InteractiveFly: GeneBrief

flapwing: Biological Overview | References

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 Sqh^A20A21, coupled with the enhancement of flw¹ by Sqh^E20E21, 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-75D^F117A, 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 sqh^A20A21, 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 flw^FP41 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 flw^FP41, 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 flw^FP41 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 flw^FP41 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 flw^FP41 mutant PFCs. It is therefore likely that the defective Notch activity in flw^FP41 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 flw^FP41 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 flw^FP41 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 flw^FP41, 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 flw^FP41 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 flw^FP41, 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 flw^FP41 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).

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).

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 iDelta^20' 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 iDelta^20' 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>Neur^RNAi 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>Neur^RNAi, Sara¹²/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>Neur^RNAi 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>Neur^RNAi, 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>Neur^RNAi 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>Neur^RNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>Neur^RNAi, Sara¹²/Df(2R)48 and Pnr>Neur^RNAi, 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).

The ArfGAP Drongo promotes actomyosin contractility during collective cell migration by releasing Myosin phosphatase from the trailing edge

Collective cell migration is involved in various developmental and pathological processes, including the dissemination of various cancer cells. During Drosophila melanogaster oogenesis, a group of cells called border cells migrate collectively toward the oocyte. This study shows that members of the Arf family of small GTPases and some of their regulators are required for normal border cell migration. Notably, it was found that the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. Drongo controls the localization of the myosin phosphatase Flapwing in order to regulate myosin II activity at the back of the cluster. Moreover, toward the class III Arf, Drongo acts antagonistically to the guanine exchange factor Steppke. Overall, this work describes a mechanistic pathway that promotes the local actomyosin contractility necessary for border cell detachment (Zeledon, 2019).

Cell migration requires the precise spatiotemporal control of various determinants. In particular, motility-driving forces require the coordination of both actomyosin contractility, to generate traction forces in protrusions, and propulsive forces at the back of the cell. This spatiotemporal control is even more complex during collective cell migrations in which cells maintain contacts while migrating. Indeed, in these particular migrations, these processes need to be coordinated across the group of migrating cells. Border cell migration in the Drosophila ovary is a powerful model to investigate the mechanisms that regulate collective cell migration. Border cells (BCs) detach from the follicle epithelium surrounding the egg chambers and form a small cluster of six to ten cells that migrates invasively between the giant nurse cells that compose the center of the egg chamber, toward the oocyte. Border cells use E-cadherin to maintain cluster cohesion as well as to interact with the nurse cells. Their migration is guided by receptor tyrosine kinase (RTK) ligands that are secreted by the oocyte. During border cell migration, vesicular trafficking regulators have been involved in regulating the localization of E-cadherin between border cells, in the maintenance of active RTKs at the leading edge of the cluster, and in a cell-cell communication mechanism that restrains protrusive ability to the leader cell (Zeledon, 2019).

Vesicular transport is thus critical for the spatiotemporal control of migration determinants during border cell migration. Although previous work has focused mainly on the role of small GTPases of the Rab family, the role of Arf GTPases and their regulators during border cell migration is unknown (Zeledon, 2019).

Arf GTPases regulate the formation of vesicular transport intermediates by interacting with coatomers to bend the membrane of the donor compartment. They are grouped in three classes on the basis of amino acid similarity. Although mammals have multiple class I and class II Arfs, Drosophila possess only one Arf per class: Arf79F (class I), Arf102F (class II), and Arf51F (class III). Furthermore, another small GTPase, Sar1, is structurally related to Arfs and has also been involved in vesicular transport. In addition, there are Arl (Arf-like) proteins that are closely related to Arfs but have diverse functions. The regulation of Arfs and Arls is similar to that of other small GTPases: GDP/GTP exchange factors (GEFs) promotes their activation, while GTPase-activating proteins (GAPs) are responsible for their inactivation (Zeledon, 2019).

Class I and II Arfs and Sar1 are involved mainly in bidirectional transport within the secretory pathway. However, both class I and class II Arfs can also promote trafficking steps in the endocytic pathway. The single class III Arf (ARF6 in mammals) is present at the plasma membrane and in endosomes, where it regulates recycling to the plasma membrane (Zeledon, 2019).

Arf proteins regulate cell migration in various contexts. Notably, ARF6 regulates the recycling of integrins from dissociating focal adhesions to nascent one at the leading edge and the transport of active Rac to the plasma membrane. In mammals, a class I Arf (ARF1) regulates the formation of motile structures such as podosomes and generates actomyosin contractility by acting on different RhoGTPase. Intriguingly, these functions might be independent of the role of ARF1 in vesicular transport. Similarly, Arf regulators, in particular ArfGAPs, regulate cell migration independently of vesicular transport (Zeledon, 2019).

In Drosophila, Arf79F is required for lamellipodia formation in S2R+ cells, independently of Rac, and also in epithelial tube expansion. In the latter, the GEF Gartenzwerg (Garz) and the GAP ArfGAP1 regulate its activity. The sole member of the cytohesin family of GEFs in flies, Steppke (Step), regulates actomyosin contractility during dorsal closure. Interestingly, Step might act on both class I and III Arfs in this process (Zeledon, 2019).

To improve understanding of the vesicular machinery regulating border cell migration, an RNAi screen was performed targeting Arfs, Arls, and their potential regulators. Depletion of class I and II Arfs induced strong pleiotropic effects, while neither the expression of double-stranded RNAs (dsRNAs) against class III Arf nor against Arf-like proteins induced significant border cell migration delays. Furthermore, it was found that the depletion of several Arf regulators induced migration defects. This study focused on the ArfGAP Drongo, as it seemed to specifically affect the detachment of the border cell cluster at the onset of migration. Drongo is the ortholog of mammalian AGFG1 and was shown to be required for normal egg chamber development (Zeledon, 2019).

Drongo was found to inactivate the class III Arf at the back of the border cell cluster at the onset of border cell migration. This leads to a local decrease in the levels of myosin phosphatase and a subsequent increase in myosin II activity. In turn, this promotes contractility and allows the detachment of the border cluster from the follicle epithelium. Interestingly, it was found that Drongo acts in opposition to Step. Furthermore, it was found that this pathway seems to act independently of the kinase Par-1, which was shown to inactivate myosin phosphatase at the back of the border cell clusters. Overall, this work identifies Drongo as part of a molecular cascade promoting local actomyosin contractility by clearing the back of the cluster of the myosin phosphatase (Zeledon, 2019).

This study has shown that Arfs and several of their regulators are required for border cell migration. Although RNAi lines against Arf-like proteins and numerous regulators did not induce a phenotype, it cannot be concluded that they are not involved in border cell migration, as this study has not tested the efficiency of depletion in border cell of each potential false negatives. Downregulation of either class I or class II Arfs or of the GEF Garz in border cells induces pleiotropic effects, making it difficult to ascertain their specific role in border cell migration. However, it was found that Drongo has a specific function at the initiation of border cell migration, which requires its ArfGAP activity (Zeledon, 2019).

The results indicate that Drongo regulates contractility at the back of the cluster by controlling the localization of the myosin phosphatase. In the absence of Drongo, myosin phosphatase levels increase at the back of the cluster and consequently reduce the activity of myosin II, which is required for the detachment of the cluster. Furthermore, Drongo localizes at the trailing edge at the time of detachment, suggesting a direct role in the removal of myosin phosphatase from the back of the cluster (Zeledon, 2019).

In addition to its role in regulating myosin phosphatase at the back of the cluster during detachment, Drongo might be involved in the migration process per se. Indeed, it was found in the rescue experiments that when the Drosophila melanogaster form of Drongo that is still targeted by the interfering RNA was expressed the activity of myosin II is restored at the back of the cluster, but the migration of border cell is still incomplete. Interestingly, Drongo colocalizes partially with active myosin II (p-Sqh) both at detachment and during the migration of border cells (Zeledon, 2019).

The mechanisms by which Drongo acts on myosin phosphatase are not clear. Previous work showed that the detachment of the border cell cluster requires Notch signaling and that the polarity protein Par-1 regulates myosin phosphatase activity through the direct phosphorylation of Mbs by Par-1 (Zeledon, 2019).

Drongo depletion has no effects on Par-1 and Par-3 and it does not regulate Notch activity. As Par-1 acts directly on Mbs, it is concluded that Drongo acts in parallel to Par-1 and Par-3 by regulating the localization of myosin phosphatase. These results also indicate that Drongo is not acting upstream of Notch. It could be interesting to determine if Notch regulates drongo expression. Indeed, border cells express higher levels of drongo compared with the rest of the follicle cells (Borghese, 2006), and its human ortholog AGFG1 was found to be a direct transcriptional target of Notch1 in T cell acute lymphoblastic leukemia. Hence, drongo might be part of a subset of genes regulated by Notch to ensure the detachment of the border cell cluster (Zeledon, 2019).

Several ArfGAPs have been described as regulators of the actin cytoskeleton. Some act through direct binding of actin regulators and effectors, independently of their GAP activity. For example, ASAP1 directly interact with non-muscle myosin IIA to promote cell migration. The current results indicate that the ArfGAP activity of Drongo toward Arf51F is required for border cell migration. Furthermore, it was found that Drongo functions in opposition to the ArfGEF Step. Thus, Drongo might promote contractility by maintaining Arf51F in an inactive state to keep the rear of the cluster free of myosin phosphatase. Interestingly, Step was shown to inhibit actomyosin contractility during developmental cellularization and dorsal closure. It would be interesting to determine if Drongo could also counterbalance the activity of Step to regulate contractility during these two processes (Zeledon, 2019).

The way in which the balance between Drongo and Step regulates the localization of myosin phosphatase remains unknown. As Arf51F is involved, it is appealing to hypothesize that a specific vesicular transport event might regulate the localization of myosin phosphatase. However, no evidence was obtained that myosin phosphatase is transported to or cleared from the back of the cluster through vesicular trafficking. In particular, Mbs localized in vesicular structure was not observed, neither in control conditions nor after depletion of Drongo. Still, this does not rule out that trafficking might regulate myosin phosphatase. Indeed, this study might have overlooked the trafficking of the myosin phosphatase subunits because of technical limitations. Alternatively, it is possible that a regulator of myosin phosphatase activity is trafficked or that Arf51F might directly recruit the myosin phosphatase or a regulator of myosin phosphatase. In both cases, such a regulator is probably different than the polarity proteins Par-1 and Par-3, as their distribution was found to be unaltered after Drongo depletion. Finally, Drongo and Arf51F might remodel the protein or lipid content of the plasma membrane to allow the recruitment of Mbs. For example, ARF6, the mammalian ortholog of Arf51F, has the ability to modify the lipid composition of membranes. Further work will be necessary to discriminate among these possibilities. For example, it would be possible to try to determine if perturbing various vesicular trafficking steps by independent means affects detachment and contractility. Alternatively, it would be interesting to analyze the interactome of Arf51F in its active and inactive forms to determine if active Arf51F may directly recruit the myosin phosphatase or one of its regulators (Zeledon, 2019).

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 puc^A251, 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 hep^CA enhanced the weak viable allele flw¹, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw¹/Y ; puc^A251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw¹/Y ; puc^A251/+, 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/+ ; puc^A251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw¹/Y ; puc^A251/+. A possible difficulty with using puc^A251 (or puc^E69, 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 ; puc^A251/+. Wing imaginal disc extracts from both flw¹ and puc^A251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK. In flw¹/Y ; puc^A251/+, 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 ; puc^A251/+. 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 ; puc^A251/+ 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 ; puc^A251/+ 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 ; puc^A251 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 ; puc^A251/+ 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).

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).

Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila

In the metazoan S phase, coordinated firing of clusters of origins replicates different parts of the genome in a temporal program. Despite advances, neither the mechanism controlling timing nor that coordinating firing of multiple origins is fully understood. Rif1, an evolutionarily conserved inhibitor of DNA replication, recruits protein phosphatase 1 (PP1) and counteracts firing of origins by S-phase kinases. During the midblastula transition (MBT) in Drosophila embryos, Rif1 forms subnuclear hubs at each of the large blocks of satellite sequences and delays their replication. Each Rif1 hub disperses abruptly just prior to the replication of the associated satellite sequences. This study shows that the level of activity of the S-phase kinase, DDK, accelerated this dispersal program, and that the level of Rif1-recruited PP1 retarded it. Further, Rif1-recruited PP1 supported chromatin association of nearby Rif1. This influence of nearby Rif1 can create a “community effect” counteracting kinase-induced dissociation such that an entire hub of Rif1 undergoes switch-like dispersal at characteristic times that shift in response to the balance of Rif1-PP1 and DDK activities. A model is proposed in which the spatiotemporal program of late replication in the MBT embryo is controlled by self-stabilizing Rif1-PP1 hubs, whose abrupt dispersal synchronizes firing of associated late origins (Cho, 2022).

During a typical metazoan cell cycle, large genomic domains initiate their replication at distinct times in S phase. Cytological studies over 60 y ago revealed that DNA sequences in the compacted heterochromatin replicate later in S phase compared to euchromatin. These early studies and recent detailed analyses revealed a complex program among late replicating domains, in which different domains initiate replication with a specific delay. Execution of this stereotyped schedule occupies much of the S phase and must finish before mitosis. Despite recent advances in genomic methods for profiling global replication timing, the basis of the timing control is not yet solved, and how multiple origins are coordinated to fire together especially within repetitive DNA sequences is not known (Cho, 2022).

The Drosophila embryo offers a unique setting in which to examine the control of temporal programing of replication. In the earliest nuclear division cycles, there is no late replication, closely spaced origins throughout the genome initiate replication rapidly at the beginning of interphase, and their simultaneous action results in an extraordinarily short S phase (3.5 min). Late replication is developmentally introduced during the synchronous blastoderm nuclear division cycles, first influencing pericentric satellite sequences that form a major part of metazoan genomes (over 30% in Drosophila). Individual blocks of satellite DNA are typically several megabase pairs in length, each composed of a different simple repetitive sequence. During the 14th cell cycle at the midblastula transition (MBT), the ∼6,000 cells of the entire embryo progress synchronously through a temporal program in which the different satellites are replicated with distinctive delays (4), dramatically extending the duration of S phase (Cho, 2022).

The initial onset of late replication during development provides a simplified context in which to define its mechanism, because numerous complex features associated with replication timing have not yet been introduced. For example, chromatin states can have major impacts on replication timing. Consistent with this, late-replicating satellite sequences are usually heterochromatic, carrying the canonical molecular marks of constitutive heterochromatin (histone H3 lysine 9 methylation and HP1). During initial Drosophila embryogenesis, the satellites lack significant levels of these marks, and they replicate in sync with the rest of the genome. Surprisingly, the introduction of the delays in replication to the satellite sequences precedes a major wave of heterochromatin maturation in the blastoderm embryo. Furthermore, in a Rif1 null mutant (Rif1KO), the S phase of cycle 14 is significantly shorter, and the late replication of satellite sequences is largely absent even though HP1 recruitment appears normal. Thus, a Rif1-dependent program bears virtually full responsibility for the S-phase program at the MBT (Cho, 2022).

Rif1 is a multifunctional protein with an evolutionarily conserved role in regulating global replication timing. In species from yeast to mammals, mutation or depletion of Rif1 disrupts genome-wide replication timing. Studies in a variety of systems revealed several aspects of Rif1 function. Yeast Rif1 associates with late origins, while the Rif1 of both Drosophila and mammals binds broadly within large late-replicating domains. Rif1 has a conserved motif for interacting with protein phosphatase 1 (PP1), and mutations in the PP1-interacting motifs lead to hyperphosphorylation of MCM helicase in the prereplicative complex (pre-RC) and the disruption of global replication timing. Rif1 itself also harbors many sites recognized by S-phase kinases, including CDK and DDK, near its PP1-interacting motifs. In yeast, both a Rif1 mutant with phosphomimetic changes at these phosphorylation sites and a null mutation of Rif1 partially restore the growth defect of DDK mutants. These data suggest an interplay of Rif1 and DDK, wherein DDK acts first upstream of Rif1 phosphorylating it to disrupt its interaction with PP1, thus lowering the threshold of S-phase kinase activities required for origin firing. Second, DDK acts downstream to directly phosphorylate pre-RC and trigger origin firing. However, how these various features of Rif1 and DDK functions are integrated over large genomic regions to provide a domain-level control of replication timing remains elusive (Cho, 2022).

Studies in flies indicate that Rif1 has adopted a developmental role in governing the onset of the late replication program described above. During the early embryonic cell cycles, high Cdk1 and DDK activities jointly inhibit maternally deposited Rif1, promoting synchronous firing of origins throughout the whole genome to ensure completion of DNA replication during the short interphases. As the cell cycle begins to slow and oscillations in Cdk1 activity emerge, a transient Rif1-dependent delay in the replication of satellite sequences slightly prolongs S phase. When the embryo enters the MBT in cycle 14, abrupt down-regulation of Cdk1 more fully derepresses Rif1, which accumulates in semistable foci (hubs) at satellite DNA loci. High-resolution live microscopy reveals that different Rif1 hubs disperse abruptly at distinct times, followed by proliferating cell nuclear antigen (PCNA) recruitment as the underlying sequences replicate. Mutated Rif1 that is nonphosphorylatable at a cluster of CDK/DDK sites fails to dissociate from satellite DNA and dominantly blocks the completion of satellite DNA replication before mitosis. Conversely, ectopically increasing CDK activity in cycle 14 shortens the persistence of endogenous Rif1 foci and advances the replication program. These findings suggest that each Rif1 hub maintains a local nuclear microenvironment high in Rif1-recruited PP1 that inhibits DNA replication, and that kinase-dependent dispersal of Rif1 hubs is required to initiate the replication of satellite sequences. If it were understood what coordinates Rif1 dispersal throughout the large Rif1 hubs, this model could explain how firing of clusters of the underlying origins is coordinated and how replication of different satellites occurs at distinct times. However, the precise mechanisms controlling the dynamics of Rif1 hubs remain unclear (Cho, 2022).

Since Rif1 can recruit PP1 and form phosphatase-rich domains in the nucleus, it was hypothesized that localized PP1 counteracts kinase-induced Rif1 dissociation so that the Rif1 hubs are self-stabilizing. If this self-stabilization is communicated within each hub, a breakdown in self-stabilization would lead to a concerted collapse of the entire hub and allow origin firing throughout the associated satellite sequence. The current findings indicate that the opposing actions of phosphatase and kinase combined with communication within the hubs create a switch in which a large phosphatase-rich domain is stable until kinase activity overwhelms the phosphatase. It is proposed that for large late-replicating regions of the genome, recruitment of Rif1-PP1 creates a new upstream point of DDK-dependent regulation in which DDK triggers the collapse of the phosphatase-rich domain to create a permissive environment for kinase-induced firing of all previously repressed origins (Cho, 2022).

This study has investigated the mechanisms that control the timing of Rif1 foci dispersal from satellite sequences, which dictates the onset of late replication in the MBT embryo. Rif1-recruited PP1 was demonstrated to mediate self-stabilization of Rif1 hubs, while the S-phase kinase DDK opposes PP1 action and triggers the dispersal of Rif1 hubs. A model is proposed in which the firing of late origins is primarily controlled by a de-repression step upstream of the activation of the pre-RC. In this model, hubs of Rif1 create domains of locally high PP1 that prevent kinase activation of underlying pre-RCs. However, a changing balance of local phosphatase and kinase levels leads to the abrupt destabilization of different Rif1 hubs at distinct times (see A model for the multiple actions of PP1 in stabilizing Rif1 hubs.). This alleviates PP1 inhibition of hub-associated origins at specific times to trigger replication of the different satellites at different times. While this simple model appears sufficient to explain the late replication at its initial onset in the early Drosophila embryo, numerous other factors impact the replication program at later stages when chromatin acquires more complex features. Nonetheless, as is discussed below, the simplicity of the process in this biological context offers some insights into the more enigmatic aspects of late replication, and perhaps suggests a flexible regulatory paradigm that might be used in diverse contexts (Cho, 2022).

While the mechanism is unknown, it has long been clear that large domains of the genome behave as timing units, and that the numerous origins within such domains fire coordinately if not synchronously. The hub model of late replication control in the early embryo can explain how the firing of numerous origins within megabase pairs of satellite sequences can be coordinated in late S phase. Each Rif1 hub is associated with a locus of repetitive satellite sequence (10). Coordinated dispersal of a Rif1 hub will convert the subnuclear compartment from one restricting kinase actions to a permissive one, allowing the activation of pre-RCs throughout the associated chromatin domain. It was previously unclear what leads to the coordinated dispersal of these large hubs. This study shows that a mutant Rif1 that is deficient in binding PP1 cannot form stable hubs on its own, but it joins wild-type Rif1 in semistable hubs. Importantly, the mutant and wild-type Rif1 disperse together, showing that they respond equally to the property of the domain. It is suggested that Rif1-bound PP1 can act in trans to stabilize nearby Rif1-PP1 and that the propagation of this action coordinates the behavior of Rif1 across the entire hub (Cho, 2022).

The contribution of PP1 to the self-stabilization of Rif1 hubs might be mediated by feedback at multiple levels): 1) PP1 might activate itself by removing inhibitory phosphorylation catalyzed by Cdk1; 2) It could reverse Cdk1/DDK-mediated phosphorylation of Rif1 that disrupts PP1-recruitment; 3) It could reverse phosphorylation of Rif1 that disrupts Rif1 chromatin association; or 4) In a circuitous pathway, if the firing of origins were to promote Rif1 dissociation, PP1-dependent suppression of origin firing would stabilize the hubs. Any or all the above actions could reinforce the stability of Rif1-PP1 hubs, perhaps making different contributions in different situations and different organisms. However, regardless of the feedback route, a local dominance of PP1 will stabilize the Rif1 hubs, and rising kinase activity could erode this dominance of PP1. Upon reaching a tipping point, the local PP1 would no longer successfully stabilize the Rif1 hub, and S-phase kinases would then trigger complete dispersion and allow replication of the underlying chromatin (Cho, 2022).

A potential ability of origin firing to feedback and destabilize Rif1 hubs might explain observations in other organisms suggesting that the level of a variety of replication initiation factors can influence replication timing. For example, overexpression of four replication factors including a DDK subunit in the Xenopus embryo shortens the S phase at the MBT. While this has been interpreted as evidence for governance of replication timing by limitation for these factors, the effect may be indirect if overproduction of these factors overrides Rif1 suppression of pre-RC activation to advance the replication of late replicating regions as is seen in the fly embryo (Cho, 2022).

Importantly, the replication defects resulting from Cdc7 knockdown or inhibition of Cdc7, are suppressed in a Rif1 null mutant background. This shows that the level of DDK activity required to reverse or override Rif1 suppression of pre-RC activation is greater than the level needed for direct pre-RC activation. Thus, in a scenario in which rising levels of DDK during S-phase 14 act as a timer, genomic domains associated with Rif1 hubs would fail to replicate until DDK reached the high level required to destabilize the hub. This argues that replication timing depends on the threshold for derepression of the domain rather than on distinct thresholds for firing individual pre-RCs. It is therefore suggested that the timing of late replication is governed at the level of the upstream derepression step in Drosophila embryos, in contrast to the model proposed for other organisms according to which activation of pre-RCs are directly limited by availability of DDK and other replication factors. To produce the distinct temporal program of replication of different satellites, the current model requires domain-specific distinctions in the threshold for hub dispersal. Different satellite loci that are composed of a common repeat sequence replicate at the same time, while satellites composed of different sequences replicate at distinct times. This leads to a proposel that the sequence of repeats influences, likely indirectly, the threshold for Rif1 hub dispersal and the timing of replication (Cho, 2022).

The possible generality of the circuitry this study has defined in the cycle 14 Drosophila embryo can be considered in various ways. Focusing directly on Rif1 involvement in late replication, it is clear that Rif1 does not bare full responsibility for late replication at other stages. Nonetheless, a dosage-dependent function of Rif1 in controlling replication timing is also observed in Drosophila follicle cells during their mitotic cycles. Furthermore, in mammalian cells, ChIP-seq and microscopy showed that Rif1 interacts with large late-replicating domains but, as was seen in cycle 14 embryos, is absent once onset of replication of the underlying chromatin is detected. It is suggested that the mechanism described in this study will be one of multiple contributors to replication timing control in other biological contexts, and it is likely to be the major mode of replication timing in the rapid cycles of externally developing animal embryos (Cho, 2022).

Rif1 has other regulatory roles beyond timing control of pre-RC activation. In the follicle cells of Drosophila egg chambers, Rif1 is recruited to specialized replication forks during chorion gene amplification where it suppresses fork progression. While this action of Rif1 is dependent on its ability to associate with PP1, other possible parallels to the mechanism described in this study are not evident. Rif1 also regulates biological processes beyond replication. It is recruited to regions of DNA damage in mammals as well as to the telomeres in yeast where it has regulatory roles involving distinct interactions. Thus, Rif1 recruitment appears to trigger alternative regulatory pathways in different circumstances (Cho, 2022).

Despite the evident diversity of biological regulation, the capacity of Rif1 to form local membraneless compartments dominated by phosphatase and to abruptly dissolve in response to kinase levels might be an example of a group of flexible regulatory strategies. Many important regulatory events, such as phosphorylation, acetylation, and ubiquitination, are countered by reverse reactions. Various processes, notably the formation of liquid-like condensates, promote local accumulation of proteins. Accumulations of proteins that promote or oppose regulatory modifications could control major regulatory pathways. Furthermore, since protein accumulations could be stabilized or destabilized by the modifications they regulate, a feedback mechanism could control the formation and destabilization of a compartment to give precise spatiotemporal control, as exemplified by the behavior of the Rif1 hubs in the cycle 14 Drosophila embryo (Cho, 2022).

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).

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

Cho, C. Y., Seller, C. A. and O'Farrell, P. H. (2022). Temporal control of late replication and coordination of origin firing by self-stabilizing Rif1-PP1 hubs in Drosophila. Proc Natl Acad Sci U S A 119(26): e2200780119. PubMed ID: 35733247

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

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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

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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

Zeledon, C., Sun, X., Plutoni, C. and Emery, G. (2019). The ArfGAP Drongo promotes actomyosin contractility during collective cell migration by releasing Myosin phosphatase from the trailing edge. Cell Rep 28(12): 3238-3248. PubMed ID: 31533044

date revised: 25 April 2024

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