whereas the septate junction marker anti-Coracle staining is not changed in intensity over the whole disc. This suggests that as a result of changes in Mer and Moe phosphorylation there are changes in links to the actin cytoskeleton and adherens junctions where both Mer and Moe play roles in wild type cells. Previous studies have demonstrated that phosphorylated Mer is more tightly associated with the plasma membrane. In agreement with data from Drosophila, mammalian cells also show increased plasma membrane association of a phosphomimic form of moesin or the related protein ezrin whereas dephosphorylated ERM proteins are less associated with the plasma membrane. Following flw knockdown in selected cells in the wing epithelium, cells within the boundary between cells with reduced flw expression levels and cells with wild type flw expression levels undergo the greatest amount of change in terms of epithelial integrity. The loss of polarity leads to increased apoptosis in these cells. These effects are observed when flw expression is reduced in only a few cells such as using the ptc Gal4 driver or in the entire dorsal compartment of the wing such as using the apterous Gal4 driver. The cells along the boundary region appear to fold inwards and detach from the rest of epithelium. This is likely the direct result of the difference in adhesion between cells that have reduced flw expression and cells which express wild type levels of Flw protein. As Mer and Moe appear to be direct targets of Flw, and both Mer and Moe have roles in adhesion, the changes in the adhesion of wing epithelium upon reduction of flw are likely a result of changes in Mer and Moe phosphorylation and thus activity. The combination of excess active Moe and excess inactive Mer would affect the balance between maintenance and loss of stabilization of adherens junctions leading to the changes in adhesion and deformation of the wing epithelia that were observed. These adhesion differences could account for the formation of the large folds along the boundary of the ptc expression domain, since cells of similar adhesion are more likely to adhere to themselves (Yang, 2012).

The deformation of the wing imaginal tissue appears to be progressive, since in pre-pupal wing discs (10 h after pupariation) deep holes are observed that extend from the apical surface basally indicating that cells at the apical surface have left the epithelium and are forming balls of cells basally within the disc. In further support of the results, the loss of sds22, a PP1 regulatory subunit, in clonal analysis shows that in large clones in wing discs there is infolding of the mutant tissue with cells being extruded from the epithelium. Cells with loss of function Sds22 also exhibit Moe hyper-phosphorylation. Notably, this is reminiscent of what was observe with reduction of flw expression and overexpression of phosphomimic or nonphsophorylatable Mer or Moe (Yang, 2012).

While a likely cause of some of the changes seen in functional assays are due to changes in Mer and Moe phosphorylation as a result of changes in flw expression, the possibility remains that the level of analysis and resolution of the functional assays in both larval and pupal imaginal wing discs may be insufficient to clearly show subtle differences in the subcellular localization on the membrane of Mer, Moe and apical markers. Thus, it cannot be concluded that the defects associated with flw are due solely to defects in Mer and Moe activity (Yang, 2012).

The ability to partially recapitulate the loss of flw phenotype in ptc expressing cells by the over-expression of either a phosphomimic or nonphsophorylatable Mer or Moe also strongly suggests that this phenotype is, in part, due to the differences in the ratios of active Mer or Moe to inactive Mer or Moe which lead to the corresponding changes in apical epithelial integrity, in third instar discs. This is exemplified by the observation that often with overexpression of either the phosphomimic or nonphosphorylatable Mer or Moe, the formation of a fold is most apparent at the edge of ptc expression at the boundary where the difference in the expression of Mer or Moe within the ptc expressing cells and the neighbouring wild type cells would be greatest. In this way it is not unexpected that the overall effect on the tissue deformation and adhesion is the same with phosphomimic or nonphosphorylatable Mer or Moe, although it is possible that the underlying causes are different due to the predicted opposite activities of the transgenes (Yang, 2012).

Within or directly beside the edge of the ptc expression domain in wing imaginal discs, significantly more cells stain positively for activated Caspase 3. This suggests that cells in these affected domains are undergoing increased levels of apoptosis. These phenotypes are again reminiscent of what is observed in loss of function clones of Sds22, which exhibit an increase in the number of apoptotic cells in the wing discs (Yang, 2012).

It was also demonstrated that Flw binds to the scaffold protein Sip1. It functions with the kinase Slik to regulate Moe activity to maintain epithelial cell integrity. Therefore, the findings suggest that Mer, Moe, Flw, and Sip1 function within a protein complex. This coordinate incorporation within a regulated protein complex is necessary to coordinate cellular response to changing epithelial integrity. This might also explain why the overexpression of flw does not have a strong effect on epithelial integrity. If Mer and Moe need to be part of a complex with Flw and Sip1 in order to regulate epithelial integrity and proliferation, then expression of excess phosphatase outside the complex would have no effect on tissue morphology and growth. In contrast, loss of the phosphatase would have a direct effect since there would be reduced levels of functional protein complex (Yang, 2012).

Future studies are required to determine additional members of this regulatory complex, such as the likely candidates Sds22 and MYPT-75D. The similarity in phenotypes between Sds22 mutant cells and the results of knockdown of flw function would also support a role of Sds22 to interact with Flw in regulating Moe function (Yang, 2012).

This study has shown that the Mer and Moe proteins are direct targets of the catalytic subunit of the PP1 phosphatase Flw. This identifies another important player in the regulation of both Mer and Moe in Drosophila. This is the first identification of a phosphatase coordinately regulating both Mer and Moe activity in vivo. What remains to be determined is how Flw is targeted to regulate Mer and Moe function and what downstream pathways may be affected by these interactions (Yang, 2012).

Protein phosphatase 1β is required for the maintenance of muscle attachments

Type 1 serine/threonine protein phosphatases (PP1) are important regulators of many cellular and developmental processes, including glycogen metabolism, muscle contraction, and the cell cycle. Drosophila and humans both have multiple genes encoding PP1 isoforms; each has one β and several α isoform genes (α1, α2, α3 in flies, α and γ in humans; mammalian PP1β is also known as PP1δ). The α/β subtype differences are highly conserved between flies and mammals. Though all these proteins are >85% identical to each other and have indistinguishable activities in vitro, this study shows that the Drosophila β isoform has a distinct biological role. PP1β9C corresponds to flapwing (flw), previously identified mutants of which are viable but flightless because of defects in indirect flight muscles (IFMs). A new, semi-lethal flw allele has been islated that shows a range of defects, especially in muscles, which break away from their attachment sites and degenerate (Raghavan, 2000; full text of article).

Though Drosophila has four genes encoding PP1 isoforms, one of these (PP1α87B) encodes 80% of the total PP1 activity. Another PP1 gene, PP1β9C, is much more closely related (94% identity) to mammalian PP1β than it is to other PP1 genes from flies or mammals (85% identity), suggesting that it has a conserved function distinct from that of PP1α. This study analysed the intron/exon structure of PP1β9C and it was found that three of the four introns in its coding region correspond exactly to three of the seven introns in the human gene, confirming the ancestral origin of this isoform. The α and β isoforms therefore appear to have been preserved in each lineage since their divergence >500 million years ago, despite their extreme sequence identity (Raghavan, 2000).

The PP1 isoforms have indistinguishable activity in vitro, so attempts were made to identify the non-redundant role(s) of PP1β by mutational analysis. It was found that PP1β9C corresponds to the flightless mutant flapwing (flw), which was originally isolated in a screen for viable, flightless mutants. The conservation of PP1β between flies and mammals suggests an essential role. Therefore lethal alleles of flw were screened for using the chemical mutagen ethyl methane sulphonate (EMS). flw6, a new, semi-lethal allele of flw, was successfully isolated. This demonstrates for the first time that the α and β isoforms are both essential, despite their 85% sequence identity. The flw1/flw6 flies are completely flightless and their muscle and wing phenotypes resemble combinations of flw1 with deficiencies covering the flw region (flw1/Df(1)N110 and flw1/Df(1)Hk), so flw6 appears to act as an amorphic allele (Raghavan, 2000).

The IFMs comprise two sets of muscles, the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). Alternating contraction of these two sets of muscles resonates the thorax, which in turn drives the wings. The development of the IFMs has been analysed in some detail. They are constructed during pupation from myoblasts, previously sequestered in the wing imaginal disk. The DLMs are built on a template of the larval oblique muscles, which are spared from the general histolysis of larval muscles, whereas the DVMs are formed by de novo fusion of myoblasts. The IFM defect was investigated in flw mutants using polarised light microscopy, dissection and plastic sections. The flw1 flies show a variable phenotype ranging from normal, through disorganised to absent IFMs. This phenotypic range was equally broad in an isogenic derivative line. IFMs were never present in flw1/Df(1)N110, flw1/Df(1)Hk or flw1/flw6 flies, but the jump muscle (tergal depressor of trochanter muscle or TDT) appeared normal. In the few (<1%) flw6/Y males that eclosed, the IFMs were absent and the TDT disorganised. All aspects of the flw6/Y phenotype were fully rescued by a PP1βC cDNA expression construct, demonstrating that flapwing corresponds to PP1β9C (Raghavan, 2000).

IFM development was analyzed in flw1/Df(1)Hk pupae. The early development of the IFMs appeared normal: the pre-templates on which the DLMs form were present, the DVMs also developed normally. Muscle defects became apparent at about 28 h after puparium formation. In the wild type, the muscles shorten and then elongate to form the final structure as they send out processes to their attachment sites. In the mutant, most of the muscles broke away from their posterior attachment sites and were found as ball-like structures at the anterior segment boundaries. These clumps were dissected and examined by electron microscopy. The sarcomere organisation appeared normal (Raghavan, 2000).

Less than 1% of flw6/Y animals survive to adult at 25°C. The period of lethality extends from the second larval instar onwards. Dying larvae are very sluggish, suggesting a possible defect in the larval body wall musculature. In the abdominal segments of the larva (A1-A7), each hemisegment contains a stereotyped set of 30 muscles. The musculature of flw6/Y was examined under polarised light and by using a muscle-specific LacZ marker. The muscle pattern in these larvae was clearly disrupted. Many muscles are missing, and others floated away during dissection, indicating very weak attachment. It is suggested that this muscle attachment defect leads directly to the semi-paralysed phenotype of the dying larvae and causes death by an inability to feed. Adult escapers also have very poor ambulatory ability, but the muscle attachments at this stage were not examined. Since the muscles appear to develop normally, but then detach, it is suggested that PP1β9C is required for the maintenance, rather than the formation of muscle attachments (Raghavan, 2000).

PP1β is the major isoform in rabbit skeletal muscle and so is the main isoform complexed to the myosin-targeting or M subunit of PP1. This complex is thought to dephosphorylate myosin regulatory light chain (MLC), antagonising myosin regulatory light chain kinase (MLCK). It was considered whether the phenotype of flw mutants might be due to a failure to dephosphorylate Mlc-2, but Mlc-2 mutants do not show defects in muscle attachment. A S66A/S67A non-phosphorylatable mutant of Mlc-2 does not suppress any flw mutant phenotypes (Raghavan, 2000).

Though PP1α87B is the major isoform overall, it might be that PP1β9C is the only or major isoform present in muscles. The flw phenotype might then be due to a reduction in the overall PP1 activity in the muscle, rather than any isoform-specific role of PP1β9C. Therefore the PP1 activity of muscle extracts were measured from various PP1 mutants. The total PP1 activity of larvae homozygous for Pp1-87B1 (also known as Su-var(3)601) is correspondingly reduced to about 20% of that of wild type and that of heterozygotes to about 50% of wild type. It was found that the PP1 activity from extracts of larval body wall muscles of these genotypes was reduced by a similar proportion, showing that PP1α87B is the major PP1 gene in these muscles. In contrast, the PP1 activity of equivalent extracts from flw1/Y or flw6/Y larval body walls were only slightly lower than wild-type controls (92% and 84% of wild type). Similar results were obtained from IFM extracts from these genotypes. Pp1-87B1 homozygous and heterozygous larvae do not show the muscle detachment phenotype of flw6/Y larvae, nor do the adults show the IFM loss of flw1 and flw6 . These phenotypes are therefore due to a specific requirement for PP1β9C and not to loss of overall PP1 activity in the affected muscles (Raghavan, 2000).

PP1β9C is required for the maintenance of muscle attachments. Whether PP1β9C is required in the muscle, the epidermis or elsewhere, was investigated. Using the GAL4-UAS expression system, a PP1β9C cDNA was placed under the control of a muscle-specific or an epidermis-specific promoter (Gal4-24B and Gal4-69B respectively. Neither of these rescues flw6, but a combination of the two does. This suggests that PP1β9C is required on both sides of the muscle attachment site (Raghavan, 2000).

In addition to the muscle attachment defects, strong flw mutants show extensive blistering and/or crumpling of the wing. The wing forms from two sheets of cells; blistering is due to a failure of these two sheets to adhere to each other. PP1β is therefore required for cell adhesion in non-muscle tissues as well as in the maintenance of muscle attachments (Raghavan, 2000).

Normal levels of PP1β9C protein are produced in flw1 and flw6. Therefore the organisation of the PP1β9C gene was analyzed, and the coding region and intron/exon boundaries in each mutant was sequenced. Each has a single amino acid change. Both mutations affect amino acids that are completely conserved across all four fly PP1 genes, and indeed through to mammals. Based on the published X-ray structure of PP1, Y133 forms a hydrogen bond to the peptide backbone of the substrate. Flw6 protein is therefore predicted to bind all substrates with reduced affinity. V284 is in a hydrophobic pocket adjacent to the binding site for a set of regulatory proteins. Flw1 protein is therefore predicted to have altered affinity for these regulatory proteins (Raghavan, 2000).

The PP1 gene family encodes a set of closely related proteins. Despite their similarity in sequence and in vitro activity, subtle sequence differences between different isoforms are conserved between flies and humans. Of the mammalian genes, functional analysis by gene knockout has so far only been performed for one PP1 isoform, PP1γ. This knockout eliminated both the widely expressed PP1γ1 and the testis-specific PP1γ2. Homozygous mutant female mice were viable and fertile; homozygous mutant males were viable but sterile, with a range of defects in spermatogenesis. Presumably the somatic functions of PP1γ are redundant with PP1α and/or the less closely related PP1β. This study has shown an essential, in vivo role for a minor PP1 isoform. The recent demonstration of differential subcellular distribution of the mammalian isoforms suggests that this may be mediated by isoform-specific targeting and regulatory subunits, localising the isoforms to different subcellular compartments and substrates (Raghavan, 2000).


Functions of Flapwing orthologs in other species

Protein phosphatase 1 recruitment by Rif1 regulates DNA replication origin firing by counteracting DDK activity

The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. This function of Rif1 is shown to depend on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. These data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing (Dave, 2014).

Protein phosphatase 1 down regulates ZYG-1 levels to limit centriole duplication

In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1 (see Drosophila SAK), is required for centriole duplication, but the understanding of how ZYG-1 levels are regulated remains incomplete. This study identified the two PP1 orthologs, GSP-1 (see Drosophila flw) and GSP-2 (see Drosophila Pp1-87B), and their regulators I-2SZY-2 (see Drosophila I-2) and SDS-22 (see Drosophila sds22) as key regulators of ZYG-1 protein levels. Down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication (see Drosophila centrioles) associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. These results thus define a new pathway that limits the number of daughter centrioles produced each cycle (Peel, 2017).


REFERENCES

Search PubMed for articles about Drosophila Flap wing

Axton, J. M., Dombradi, V., Cohen, P. T., and Glover, D. M. (1990). One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63: 33-46. PubMed ID: 2170019

Baksa, K. et al., (1993). Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster. Genetics 135: 117-125. PubMed ID: 8224813

Bollen, M, (2001). Combinatorial control of protein phosphatase-1.Trends Biochem. Sci. 26: 426-431. PubMed ID: 11440854

Bresnick, A, (1999). Molecular mechanisms of nonmuscle myosin-II regulation. Curr. Opin. Cell Biol. 11: 26-33. PubMed ID: 10047526

Cohen, P, (2002). Protein phosphatase 1 - targeted in many directions. J. Cell Sci. 115: 241-256. PubMed ID: 11839776

Dave, A., Cooley, C., Garg, M. and Bianchi, A. (2014). Protein phosphatase 1 recruitment by Rif1 regulates DNA replication origin firing by counteracting DDK activity. Cell Rep 7: 53-61. PubMed ID: 24656819

Dombrádi, V., Axton, J. M., Barker, H. M., and Cohen, P. T. (1990a). Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation. FEBS Lett. 275: 39-43. PubMed ID: 2175717

Dombrádi, V., Axton, J. M., Brewis, N. D., da Cruz e Silva, E. F., Alphey, L., and Cohen, P. T. W. (1990b). Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1. Eur. J. Biochem. 194: 739-745. PubMed ID: 2176604

Dombrádi, V., Mann, D. J., Saunders, R. D. C., and Cohen, P. T. W. (1993). Cloning of the fourth functional gene for protein phosphatase 1 in Drosophila melanogaster from its chromosomal location. Eur. J. Biochem. 212: 177-183. PubMed ID: 8383037

Hartshorne, D, (1998). Myosin phosphatase: subunits and interactions. Acta Physiol. Scand. 164: 483-493. PubMed ID: 9887971

Kaibuchi, K., Kuroda, S., and Amano, M, (1999). Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68: 459-486. PubMed ID: 10872457

Kirchner, J., Gross, S., Bennett, D. and Alphey, L. (2007). The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-Terminal kinase in wing imaginal discs of Drosophila. Genetics 175(4): 1741-1749. PubMed ID: 17277363

Loubery, S., Seum, C., Moraleda, A., Daeden, A., Furthauer, M. and Gonzalez-Gaitan, M. (2014). Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division. Curr Biol 24(18): 2142-2148. PubMed ID: 25155514

Majumder, P., Aranjuez, G., Amick, J. and McDonald, J. A. (2012). Par-1 controls myosin-II activity through myosin phosphatase to regulate border cell migration. Curr. Biol. 22(5): 363-72. PubMed ID: 22326025

Mizuno, T., Tsutsui, K., and Nishida, Y, (2002). Drosophila myosin phosphatase and its role in dorsal closure. Development 129: 1215-1223. PubMed ID: 11874917

Peel, N., Iyer, J., Naik, A., Dougherty, M.P., Decker, M. and O'Connell, K.F. (2017). Protein phosphatase 1 down regulates ZYG-1 levels to limit centriole duplication. PLoS Genet [Epub ahead of print]. PubMed ID: 28103229

Raghavan, S. et al, (2000). Protein phosphatase 1beta is required for the maintenance of muscle attachments. Curr. Biol. 10: 269-272. PubMed ID: 10712908

Rudenko, A., Bennett, D. and Alphey, L. (2004). PP1β9C interacts with Trithorax in Drosophila wing development. Dev Dyn 231: 336-341. PubMed ID: 15366010

Skinner, J. and Saltiel, A. (2001). Cloning and identification of MYPT 3, a prenylatable myosin targetting subunit of protein phosphatase 1. Biochem. J. 356: 257-267. PubMed ID: 11336659

Somlyo, A. and Somlyo, A. (2000). Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J. Physiol. 522: 177-185. PubMed ID: 10639096

Sun, Y., Yan, Y., Denef, N. and Sch├╝pbach, T. (2011). Regulation of somatic myosin activity by protein phosphatase 1β controls Drosophila oocyte polarization. Development 138(10): 1991-2001. PubMed ID: 21490061

Tan, C., Stronach, B. and Perrimon, N. (2003). Roles of myosin phosphatase during Drosophila development. Development 130: 671-681. PubMed ID: 12505998

Varmuza, S., Jurisicova, A., Okano, K., Hudson, J., Boekelheide, K. and Shipp, E. B. (1999). Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1cγ gene. Dev. Biol. 205: 98-110. PubMed ID: 9882500

Vereshchagina, N., et al. (2004). The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin. Mol. Biol. Cell 15(10): 4395-405. PubMed ID: 15269282

Yang, Y., et al. (2012). The PP1 phosphatase flapwing regulates the activity of Merlin and Moesin in Drosophila. Dev. Biol. 361(2): 412-26. PubMed ID: 22133918


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date revised: 22 December 2018

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