InteractiveFly: GeneBrief

spaghetti squash: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila egg chamber is an organ composed of a somatic epithelium that covers a germline cyst. After egg-chamber formation, the germline cells grow rapidly without dividing while the surface of the epithelium expands by cell proliferation. The mechanisms that coordinate growth and morphogenesis of the two tissues are not known. This study identifies a role for the actomyosin cytoskeleton in this process. Myosin activity is restricted to the epithelium's apical surface, which is facing the growing cyst. The epithelium collapses in the absence of myosin activity; the force that deforms the epithelium originates from the growing cyst. Thus, myosin activity maintains epithelial shape by balancing the force emanating from cyst growth. Further, these data indicate that cyst growth induces cell division in the epithelium. In addition, apical restriction of myosin activity is controlled. Myosin is activated at the apical cortex by localized Rho kinase and inhibited at the basolateral cortex by PP1β9C. In addition, these data indicate that active myosin is apically anchored by the Bazooka/Par-6/aPKC complex (Wang, 2007).

To analyze the correlation between cyst growth and follicle cell division, dividing cells in the follicular epithelium were counted. Within the first 56 hr that are required to form a stage 3 egg chamber, cell-division rates are very low. In the 14 hr period between stage 4 and stage 6, however, cell-division rates continuously increase. During this time, the volume of the cyst increases approximately 11-fold. This parallel increase in mitosis and cyst growth reflects how the growth of the inner cyst is compensated by cell division in the outer follicular epithelium. After stage 7, the follicle cells stop dividing and undergo diverse morphological changes (Wang, 2007).

Newly formed egg chambers are round and change their shape to ellipsoid during early oogenesis. After stage 7, the process of egg-chamber elongation, which is mediated by a polarized actin cytoskeleton within the follicular epithelium, starts. Actin fibers at the basal cortex of the follicle cells run perpendicular to the anterior-posterior axis of the egg chamber, and their contraction leads to an axis expansion. The mechanisms that shape the egg chamber before elongation takes place are unknown. The simultaneous and rapid growth of cyst and epithelium after stage 3 indicates that the development of the two tissues is precisely coordinated. It is, however, unclear how epithelial morphogenesis and proliferation are coupled to the growth of the cyst (Wang, 2007).

The actin cytoskeleton is central for the cell shape, and is thus a possible candidate involved in a controlled epithelial response to the cyst growth. The activity in the actomyosin cytoskeleton was examined by using an antibody specific for the activated form of the regulatory light chain of nonmuscle myosin II (RMLC; Spaghetti squash). The phospho-specific RMLC antibody binds to phosphoSerine21 and reveals myosin in its active state. Around stage 3 of oogenesis, myosin activity restricts to the apical cortex of the follicle cells, where it is maintained until late oogenesis. After stage 7, myosin activity is also present at the basal cortex of the follicle cells in the actin bundles required for egg chamber elongation. Optical confocal sections reveal a pattern of myosin activity in these long parallel bundles that is reminiscent of stress fibers. In contrast, at the apical cortex, myosin is active in short fibers with random orientation reminiscent of a web (Wang, 2007).

The membrane domains of the follicle cells are established before myosin activity restricts to the apical cortex at stage 3. To examine how apical myosin activation relates to follicle cell polarity, mutants affecting epithelial polarity were examined. To avoid perdurance of the wild-type protein after clone induction, focus was placed on large clones, or clones spanning the whole epithelium. The adherence junctions are central for the organization of the apical actin cytoskeleton, and the domain of myosin activity extends into the region where they localize. Therefore null mutants of armadillo (arm), which encodes Drosophila β-catenin, were examined. It has been shown that the adherence junctions are disrupted in arm follicle cell clones since neither DE- or DN-cadherin are detectable. As a result, arm mutant cells exhibit strong cell-shape defects. Surprisingly, it was found that myosin activity is clearly restricted to the apical membrane in arm follicle cell clones. Thus, myosin activity restricts apically in the absence of adherence junctions (Wang, 2007).

The apical membrane domain is established by the Crumbs (Crb)/Stardust (Sdt)/Patj complex and the Bazooka (Baz)/Par-6/aPKC complex. All these proteins localize, like pRMLC, to the apical membrane of the follicular epithelium. In epithelia lacking crb, myosin restriction is affected as revealed by the interrupted apical pRMLC pattern and by ectopic activity at the basal membrane. However, apical myosin activity is not completely disrupted as broad regions of the epithelium still concentrate higher levels of pRMLC at the apical compared to the basal cortex. In contrast, par-6, aPKC and baz mutants abolish the formation of the apical myosin activity. In these mutants, apical pRMLC restriction is lost, and ectopic myosin activity is detectable in the cytoplasm and at the basal cortex. To test whether the two apical complexes cooperate in apical myosin restriction, baz sdt double mutants were examined. The phenotype of the double mutants is, however, very similar to that of the baz single mutants, suggesting that apical myosin activity is controlled by the Baz/Par-6/aPKC complex (Wang, 2007).

To further analyze this interaction, the Baz/Par-6/aPKC complex was immunoprecipitated from ovaries using an antibody against Baz. Western-blot analysis of the precipitated protein complex reveals a strong enrichment of Baz and aPKC. Notably, pRMLC is also present in the precipitated protein complex, indicating an association of Baz and active myosin. Taken together, these genetic data show that baz, par-6, and aPKC are required for apical myosin restriction, and biochemical data show that Baz associates with pRMLC. This suggests the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex. To further analyze the role of the complex in the apical restriction of myosin activity, its localization was examined in mutants that affect pRMLC localization. Consistent with a function in the anchoring of active myosin, it was found that apical aPKC localization is not affected in arm mutants, in which pRMLC is apically restricted. Further, apical aPKC localization is interrupted in crb mutants, in which pRMLC localization is also interrupted. In summary, the data suggest that the Baz/Par-6/aPKC complex anchors active myosin at the apical cortex independently of the adherence junctions (Wang, 2007).

To examine how myosin activity is inhibited during early oogenesis at the basal and lateral cortex, the localization and function of PP1β9C, the phosphatase that deactivates phosphorylated RMLC (Vereshchagina, 2004) was examined. In follicle cells, PP1β9C is ubiquitously distributed as revealed by a hemagglutinin (HA) fusion protein. PP1β9C is encoded by flap wing (flw). Western-blot analysis of the viable flw¹ allele showed that the total pRMLC levels in ovaries are increased 2.8-fold compared to those of the wild-type. The total increase is the result of ectopic myosin activity in the follicular epithelium. This is revealed by flw⁶ follicle cell clones and homozygous flw¹ mutant egg chambers, which show pRMLC staining at the basal and lateral cortex. Interestingly, the ectopic Myosin activity is accompanied by an irregular and wavy appearance of the apical surface of the epithelium. In addition, flw mutant egg chambers are not round or ellipsoid like the wild-type but are either stretched or develop bulges. The coincidence of the altered shape with the ectopic pRMLC staining in the follicular epithelium suggests that the abnormal shape is the result of ectopic myosin activity. This is confirmed by the finding that the expression of constitutively active RMLC results in a very similar phenotype. The defects in flw mutants are not secondary effects of mislocalization of the Baz/Par-6/aPKC complex as the localization of aPKC is indistinguishable from that of the wild-type. In summary, these results show that PP1β9C activity is required to prevent myosin activity at the basal and lateral cortex. They further suggest that during early oogenesis, myosin activity has to be restricted to the apical cortex to ensure the development of normally shaped egg chambers (Wang, 2007).

To investigate how myosin is activated at the apical cortex, the function of Rok, which has been shown to regulate myosin phosphorylation, was analyzed. Myosin phosphorylation is greatly reduced but still detectable in rok mutant follicle cell clones. This confirms that Rok phosphorylates myosin in the follicular epithelium, but also indicates that Rok is not the only kinase involved in myosin activation. A HA-Rok fusion protein accumulates in particles at the apical cortex of the follicle cells, which are in close proximity to the web-like myosin fibers. Thus, localized Rok activates myosin in the follicular epithelium (Wang, 2007).

Because RMLC phosphorylation is strongly reduced in rok mutant cells, rok clones were used to examine the function of apical myosin activity. rok mutant follicle cells divide normally, form a monolayered follicular epithelium, and retain polarity. However, rok mutant cells fail to adopt a normal shape. As a consequence, the epithelium is flatter in these regions than it is in regions with rok activity. Optical sections at the level of the zonula adherens show that rok mutant cells are also stretched compared to neighboring wild-type cells. Furthermore, egg chambers with large follicle cell clones develop abnormal shapes as the cyst bulges outwards in the area of the clones. These results show that rok is required for follicle cell and egg-chamber shape, and indicate that the function of the apical myosin activity is to prevent flattening and stretching of epithelial cells (Wang, 2007).

To test the function of the apical myosin activity directly, follicle cell clones were generated using a null mutation for spaghetti squash (sqh). sqh encodes RMLC and was previously shown to be required for other aspects of egg-chamber morphogenesis, like cyst separation and follicle cell migration (Karess, 1991; Edwards, 1996). Follicle cells lacking RMLC activity are extremely flat and appear stretched. In many egg chambers with sqh clones, gaps were found in the follicular epithelium, suggesting that stretching of the follicle cells eventually disrupts the monolayer. The flat sqh mutant cells retain polarity, as revealed by the localization of Discs large (Dlg), a marker for the region where the septate junctions are formed, and the localization of the apical marker aPKC. The change in the shape of the follicle cells is accompanied by a change in the morphology of the egg chamber. Although those regions of the egg chamber covered by wild-type follicle cells retain a normal shape, the germline cyst bulges out in regions covered by sqh mutant cells. In summary, the morphological defects in the sqh clones are very similar to the defects in the rok mutant clones, although the sqh phenotype is much stronger. The stronger morphological defects in sqh mutants are consistent with the finding that RMLC activity is only reduced in rok, whereas it is abolished in sqh mutant cells (Wang, 2007).

sqh function is also required for cytokinesis (Jordan, 1997), and, consistent with this, epithelia with sqh mutant clones show a reduced number of phospho-Histone H3-positive cells, huge nuclei, and abnormally large cells. To examine whether these defects contribute to the morphological defects, epithelia with clones mutant for diaphanous (dia), another gene required for cytokinesis, were examined. Using the weak allele dia⁵, follicle cell clones were identifed showing cytokinesis defects in the presence of a normal actin cortex. During early oogenesis, these clones retain a rectangular shape, do not flatten, and the underlying cyst bulges out only very mildly. Late clones show no outward bulging over the growing oocyte and maintain a normal cell shape, with the exception that the cells are bigger because of the absence of cytokinesis. Thus, cytokinesis defects alone do not affect the rectangular shape of the follicle cells, and they affect the shape of the egg chamber only mildly and only during early oogenesis. Importantly, the morphological defects are fully penetrant in sqh mutant follicle cell clones. It is therefore concluded that the morphological defects in egg chambers with sqh clones are the result of the loss of apical myosin activity (Wang, 2007).

The epithelial deformations in sqh clones suggest a stress that is acting on the epithelium. The outward bulging of the cyst further suggests that the origin of this stress is the volume increase of the growing cyst. Wild-type cells might resist this stress because of the myosin activity at the apical cortex that is facing the cyst, whereas sqh mutant cells collapse. To test this hypothesis, cyst growth was blocked by using a chromosome carrying an ovo^D1 mutation. ovo^D1 is a dominant female-sterile mutation that is normally applied in germline mosaics. Importantly, the ovo^D1 phenotype is restricted to the germline and does not affect the somatic epithelium. The ovo^D1 harbouring chromosome that was used in this experiment leads to a growth arrest after stage 4 resulting in small stage 6 egg chambers, which later degenerate (Wang, 2007).

sqh follicle cell clones were genrated in parallel in wild-type and in ovo^D1 mutant backgrounds and cyst and epithelial shape was analyzed. Strikingly, sqh mutant follicle cells maintain their rectangular shape when cyst growth is blocked, whereas sqh cells are deformed when the cyst grows. Moreover, the cyst bulges out underneath the sqh clones only in the wild-type background, but not in the ovo^D1 mutant cysts. Thus, myosin activity is required for epithelial and egg-chamber shape only if the cyst is growing. It is therefore concluded that epithelial myosin activity counteracts the force from the growing cyst (Wang, 2007).

How could myosin activity counteract stress from the growing cyst mechanistically? In Dictyostelium, it has been shown that the cell membrane is able to resist deformations induced by a cell poker, revealing stiffness of the cortex. In myosin mutants, the cortical stiffness is greatly reduced, indicating that stiffness is generated by myosin-mediated contractions within the actin cortex. Consistent with this, in vitro studies demonstrated that myosin activity increases the stiffness of crosslinked actin filaments by a factor of 100. Stiffness is generated by diminishing thermal fluctuations within a crosslinked actin network. Myosin is able to suppress these fluctuations by mediating contractions of actin filaments between crosslink points. It is proposed that stiffness is a crucial feature of the apical epithelial cortex in response to the stress emanating from the growing cyst, and that myosin regulates the stiffness by generating tension between actin crosslink points (Wang, 2007).

The pattern of myosin activity reflects the organization of the actin cytoskeleton. The stress fiber-like pattern at the basal cortex reveals activity in the parallel actin arrays, and this activity leads to egg-chamber elongation. In contrast to this polarized pattern of myosin, the apical pattern shows no uniform direction, indicating that actin filaments of all orientations contract. This suggests that the actin filaments at the apical cortex are crosslinked like a net. Thermal fluctuations are higher in actin networks compared to bundled actin filaments. A netlike organization of the actin filaments is therefore consistent with the model, in which myosin-mediated contractions increase cortical stiffness by suppressing thermal fluctuations within the net (Wang, 2007).

The follicular epithelium responds to the cyst growth by increasing the epithelial surface by cell proliferation. The signal that induces mitosis is unknown. These data raise the possibility that the actomyosin cytoskeleton is involved in the coordination of cyst growth and epithelial proliferation. It is likely that the apical cortex, which is stiffened by myosin, perceives the volume increase of the growing cyst as a further tension increase in the crosslinked actin filaments. It is speculated that tension increase above a certain threshold triggers mitosis in the epithelium. The resulting cell divisions lead to an enlarged epithelial surface and thereby to a tension decrease at the apical cortex. The coupling of tension increase and cell proliferation adapts the growth of the epithelium to the volume increase of the cyst and prevents epithelial rupture. The role of tension in regulating cell growth was proposed in the past and has been demonstrated recently in cell culture experiments (Wang, 2007).

If cyst growth and epithelial proliferation are coupled, follicle cell division should be reduced when the cyst volume does not increase. Notably, a dramatic reduction was found in cell division in ovo^D1 mutant ovarioles, in which growth is blocked. Consistent with this, it has been reported that block of cyst growth induced by germline clones mutant for the Drosophila Insulin receptor and dMyc does not result in excess follicle cells. These results show that cyst growth and epithelial growth are coupled. However, they allow no conclusion about the coupling mechanism (Wang, 2007).

The restriction of myosin activity to the apical cortex of the epithelium is mediated by at least three different mechanisms. First, myosin phosphorylation at the apical cortex is achieved by apical localization of Rok. Rok is also regulated by the small GTPase Rho1. rho1 mutant follicle cell clones show reduced apical myosin phosphorylation and cell flattening, suggesting that Rho1 binding enables Rok to phosphorylate myosin. In contrast to rok mutant clones, rho1 mutant cells have large nuclei and an increased cell size, indicating that Rho1 is also required for cytokinesis. The second mechanism that restricts myosin activity to the apical cortex is the anchoring of active myosin by the Baz/aPKC/Par-6 complex. The third mechanism is the inhibition of myosin at the lateral and basal cortex via PP1β9C-mediated dephosphorylation. In the future, it will be important to find additional components regulating apical myosin activity, and to find out whether myosin activity is also in other epithelia restricted to certain domains (Wang, 2007).

Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1

Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. This study shows that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, evidence is provided that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, it was demonstrated that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. These results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC (Widmann, 2009).

Cell extrusion was observed when Dpp signaling was locally reduced in tkv^a12 bsk^- clones, but not when it was reduced throughout the dorsal compartment by expression of Dad. This indicates that cell extrusion is a consequence of the sharp boundary of Dpp signaling at the clone border. One of the first morphological consequences of the loss of Dpp signaling in tkv^a12 bsk^- clones was the apical constriction of mutant cells and surrounding control cells. Apical constriction correlated with increased staining intensities of F-actin and P-MRLC, a marker for active non-muscle myosin II, at the apicolateral side of tkv^a12 bsk^- and neighboring wild-type cells. The formation of a similar actin-myosin ring has been previously demonstrated during the extrusion of apoptotic cells, and it has been proposed that contraction of this ring squeezes cells out of the epithelium. It is currently unclear whether these increased staining intensities reflect an increase in the total amount of F-actin and P-MRLC in tkv^a12 bsk^- mutant clones, or whether they are instead merely a consequence of the apical constriction of cells. Nevertheless, these findings are consistent with the view that contraction of an actin-myosin ring might contribute to the extrusion of tkv^a12 bsk^- cells. Apical cell constriction was paralleled with cell shortening along the apical-basal axis. Based on the observation that reduction in Dpp signaling throughout the wing disc pouch resulted in apical-basal cell shortening, but not in apical cell constriction, it is speculated that cell shortening, and thus the formation of an inappropriate cell shape, might be an initial event leading to the extrusion of tkv^a12 bsk^- cells. If so, cell extrusion might not represent a specific response to eliminate slow-growing or apoptotic cells, but rather represents a general response to inappropriate cell function or morphology. In the wild type, cell extrusion might be instrumental in maximizing tissue fitness by removing cells with inappropriate function or morphology (Widmann, 2009).

The basal membrane of tkv^a12 bsk^- cells and neighboring control cells, identified by PSβ-integrin labeling, became apposed. Since this led to a reduction in the lateral contact between mutant and neighboring control cells, this apposition might help to dislodge tkv^a12 bsk^- cells from the remaining epithelium, and thereby, might aid the extrusion process. It is also noted that extruded tkv^a12 bsk^- cells displayed features reminiscent of epithelial-to-mesenchymal transition (EMT). In particular, a strong decrease in E-cadherin, a hallmark of EMT and actin-rich processes were observed in extruded tkv^a12 bsk^- cells. Interestingly, a role for Dpp/BMPs in preventing EMT has also been identified in vertebrates. Mouse BMP7, which is related to Dpp, for example, is required for counteracting EMT associated with renal fibrosis. Decreased E-cadherin levels have also recently been reported following the extrusion of cells deficient for C-terminal Src kinase from Drosophila epithelia, indicating that this might be a more common consequence of cell extrusion (Widmann, 2009).

Reduced apical-basal cell length was observed when Dpp signaling was severely reduced, either in clones or throughout the wing disc pouch; however, apical cell constriction, fold formation and cell extrusion were only detected by clonal reduction of Dpp signaling. Instead, cells were apically widened and did not extrude when Dpp signaling was reduced throughout the dorsal compartment. These experiments therefore allowed the effects of sharp boundaries of Dpp signaling at clone borders to be separated from cell-autonomous functions of Dpp signaling. They demonstrate that the cell-autonomous function of Dpp signaling is not to prevent apical cell constriction, folding and cell extrusion, but rather to maintain proper columnar cell shape. Moreover, three further observations suggest that Dpp signaling has an instructive role that drives cell elongation. (1) In the wild type, an increase in Dpp signal transduction activity correlated with apical-basal cell elongation in second instar larval discs. (2) In wing discs of late third instar larvae, Dpp signal transduction activity correlated with apical-basal cell length along the anteroposterior axis. (3) Activation of Dpp signaling, by expressing the constitutively active Dpp receptor Tkv^Q-D, resulted in precocious cell elongation and apical cell narrowing during early larval development. These findings indicate that Dpp signaling is an important trigger for the cuboidal-to-columnar transition in cell shape that occurs during mid-larval development (Widmann, 2009).

How does Dpp signaling promote the apical-basal elongation of wing disc cells? Compartmentalization of Rho1 activity has been recognized as being important for shaping cells and tissues. In the wild-type wing disc, Rho1 protein is enriched and the activity of the Rho1 sensor is increased at the apicolateral side, and more moderately at the basal side, of elongated cells. By contrast, Rho1 activity is more uniform in cuboidal cells, and overexpression of RhoGEF2, which leads to uniform distribution of this protein and presumably also uniform Rho1 activity, resulted in a cuboidal cell shape. Rho1, when present at the apicolateral side of cells, might therefore have a function in stabilizing or promoting cell elongation. Since the apicolateral increase in Rho1 sensor activity correlated with an increase of P-MRLC at a similar location, this function of Rho1 might be mediated by myosin II. The observation that a decrease in the bulk of Rho1 activity, either through expression of Rho1^N19 or rho1^dsRNA, resulted in cell elongation rather than in cell shortening, further suggests that the compartmentalization of Rho1 activity is important for shaping wing disc cells. Future studies will need to examine the morphogenetic consequences of locally modulating the activity of Rho1 (Widmann, 2009).

The results provide strong evidence for a functional link between Dpp signaling and Rho1-myosin II. Shortening of cells with compromised Dpp signaling could be rescued by a decrease in Rho1 or MRLC activity. In particular, the expression of Mbs^N300, an activated form of a subunit of myosin light chain phosphatase, which in wild-type wing discs did not significantly alter cell length, did rescue the shortening of Dpp-compromised cells. This indicates a specific interaction between Dpp signaling and Mbs-myosin II. The data further suggest that Dpp signaling controls apical-basal cell length by compartmentalizing Rho1 protein abundance and/or activity. (1) In late third instar wing discs, apicolateral enrichment of Rho1 protein and Rho1 sensor activity directly correlated with the local level of Dpp signal transduction activity. (2) Rho1 protein abundance and Rho1 sensor activity were decreased at the apicolateral side of cells when Dpp signal transduction was compromised by expression of Dad. (3) Rho1 protein and Rho1 sensor activity were increased at the apicolateral side and also at the basal side of cells when Dpp signal transduction was activated during early development by expression of Tkv^Q-D (Widmann, 2009).

Local activation of Rho1 and myosin II can lead to contraction of actin-myosin filaments, which can increase the cortical tension that is important for the shaping of cells during various developmental processes. By compartmentalizing Rho1 activity, Dpp signaling might promote both apical-basal cell elongation and apical cell narrowing. An increase in tension at the apicolateral cell cortex might promote apical cell narrowing. At the same time, a relative decrease in cortical tension laterally, compared with that on the apicolateral side, might allow cells to elongate through intrinsic cytoskeletal forces and/or extrinsic forces imposed by the growth of the epithelium. In this model, Dpp signaling directs the cuboidal-to-columnar shape transition of wing disc cells by increasing the Rho1 and myosin II activities at the apicolateral side of cells. The local increase of Rho1 and myosin II activities might then shift the balance of tension between the apicolateral cell cortex and the lateral cell cortex towards an increased tension at the apicolateral cell cortex (Widmann, 2009).

The results identify a Dpp-Brk-Rho1-myosin II pathway controlling cell shape in the wing disc epithelium. The elimination of Brk function in mad^- mutant cells allowed these cells to maintain a normal columnar cell shape, indicating that Dpp controls epithelial morphogenesis through repression of Brk. Since Brk acts as a transcriptional repressor, the link between Brk and Rho1 is most probably established through an unknown Brk-repressible gene. The identification of genes transcriptionally repressed by Brk will thus be important for determination of how Dpp signaling controls Rho1 and thereby, epithelial cell shape. The finding that Dpp signaling has a cell-autonomous morphogenetic function indicates that Dpp signaling provides a connection between cell-fate specification, cell growth and the control of morphogenesis. It, thereby, might help to facilitate the coordination of these processes during wing disc development (Widmann, 2009).

Given the evolutionary conserved functions of Rho and myosin II, it is anticipated that the mechanisms regulating columnar cell shape, which are describe in this study for the wing disc, will also operate in a wide range of other epithelia. Moreover, the role of TGFβ/Dpp signaling in patterned morphogenesis appears to be conserved in vertebrates, raising the possibility that Rho and myosin II are common mediators of TGFβ/Dpp signaling (Widmann, 2009).

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).

Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis

Apical constriction is a cell shape change that promotes epithelial bending. Activation of nonmuscle myosin II (Myo-II) by kinases such as Rho-associated kinase (Rok) is important to generate contractile force during apical constriction. Cycles of Myo-II assembly and disassembly, or pulses, are associated with apical constriction during Drosophila melanogaster gastrulation. It is not understood whether Myo-II phosphoregulation organizes contractile pulses or whether pulses are important for tissue morphogenesis. This study shows that Myo-II pulses are associated with pulses of apical Rok. Mutants that mimic Myo-II light chain phosphorylation or depletion of myosin phosphatase inhibit Myo-II contractile pulses, disrupting both actomyosin coalescence into apical foci and cycles of Myo-II assembly/disassembly. Thus, coupling dynamic Myo-II phosphorylation to upstream signals organizes contractile Myo-II pulses in both space and time. Mutants that mimic Myo-II phosphorylation undergo continuous, rather than incremental, apical constriction. These mutants fail to maintain intercellular actomyosin network connections during tissue invagination, suggesting that Myo-II pulses are required for tissue integrity during morphogenesis (Vasquez, 2014).

Recent studies demonstrated that pulsatile Myo-II contractions drive diverse morphogenetic processes, including Caenorhabditis elegans embryo polarization, Drosophila gastrulation, dorsal closure, germband extension, oocyte elongation, and Xenopus laevis convergent extension. Although Rok, and likely Myo-II activation via Rok phosphorylation, is required for contraction, it was not clear whether Myo-II activation simply regulates cortical Myo-II levels or whether coupling between Myo-II activity and its regulators organizes contractile pulses in space and time. Furthermore, why cells undergo pulsatile, rather than continuous, contraction to drive tissue morphogenesis was unknown. This study was able to answer these questions by visualizing the consequences of uncoupling Myo-II activation from upstream signaling pathways on cell and tissue dynamics (Vasquez, 2014).

This study identified dynamic Myo-II phosphorylation as a key mechanism that regulates contractile pulses. Myo-II pulses are associated with dynamic medioapical Rok foci and myosin phosphatase. In addition, the phosphomimetic sqh-AE and sqh-EE mutants, which exhibited constitutive cytoplasmic Myo-II assembly in vivo, exhibited defects in two properties of contractile pulses. First, phosphomimetic mutants did not initially condense apical Myo-II or F-actin into medioapical foci, resulting in Myo-II accumulation across the apical domain and thus a defect in Myo-II radial cell polarity. Second, phosphomimetic mutants continuously accumulated Myo-II in the apical cortex, lacking clear cycles of Myo-II remodeling that are observed in wild-type embryos. Although the phosphomimetic alleles are predicted to partially activate the Myo-II motor’s ATPase activity compared with normal phosphorylation, the similarity of the Myosin binding subunit (MBS) (see Lee, 2004) knockdown phenotype suggests that the changes in Myo-II organization and dynamics in phosphomimetic mutants reflect defects in the control over Myo-II dynamics rather than a reduction in motor activity. The consequence of persistent Myo-II assembly across the apical surface in phosphomimetic mutants and MBS knockdown is a more continuous, rather than incremental, apical constriction, demonstrating that pulsatile cell shape change results from temporal and spatial regulation of Myo-II activity via a balance between kinase (Rok) and phosphatase (myosin phosphatase) activity (Vasquez, 2014).

Mutants that decrease Myo-II phosphorylation affected contractile pulses in a manner that was distinct from the phosphomimetic alleles. Both the sqh-AA and the sqh-TA mutants exhibited Myo-II assembly into apical foci, potentially mediated by phosphorylation of low levels of endogenous Sqh or phosphorylation of threonine-20, respectively. For the sqh-TA mutant, Myo-II assembly was correlated with constriction, suggesting that Myo-II motor activity is not rate limiting to initiate a contractile pulse. However, Myo-II foci in sqh-TA and sqh-AA mutants were not efficiently remodeled after assembly and coalescence. The persistence of cortical Myo-II foci in sqh-AA and sqh-TA mutants was surprising given that rok mutants and injection of Rok inhibitor reduce cortical localization of Myo-II. One explanation is that high levels of Myo-II activity induce actomyosin turnover and thus could be required to remodel the actomyosin network after contraction. Alternatively, apical recruitment of myosin phosphatase or proteins that negatively regulate Rok could depend on Myo-II phosphorylation or actomyosin contraction. Although future work is needed to address the role of Myo-II motor activity in contractile pulses, the phenotypes of alleles that constitutively reduce phosphorylation further suggest that cycling between high and low phosphorylation states is required for proper Myo-II pulses (Vasquez, 2014).

A model is proposed for contractile pulses in the ventral furrow where, in combination with unknown cortical cues that apically localize Myo-II, local pulses of apical Rok activity within the medioapical cortex polarize Myo-II assembly and coalescence. Rok foci could polarize actomyosin condensation by generating an intracellular gradient of minifilament assembly and tension that results in inward centripetal actomyosin network flow. In addition, local Myo-II activation by Rok foci combined with broader myosin phosphatase activity throughout the apical cytoplasm could generate a gradient of Myo-II turnover that will concentrate Myo-II into medioapical foci. MBS is required to restrict phosphorylated Myo-II to specific cell–cell interfaces during dorsal closure, demonstrating that the balance between Myo-II kinases and phosphatase can generate spatial patterns of Myo-II activation in epithelial cells. Myo-II remodeling after coalescence could result from local decreases in Rok activity and enrichment of apical myosin phosphatase with Myo-II structures. Thus, coupling Myo-II activation to dynamic signals that regulate Myo-II phosphorylation organizes contractile pulses in space and time to drive incremental apical constriction (Vasquez, 2014).

Polarized actomyosin contraction, pulses, and flows generate force and organize the actin cortex in a variety of cellular and developmental. In contrast to the ratchet-like constriction of ventral furrow cells, some cell types undergo extended periods of actomyosin pulsing and area fluctuations without net reduction in area. Furthermore, directional rearrangement of cell contacts, such as during convergent extension in the Drosophila germband, can be achieved through planar polarized accumulation of junctional Rok and Myo-II in conjunction with planar polarized medioapical actomyosin flows. Modulating the spatial and temporal regulation of Myo-II phosphorylation and dephosphorylation provides a possible mechanism to tune contractile dynamics and organization to generate diverse cell shape changes. Consistent with this organizational role, phosphomimetic RLC mutants also disrupt the planar polarized localization of junctional Myo-II in the Drosophila germband. Thus, it will be important to define the principles that control Myo-II activity and dynamics and how tuning Myo-II dynamics impacts force generation and tissue movement (Vasquez, 2014).

Myo-II phosphomutants resulted in a more continuous apical Myo-II assembly and apical constriction, enabling investigation of the role of pulsation during tissue morphogenesis. Continuous Myo-II assembly and contraction in the sqh-AE mutant resulted in a slower mean rate of apical constriction and thus delayed tissue invagination. This delay suggested that pulsing might be important for the efficiency of apical constriction. However, phosphomimetic mutants might not fully recapitulate the ATPase activity of phosphorylated Myo-II. The sqh-TA mutant, which also perturbs Myo-II remodeling, constricted ventral furrow cells at a rate that is only slightly slower than wild type. In addition, MBS knockdown, which disrupted Myo-II pulses, exhibited a more variable constriction rate, but with a mean rate comparable to control embryos. The current finding is distinct from studies in other cell types where loss of MBS results in excessive phosphorylated Myo-II accumulation and cell invagination. Thus, MBS can regulate Myo-II organization and dynamics without causing a significant increase in apical Myo-II levels. It is concluded that Myo-II pulses are not absolutely required for individual cell apical constriction (Vasquez, 2014).

Although phosphomimetic mutant cells constrict and undergo tissue invagination, the coordination of invagination and the stability of the supracellular actomyosin meshwork were perturbed. Continuous apical constriction was associated with abnormal separation events between Myo-II structures in adjacent cells, resulting in gaps or holes in the supracellular Myo-II meshwork. Thus, continuous Myo-II assembly and a lack of Myo-II dynamics during apical constriction appear to sensitize the tissue to loss of intercellular cytoskeletal integrity during morphogenesis. Although loss of cytoskeletal continuity in phosphomimetic mutants does not block tissue invagination, it is speculated that dynamic Myo-II pulses are important to make tissue invagination robust to changes in tensile stress. One possible function of Myo-II pulses is to attenuate tissue tension or stiffness during morphogenetic movements. Because pulsed Myo-II contractions are staggered between neighboring cells, pulsation could serve as a mechanism to coordinate contractile force generation across the tissue such that intercellular connections are buffered from high levels of tension. Indeed, reducing adherens junction proteins sensitizes the intercellular connections between cytoskeletal networks to tensile forces generated in ventral furrow cells. Alternatively, remodeling of actomyosin networks that occurs during pulses could be required to adapt the cytoskeletal organization such that forces transmitted between cells accommodate the changing pattern of tissue-scale forces during the course of morphogenesis. In either case, the current data suggest that Myo-II pulsing and remodeling are important for collective cell behavior by ensuring proper force transmission between cells in a tissue undergoing morphogenesis (Vasquez, 2014).

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC; Spaghetti squash) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. This study shows that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis (Kasza, 2014).

Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy

Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. This study shows that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, it was found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9; see Drosophila Atg9) trafficking when cells were deprived of nutrients. These findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein (Tang, 2011).

Myosin II is a conventional two-headed myosin composed of two heavy chains, two essential light chains, and two regulatory light chains. Myosin II activation is regulated by the phosphorylation of its regulatory light chain via MLCKs. Rho GTPase and Rho kinase have been implicated in the regulation of myosin activation. However, this study found that neither RNA-mediated knockdown of dRok nor mutations in Rho1 or dRhoGEF2 could suppress the Atg1-induced wing defects. Instead, it was found that depletion of Sqa rescued Atg1-induced wing defects. This epistasis analysis showed that Sqa functioned downstream of Atg1. Moreover, it was found that Sqa but not Atg1 could directly phosphorylate Spaghetti squash (Sqh) in the in vitro kinase assay, suggesting that Atg1 stimulates myosin activity via Sqa. Importantly, Atg1 phosphorylates and interacts with Sqa, indicating that Atg1-Sqa functions in a kinase cascade to regulate myosin II activation. Moreover, Atg1 has been found to have a critical role in the regulation of autophagy induction under stress conditions in yeast, Drosophila, and mammalian cells. These results provide the first evidence that nutrient starvation stimulates myosin II activation in an Atg1-Sqa-dependent manner. Most significantly, a dramatic decrease was found in the size and number of autophagosomes in cells expressing Sqa-T279A, Sqa-RNAi, and SqhA20A21 on nutrient deprivation, indicating that Atg1-Sqa-mediated actomyosin activation has a critical role in autophagy (Tang, 2011).

The kinase domain of Sqa is also highly homologous to that of the mammalian DAPK family proteins. Recent studies have indicated that DAPK1 regulates autophagy through its association with MAP1B and Beclin1, or by modulating the Tor signalling pathway. As DAPK family proteins also regulate myosin II phosphorylation, one might speculate that Sqa may be the Drosophila counterpart of DAPK protein. Indeed, although overexpression of Sqa does not induce cell death, Sqa shares several characteristics with DAPK3/ZIPK. First, unlike MLCK family proteins, both Sqa and ZIPK contain an amino-terminal kinase domain that has 42% sequence identity and 61% similarity. Moreover, like ZIPK, recent sequence analysis from FlyBase identified a Sqa isoform that also contains a leucine-zipper domain. Second, as phosphorylation of Thr-265 in ZIPK is essential for its kinase activity, this study found that Atg1 phosphorylates Sqa at the corresponding Thr-279, and is critical for Sqa activity. Third, just as Sqa specifically associates with kinase-inactive Atg1, the results indicate a similar interaction between ZIPK and Ulk1. Importantly, depletion of Sqa and ZIPK resulted in autophagic defects in response to nutrient deprivation. These findings together suggest that ZIPK may act as a mammalian homolog of Sqa during starvation-induced autophagy. Further investigation is needed to determine whether the mammalian Atg1 (Ulk1) directly phosphorylates ZIPK at Thr-265, and the role of this regulation in autophagy (Tang, 2011).

In autophagy, the source of the autophagosomal membrane and dynamics of autophagosome formation are fundamental questions. Studies in yeast and mammalian cells have identified several intracellular compartments as potential sources for the PAS (also termed isolation membrane/phagophore). Formation of PI(3)P-enriched ER subdomains (omegasomes) has been reported during nutrient starvation and autophagy induction, and a direct connection has been observed between ER and the phagophore using the 3D electron tomography. In addition, recent studies in yeast cells have suggested Atg9 and the Golgi complex have a role in the formation of autophagosomes. It has been proposed that the integral membrane protein Atg9 may respond to the induction signal in promoting lipid transport to the forming autophagosomes. The mAtg9 has been found to localize on the TGN and the endosomes in nutrient-rich conditions and translocate to LC3-positive autophagosomes on nutrient deprivation. Although several proteins, including Ulk1, mAtg13, and p38IP, have been found to regulate starvation-induced mAtg9 trafficking, the molecular motor that controls the movement of mAtg9 between different subcellular compartments remains unknown (Tang, 2011).

The finding that myosin II redistributes from peripheral to the perinuclear region of cells on starvation suggests that myosin II has a role in membrane trafficking. In fact, it has been reported that myosin II is required for the trafficking of major histocompatibility complex (MHC) class II molecules and antigen presentation in B lymphocytes. Myosin II has also been found to be involved in the protein transport between ER and Golgi. This study has shown that there here is a molecule link between mAtg9 and the actomyosin network, indicating that myosin II may function as a motor protein for mAtg9 trafficking during early autophagosome formation. In conclusion, this work has unravelled a regulatory mechanism between Atg1 activity and the Atg9-mediated formation of autophagosomes. Further studies are needed to determine the involvement of this signalling process in other stress-induced or developmentally regulated autophagy (Tang, 2011).

The regulatory light chain of nonmuscle myosin is encoded for by spaghetti squash, a gene required for cytokinesis in Drosophila

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

Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos

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

Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis

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

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

Rho-associated kinase indirectly affects the activity of myosin II in the development of planar cell polarity

Drosophila Rho-associated kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh¹ multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh¹ mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh¹ phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh¹ background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqh^AX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

MRLC phosphorylation in response to Rok activation would be predicted to modify the conformation and elevate the catalytic activity of its associated heavy chain, Zipper (Zip). Does Zip also participate in regulating actin distribution/wing hair number in response to Fz/Dsh? Loss of one copy of the zip gene enhances the dsh¹ phenotype by 4.5-fold, consistent with the genetic interaction data between fz/dsh and sqh. These results suggest that myosin II functions positively downstream of Fz/Dsh in regulating actin prehair development (Winter, 2001).

The localization of Zip protein in wing cells further supports its role downstream of Fz/Dsh. At the apical surface of the pupal wing cell, Zip is asymmetrically localized to the distal portion of the cell, where prehair growth initiates. This distal localization appears to coincide, temporally, with prehair initiation. To test whether Zip localization could be modified by Fz/Dsh signaling, Zip distribution at the apical surface was examined in dsh¹ mutants. Instead of being concentrated in the distal region of the cell, Zip is concentrated in the center of the cell, where prehairs form in dsh¹ mutants (Winter, 2001).

Does reduction in myosin II/Zip activity also result in the multihair phenotype? Use was made of the hypomorphic zip⁰²⁹⁵⁷, since zip and sqh null mutations appear to be cell lethal in the wing. As is the case with rok, some homozygous zip⁰²⁹⁵⁷ wing cells possess multiple F-actin prehairs (Winter, 2001).

Tests were performed to see if the gene crinkled (ck) is involved in the Fz/Dsh signaling pathway regulating wing hair number because (1) ck mutant cells in the wing lead to multiple hair and split hair phenotypes, and (2) ck encodes the Drosophila myosin VIIA protein. Mutations in mouse myosinVIIA lead to stereocilia disorganization and the formation of multiple bundles of stereocilia (Winter, 2001 and references therein).

Reduction of ck activity potently suppresses the dsh¹ multiple hair phenotype. This result contrasts with the result that zip¹ enhances the dsh¹ multiple hair phenotype, and suggests that the two myosin heavy chains have opposing effects in regulating prehair assembly (Winter, 2001).

Both myosin heavy chain genes were tested for their ability to interact with the hs-fz induced multiple hair phenotype, and again it was found that they have opposing effects. Surprisingly, loss of one copy of zip slightly but significantly enhances the late hs-fz multiple hair phenotype, while loss of one copy of ck markedly suppresses this phenotype. These results are the reverse of what one would expect based on their interactions with dsh¹, and suggests the possibility that there is a signal from Fz to Ck that is independent of Dsh, or that the multiple hair phenotypes resulting from hypo- or hyper-activity of the Fz/Dsh pathway arise via distinct biochemical mechanisms (Winter, 2001).

To further assess the nature of the relationship between the two myosins, the effect of raising or lowering the activity of MRLC on the ck phenotype was tested. The multiple hair phenotype in animals homozygous for a weak ck mutation is enhanced by one copy of the sqhE20E21 transgene (and hence, a probable increase in myosin II activity), but not by a sqhA20A21 transgene. Taken together, these experiments suggest that a balance between the activities of myosin II and myosin VIIA is important in regulating wing hair number (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok² clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh¹, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh¹ mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh¹. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

The data presented in this study suggest that the Rok/myosin II pathway is involved in regulating the number -- but not orientation -- of the wing hair. What then are the components that regulate wing hair orientation? One possibility is that a bifurcation of the pathway occurs at the level of RhoA, with a separate effector pathway regulating wing hair orientation. In the eye, the JNK pathway has been implicated in functioning downstream of RhoA in regulating ommatidial polarity. However, the function of the JNK pathway in the wing has not been described, and a signaling pathway that regulates transcription is unlikely to encode the requisite spatial information necessary for selection of the site of prehair initiation. Therefore, it is likely that a separate signal from or upstream of RhoA may control the selection of the F-actin assembly site, and therefore the orientation of the wing hair (Winter, 2001 and references therein).

By what mechanism do myosins restrict F-actin bundle formation? In light of the finding that myosin II is concentrated at the site of prehair formation, it seems plausible that myosin II is actively involved in either the recruitment of F-actin to the prehair site, or that it directly participates in the assembly of actin bundles, or both. Studies of mammalian myosin II provide a precedent for a role in the formation of F-actin bundles. Phosphorylation of MRLC promotes a conformational change in myosin II from a folded to an extended state that readily forms multivalent bipolar filaments capable of binding multiple actin filaments. This is thought to result in F-actin bundling and stress fiber formation (Winter, 2001 and references therein).

It appears that in the developing wing, the level of MRLC phosphorylation/myosin II activity must be within an optimal range to establish the formation of a single hair. It is possible that the efficiency of F-actin bundle formation is regulated by MRLC phosphorylation in a manner similar to the control of stress fiber formation. If one further assumes that there are certain bundling substrates present only at limiting concentrations (e.g., F-actin itself), then one would predict that the assembly of one F-actin bundle would reduce the probability of forming a second bundle. When MRLC phosphorylation falls below some threshold level (e.g., in rok mutant cells), the efficiency of primary bundle formation is reduced, and thus the concentration of the limiting substrate remains at sufficient levels to support the assembly of secondary bundles/prehairs. Conversely, if MRLC is hyperphosphorylated (e.g., in Fz-overexpressing cells), the bundling efficiency may increase such that the threshold concentration for bundle formation would be reduced, thereby increasing the probability of assembling multiple bundles/prehairs. Future studies will be required to determine the detailed mechanisms involved (Winter, 2001).

In addition to nonmuscle myosin II, which resembles the myosin II from skeletal muscle, there exists a large class of unconventional myosins that have different properties and potential functions in nonmuscle cells. For instance, several different classes of unconventional myosins are expressed in inner ear epithelium with different subcellular localization. Mutations in three of the unconventional myosins, myosin VI, VIIA, and XV, cause hearing/balancing defects in mice, two of which when mutated in humans result in deafness. Of particular interest in the context of this study is myosin VIIA, mutations of which are responsible for mouse shaker-1 and human Usher's syndrome 1B. Loss-of-function ck (Drosophila Myosin VIIA) mutants exhibit a multiple hair and split wing hair phenotype. ck exhibits strong genetic interactions with components of the signal transduction pathway defined in this study, and has the opposite effects as that of myosin II. The seemingly antagonistic relationship between myosin II and myosinVIIA may suggest a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process they regulate. For example, myosin II and myosin VIIA may share some common, limiting component(s) required for their activity. Thus, by reducing the myosin VIIA dose, myosin II has a larger share of the common component(s) and thus its activity is upregulated (Winter, 2001 and references therein).

Regulating axon branch stability. the role of p190 RhoGAP in repressing a retraction signaling pathway

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

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

Drosophila myosin phosphatase and its role in dorsal closure

Drosophila Myosin-binding substrate (MBS), the homolog of mammalian MBS, was identified to study the roles of myosin phosphatase in morphogenesis. Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC: Spaghetti squash). Myosin phosphatase's regulatory myosin-binding subunit, MBS, is responsible for regulating the myosin phosphatase catalytic subunit in response to upstream signals and for determining myosin phosphatase's substrate specificity (Mizuno, 2002).

Embryos defective for both maternal and zygotic MBS demonstrate a failure in dorsal closure. In the mutant embryos, the defects are mainly confined to the leading edge cells which fail to fully elongate. Ectopic accumulation of phosphorylated MRLC is detected in the lateral region of the leading edge cells, suggesting that the role of MBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-associated kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of MBS with mutations in the components of the Rho signaling cascade also indicates that MBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis (Mizuno, 2002).

To examine whether defects in the dorsal closure in the embryos lacking MBS or overexpressing wild-type Rho kinase are due to an aberrant activation of nonmuscle myosin II, the genetic interactions with zipper (zip), which encodes the heavy chain of nonmuscle myosin II, were analyzed. About 25% of the progeny from crossing the females transheterozygous with DMBS^P2 and Df(3L)th117 to the males heterozygous for DMBS^E1 are embryonically lethal. It was expected that a reduction in the gene dosage of zip⁺ would suppress the defects in the MBS mutant or Rho-kinase-expressing embryos. When DMBS^P2/Df(3L)th117 females are mated with males heterozygous for both DMBS^E1 and zip^Ebr, half of the embryos defective for both maternal and zygotic MBS should be heterozygous for zip^Ebr. As expected, the embryonic lethality was reduced to nearly half that of the corresponding cross. Similarly, the heterozygosity for zip^Ebr considerably suppresses lethality due to ectopic wild-type Rho kinase expression. These results strongly suggest that either loss of MBS⁺ or overexpression of wild-type Rho-kinase causes hyperactivation of nonmuscle myosin II through increasing the levels of phosphorylation of MRLC (Mizuno, 2002).

zip^Ebr is a point mutation reported to be highly sensitive to genetic backgrounds. About 70% of the flies transheterozygous between zip^Ebr and zip⁰²⁹⁵⁷ have malformed wings with varying degrees of severity. Although zip^Ebr is recessive, a considerable percentage of the flies heterozygous for both zip^Ebr and the mutations in the components of the Rho signaling pathway such as DRho1 and DRhoGEF2 produced similar defects. A half reduction of Drok, which encodes Rho-kinase, also dominantly enhances zip^Ebr. This indicates the involvement of the Rho signaling pathway and its effector, Rho-kinase, in the myosin function of adult wing morphogenesis. When the flies are also heterozygous for DMBS^E1, wing malformation is significantly suppressed, suggesting that MBS functions antagonistically to the Rho signaling pathway (Mizuno, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Excessive myosin activity in Mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium

Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. The function of myosin light chain phosphatase, which down-regulates myosin activity, has been characterized in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. This phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and the phenotype requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. It is suggested that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration (Lee, 2004).

Nonmuscle myosin II consists of a hexamer of two myosin heavy chains (MHC), two myosin light chains (MLC), and two myosin regulatory light chains (MRLC). Phosphorylation of key serine and threonine residues on MRLC stimulates the ATPase activity of MHC and promotes its assembly into filaments, leading to stress fiber contraction. Mutations in the Drosophila orthologs of these myosin subunits have provided insight into the developmental functions of myosin II. Mutations in zipper (zip), which encodes MHC, cause defects in cytokinesis, closure of the dorsal embryonic epidermis over the amnioserosa, axon patterning, and myofibril formation. spaghetti squash (sqh), encoding MRLC, is required for cytokinesis, oogenesis, and imaginal disc eversion (Lee, 2004 and references therein).

Actin-binding proteins of the ezrin, radixin, and moesin (ERM) family are thought to link transmembrane proteins to the actin cytoskeleton. ERM proteins are activated by phosphorylation of a conserved threonine residue, which inhibits association between the N-terminal FERM domain and C-terminal actin-binding domain of the protein, freeing them to bind to other substrates. Moesin-like (Moe) is the only representative of this family in Drosophila. Moe mutants have abnormal oocyte polarity because defects in the anchorage of actin filaments to the oocyte cortex disrupt the localization of maternal determinants. In addition, Moe mutant cells in the wing disc undergo an epithelial-to-mesenchymal transition and adopt invasive migratory behavior (Lee, 2004 and references therein).

Interestingly, genetic and biochemical studies implicate the same kinase and phosphatase in the regulation of both nonmuscle myosin II and Moesin. Rho-associated kinase (ROCK/Rok) has been shown to phosphorylate MRLC in both mammalian and Drosophila systems. Myosin light chain kinase (MLCK) also can phosphorylate and activate MRLC; MLCK seems to act at the periphery of the cell, whereas ROCK is active in more central regions. Although ERM proteins are positively regulated by Rho GTPases, it is not clear whether they are directly phosphorylated by ROCK or by phosphoinositide-regulated kinases. However, in Drosophila wing disc development Moe seems to act antagonistically to Rho1 and rok (Lee, 2004 and references therein).

A major antagonist of the Rok/myosin signaling pathway is myosin light chain phosphatase (MLCP). This serine/ threonine protein phosphatase is a heterotrimer consisting of a catalytic subunit (PP1cdelta), a 20-kDa protein of unknown function, and the myosin binding subunit (MBS) that targets MLCP to its substrates, which include both MRLC and Moesin. Phosphorylation by Rok of a specific threonine within a conserved motif in MBS has been shown to inhibit MLCP activity; this suggests that Rok can positively activate MRLC and Moesin both by direct phosphorylation of these two substrates and also by inhibition of MBS. Like zip mutants, Drosophila Myosin binding subunit (Mbs) mutants fail to complete dorsal closure, suggesting that this process requires spatially regulated myosin activation. Mbs is also required for the growth of ring canals during oogenesis, and genetic interactions suggest that it opposes the functions in imaginal disc development of zip, Rho1, and rok. Likewise, Caenorhabditis elegans mel-11, which encodes MBS, and let-502, which encodes Rok, have opposite functions in embryonic elongation (Lee, 2004 and references therein).

Photoreceptor differentiation progresses across the Drosophila eye disc from posterior to anterior and is preceded by an epithelial indentation known as the morphogenetic furrow (MF). Cells in the MF undergo a transient contraction along the apical-basal axis and constrict their apical surfaces. After emerging from the MF, some of these cells assemble into ommatidial clusters, differentiate into photoreceptors, and extend axons through the optic stalk into the brain. Mbs mutations have been identified in a screen for genes required for normal photoreceptor differentiation. Findings on the role of Mbs in photoreceptor development suggest that photoreceptor neurons require Mbs to reduce myosin activity and thus prevent their cell bodies from migrating toward their axon terminals (Lee, 2004).

Mbs exerts its effects on eye development by regulating the phosphorylation state of the Sqh MRLC subunit of nonmuscle myosin II. The level of phosphorylated Sqh is greatly increased in Mbs mutant clones in both the eye and wing discs, and nonphosphorylatable or phosphomimetic forms of Sqh strongly modulate the severity of the Mbs phenotype. In addition, the effect of zip dosage on the Mbs phenotype indicates that p-Sqh acts through Zip to control photoreceptor localization. In vivo data show that in the eye disc Mbs is not required to dephosphorylate Moe. If dephosphorylation of Moe by Mbs occurs in vivo, it may be limited to specific tissues or developmental stages (Lee, 2004).

The identity of the kinase antagonized by Mbs in the eye is less clear. Although it has been reported that Rok can phosphorylate Sqh in vitro and that p-Sqh levels are reduced in rok mutant larvae, normal levels of p-Sqh were detected in rok² eye disc clones. In addition, overexpression of Rok-CAT in the eye disc has no visible effect on photoreceptor differentiation or localization, and does not seem to enhance the Mbs phenotype. Rok may have a more significant effect on Sqh phosphorylation in other tissues; the lethality caused by overexpression of constitutively active Mbs is partially suppressed by coexpression of the catalytic domain of Rok. Myosin seems to be a downstream effector of Rho and Rok in wing and leg development, and the MEL-11 myosin phosphatase antagonizes the LET-502 Rho kinase in C. elegans development, supporting a role for Rok in phosphorylating Sqh in some cell types (Lee, 2004 and references therein).

Another kinase that might phosphorylate Sqh in the eye disc is MLCK. It has been reported that MLCK phosphorylates MRLC at the periphery of fibroblast cells, whereas ROCK acts in the central domain of these cells. Drosophila Stretchin-MLCK is a very large compound gene that produces multiple alternatively spliced transcripts, and no mutations in this gene have been identified, preventing the analysis of its interactions with Mbs. Another possible kinase is p21-activated kinase (PAK), which has been shown to increase the level of phosphorylated MRLC in cultured cells and to phosphorylate MRLC in vitro. Interestingly, overexpression of a myristylated form of PAK in Drosophila photoreceptors causes their cell bodies to detach from the eye disc epithelium and enter the brain, strongly resembling the Mbs mutant phenotype. Pak mutant photoreceptors develop normally except for axon guidance defects, suggesting that Pak is not essential for myosin activation in these cells. However, a second Pak gene, mushroom bodies tiny, is required for late photoreceptor morphogenesis and adherens junction integrity, and a third Pak gene is present in the genome, raising the possibility that these enzymes have redundant functions and complicating any analysis of their interactions with Mbs (Lee, 2004 and references therein).

The excessive myosin activity present in Mbs mutant photoreceptors causes them to adopt a more basal location in the eye disc and sometimes to enter the optic stalk. Several possible mechanisms for this phenotype have been addressed. Myosin can affect the shape of cultured cells by promoting the assembly of stress fibers and focal adhesions, and a transient accumulation of p-Sqh accompanies the apical constriction and apical-basal contraction of cells in the morphogenetic furrow. It was therefore interesting to enquire whether loss of Mbs might induce these cell shape changes in ectopic regions of the eye disc, resulting in mutant cells that formed a constitutive furrow. However, visualization of the apical surface of mutant clones by p-Tyr or phalloidin staining did not reveal any ectopic apical constriction of cells surrounding the photoreceptor clusters, suggesting that myosin phosphorylation is not sufficient to induce the cell shape changes that occur in the morphogenetic furrow. In addition, the integrity of the epithelial surface surrounding the photoreceptor clusters indicates that loss of Mbs specifically affects the localization of photoreceptor cells (Lee, 2004).

Another possibility is that Mbs mutant cells might undergo an epithelial to mesenchymal transition and become migratory. This phenotype has been reported for wing disc cells mutant for Moe, which encodes a potential substrate of Mbs. However, Mbs mutant cells in the wing disc remain within the epithelium and show no change in their apical-basal localization, although p-Sqh is up-regulated to a similar extent in both the wing and eye discs. In addition, Mbs mutant photoreceptors seem to retain some aspects of their epithelial character; they continue to express the epithelial apical junction proteins Patj, Crumbs, and E-cadherin. These proteins are present apical to mislocalized nuclei, suggesting that the entire cell is affected rather than the position of the nucleus within the cell. In contrast, the nuclei of klarsicht or Glued mutant cells are basally located within the cell due to defective dynein function (Lee, 2004).

The model that is favored is that unregulated myosin generates a traction force that pulls photoreceptor cell bodies toward their axon terminals. This would explain why the Mbs phenotype is specific to photoreceptors rather than wing disc cells or undifferentiated cells in the eye disc. It also would explain why the movement of mutant cells is directed toward the optic stalk or, in a disco background, toward the axon terminals within the eye disc. This abnormal force also might be accompanied by changes in adhesion to other cells or the substrate. Loss of Mbs could reduce the adhesion of epithelial cells to their neighbors, preventing them from withstanding the normal forces involved in axon extension. However, Mbs clones do not show the smooth borders characteristic of changes in adhesive properties (Lee, 2004).

It is not known whether the force generated by excessive myosin activity is located at the growth cone or in the cell body, although the latter model is favored because the highest levels of p-Sqh are found in apical regions of both wild-type and Mbs mutant cells. In vertebrate growth cones, two isoforms of the heavy chain of nonmuscle myosin II seem to have different locations and functions. MHCIIB is more peripheral and is required for axon outgrowth, whereas MHCIIA is central and is required for cell adhesion. Drosophila has only a single zip gene, which may perform both functions. The importance of MHCIIB in generating the traction force that allows growth cone extension suggests that this force might be increased in the absence of MLCP activity. There is a precedent for the idea that axon outgrowth can exert a pulling force on the cell body, because it has been shown that chick motor neurons will migrate out of the spinal cord along their axons if their movement is not blocked by boundary cap cells (Lee, 2004 and references therein).

The other possibility is that the actomyosin contraction takes place within the cell body, detaching it from surrounding cells and pulling it toward the growth cone. This would resemble the normal function of myosin in retracting the rear of migrating cells. Cell detachment and shrinkage has been reported for fibroblasts treated with an inhibitor of MLCP activity. Myosin light chain phosphatase activity may be specifically required in neuronal cells to allow axon extension to occur without triggering a migratory response in the cell body (Lee, 2004).

Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis

Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).

Drak is required for actomyosin organization during Drosophila cellularization

The generation of force by actomyosin contraction is critical for a variety of cellular and developmental processes. Nonmuscle myosin II is the motor that drives actomyosin contraction, and its activity is largely regulated by phosphorylation of myosin regulatory light chain. During the formation of the Drosophila cellular blastoderm, actomyosin contraction drives constriction of microfilament rings, modified cytokinesis rings. This study found that Death-associated protein kinase related (Drak) is necessary for most of the phosphorylation of myosin regulatory light chain during cellularization. Drak was shown to be required for organization of myosin II within the microfilament rings. Proper actomyosin contraction of the microfilament rings during cellularization also requires Drak activity. Constitutive activation of myosin regulatory light chain bypasses the requirement for Drak, suggesting that actomyosin organization and contraction are mediated through Drak's regulation of myosin activity. Drak also is involved in the maintenance of furrow canal structure and lateral plasma membrane integrity during cellularization. Together, these observations suggest that Drak is the primary regulator of actomyosin dynamics during cellularization (Chougule, 2016).

Tight regulation of actomyosin is likely critical for many cellular processes, but how this is accomplished is as yet poorly understood. A key input to the regulation of myosin II is through phosphorylation of the Serine-19, or the Serine-19 and Threonine-18 residues of MRLC (Spaghetti squash). The variety of MRLC kinases might allow different specific aspects of actomyosin dynamics, such as localization, organization and contraction to be regulated independently. Such a system would provide greater flexibility and control than either a single kinase, or multiple kinases acting in concert, regulating all of these functions. drak was found to be required for the organization of myosin II into contractile rings, but is not required for localization of myosin to the cellularization front. Since the majority of Sqh phosphorylation during cellularization is dependent on drak activity, Drak either regulates most aspects of myosin II dynamics during cellularization, or Drak-regulated myosin II organization is required for further function of myosin II, such as contraction (Chougule, 2016).

Myosin II is somewhat less disorganized and Sqh phosphorylation is slightly increased during late cellularization in drak mutants, suggesting that phosphorylation of myosin II by other kinases occurs during late cellularization. Thus other kinases might act synergistically with Drak to regulate actomyosin organization during late cellularization. For example, Drak function has been shown to be partially redundant with Rok function during later development. An alternative possibility is that other kinases that do not normally function in myosin II organization in the microfilament rings might phosphorylate Sqh to some degree and lead to some organization of myosin II in the absence of Drak activity (Chougule, 2016).

Myosin II has been implicated in actin bundling and F-actin organization in some contexts. Since F-actin appears to be organized normally within drak mutant microfilament rings during early cellularization, it is concluded that myosin II does not play a role in initially organizing F-actin within the microfilament rings during cellularization. F-actin is somewhat disorganized during late cellularization in drak^del mutant embryos, but not as severely as myosin II, nor does the pattern of F-actin distribution fit the pattern of myosin II distribution in drak^del mutant embryos. These observations suggest that F-actin disorganization is an indirect consequence of Drak regulation of myosin II activity, and that F-actin disorganization might be due to actomyosin contraction defects or furrow canal structural defects (Chougule, 2016).

Anillin is required for the organization of actomyosin contractile rings during cellularization and cytokinesis. scraps (scra, anillin) mutant embryos have a myosin II organization defect somewhat similar to that of drak mutant embryos: myosin II is found in discrete bars in the actomyosin network. Despite this similarity, myosin II defects differ between scra and drak mutant embryos. Myosin II becomes more disorganized during late cellularization in scra mutant embryos. Myosin II becomes slightly better organized during late cellularization in drak mutant embryos. This organizational difference is likely caused by actomyosin contraction during microfilament ring constriction occurring in a highly disorganized cytoskeleton in scra mutant embryos, and occurring in a disorganized cytoskeleton that has slightly improved during constriction in drak mutant embryos. Anillin only interacts with myosin II when MRLC is phosphorylated. Together with these results, this suggests that Drak phosphorylation of Sqh might be necessary for Anillin-mediated myosin II organization within the contractile ring (Chougule, 2016).

Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 leads to the unfolding of inactive myosin II hexamers into an open conformation that allows assembly of bipolar myosin II filaments and their association with F-actin to form actomyosin filaments. This is likely how Drak organizes myosin II. Phosphorylation of MRLC on Serine-19 or Serine-19 and Threonine-18 also leads to the activation of the Mg²⁺-ATPase activity of myosin II that slides actin filaments past each other, causing actomyosin contraction. Three aspects of the drak mutant phenotype support the requirement for Drak in actomyosin contraction: wavy cellularization fronts caused by non-uniform furrow canal depths, abnormal microfilament ring shapes, and failure of microfilament rings to constrict during late cellularization. These are the same defects that suggest an actomyosin contraction defect in src64 mutant embryos. However, src64 mutant embryos do not show myosin II organization defects. Because effective actomyosin contraction likely requires properly organized actomyosin filaments within the contractile ring apparatus, it is unclear whether Drak directly regulates actomyosin contraction or whether Drak only enables actomyosin contraction through proper organization of myosin II within the microfilament rings. One possibility is that phosphorylation of Sqh by Drak both organizes actomyosin filaments into a contractile ring apparatus and directs actomyosin contraction. An alternative possibility is that Drak is directly responsible for organizing actomyosin filaments into a contractile ring by phosphorylating Sqh, but Drak is not directly involved in its contraction and different kinases that phosphorylate Sqh regulate actomyosin contraction. Thus, Drak could be an early regulator of myosin II activity during cellularization, such that further phosphorylation of Sqh and myosin II-driven contraction is dependent on Drak-mediated organization of myosin II. At some level the regulation of actomyosin contraction diverges from the regulation of actomyosin filament organization: Src64 is required for contraction, but has no role in myosin II organization (Chougule, 2016).

Rescue of myosin II organization, actomyosin contraction and F-actin distribution defects in drak mutant embryos by the mono-phosphorylated Sqh^E21 phosphomimetic suggests that Drak-mediated mono-phosphorylation of Sqh at Serine-21 is sufficient for regulation of actomyosin dynamics during cellularization. Although the diphosphorylated Sqh^E20E21 phosphomimetic also rescues myosin II organization and actomyosin contraction defects, it does not rescue F-actin distribution defects in drak mutant embryos. These results are consistent with Drak primarily phosphorylating Sqh at Serine-21, and are consistent with reports that DAPK family members phosphorylate MRLC mainly at Serine-19 (Chougule, 2016).

The normal teardrop shape of the furrow canals in early cellularization is likely caused by actomyosin contraction in the microfilament rings. In drak mutant embryos, unexpanded early cellularization furrow canals and failure of many late cellularization furrow canals to expand further suggest that Drak is required for proper furrow canal structure. Some of the furrow canal structural defects in drak mutant embryos are similar to those of nullo mutant embryos: collapsed furrow canals and blebbing. However, nullo mutant embryos, as well as RhoGEF2 or dia mutant embryos, have other, more severe furrow canal defects: missing or regressing furrow canals and compromised lateral membrane-furrow canal compartment boundaries. Furthermore, cytochalasin treatment causes similar defects, suggesting that reduced F-actin levels in the furrow canals are responsible for these defects. Thus Nullo, RhoGEF2 and Dia regulate F-actin and its levels in furrow canals. These observations suggest that Drak regulates myosin II and thereby regulates actomyosin organization and contraction, and that these are necessary for structural integrity and expansion of the furrow canals, but not for their continued existence (Chougule, 2016).

The furrow canals of drak mutant embryos during late cellularization show extensive blebbing into the lumens. This is consistent with a defect in furrow canal membrane or cortex integrity. Blebs can be formed by local rupture of the cortical cytoskeleton or detachment of the plasma membrane from the cortical actomyosin cytoskeleton. Actomyosin contraction has been implicated in bleb formation. Therefore, it is proposed that blebbing in furrow canals is caused by aberrant localized actomyosin contraction during late cellularization in the disorganized actomyosin cytoskeleton of drak mutant embryos. Contraction is presumably driven by phosphorylation of Sqh by kinases other than Drak. Since actomyosin contraction occurs in a disorganized actomyosin cytoskeleton, it does not lead to uniform constriction of the microfilament rings, but instead leads to localized contraction that produces cytoplasmic blebs. However, other causes for furrow canal defects are possible. Plasma membrane attachment sites might not form or function properly in the disorganized furrow canal cytoskeleton in drak mutant embryos. The disorganized cytoskeleton might inhibit vesicle trafficking. Vesicle trafficking itself might be defective: mammalian DAPKs have been shown to be involved in membrane trafficking and in phosphorylation of syntaxin A1. Vesiculated lateral plasma membrane in drak mutant embryos during late cellularization suggests that the plasma membrane breaks down. Intriguingly, scra mutant embryos have lines of vesicles where the closely apposed lateral plasma membranes would have been. However in scra mutant embryos, vesiculation is observed during early cellularization, but to a lesser extent than during late cellularization. drak mutant embryos do not show lateral plasma membrane vesiculation defects until late cellularization. drak mutant defects in both the furrow canal membrane and the lateral plasma membrane might reflect a general defect in membrane integrity. It will be interesting to investigate the potential role of myosin II organization in furrow canal structure and plasma membrane integrity (Chougule, 2016).

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. The role of Dynein and Dynein-interacting proteins were studied during Drosophila muscle development, and several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, were found to contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function (Folker, 2012).

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. This study showsthat 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 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 (Vereshchagina, 2004 and references therein).

The regulation of nonmuscle myosin is thought to be broadly similar to that of vertebrate smooth muscle myosin. 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. 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 and references therein).

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). 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. In Drosophila, the four genes encoding isoforms of PP1c are named by their chromosome location and subtype: PP1β9C, PP1α13C, PP1α87B, and PP1α96A. Of these, PP1α87B contributes 80% of the total PP1 activity, therefore the phenotypes of PP1α87B loss of function mutants 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. 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 and references therein).

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. 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. 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 (also known as DMYPT). 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. 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. 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. 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, 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 and in that they both lead to the accumulation of phospho-Sqh, though DMBS mutants do not show the accumulation of myosin aggregates. 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).

Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)^Pbl-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF^Pbl-dependent control themselves. Microscopy of Drosophila S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, a separate RhoGEFPbl-dependent pathway was uncovered that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF^Pbl-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. It is proposed that such interactions occur dynamically and transiently to promote furrow stability (Hickson, 2008).

Drosophila S2 cell lines expressing anillin-GFP were generatated. The anillin-GFP fusion rescued loss of endogenous anillin and its localization paralleled that of endogenous anillin. In interphase it was nuclear, at metaphase it was uniformly cortical, and in anaphase it accumulated at the equator while being lost from the poles. In some highly expressing cells, nuclear anillin-GFP formed filaments not ordinarily seen with anillin immunofluorescence, but these disassembled upon nuclear envelope breakdown and the overexpression had no appreciable effect on the progress or success of cytokinesis (Hickson, 2008).

Tests were performed to see whether RhoGEF^Pbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEF^Pbl RNAi or Rho1 RNAi, anillin-GFP was found to be localized to the cortex in metaphase but does not relocalize to the equator during anaphase, indicating a requirement for RhoGEF^Pbl, consistent with prior analysis of fixed RhoGEF^Pbl mutant embryos. Because anillin can bind F-actin and phosphorylated myosin regulatory light chain (MRLC), RhoGEF^Pbl might regulate anillin indirectly through its control of F-actin and myosin II (Hickson, 2008).

Latrunculin A (LatA) was used to test whether F-actin was required for anillin-GFP localization. A 30-60-min incubation of 1 µg/ml LatA abolished cortical anillin-GFP localization in metaphase, indicating an F-actin requirement at this phase. However, when anillin normally relocalizes to the equator (~3-4 min after anaphase onset), anillin-GFP formed punctate structures that became progressively more filamentous over the next few minutes, reaching up to several micrometers in length and having a thickness of ~0.3 µm. These linear anillin-containing structures contained barely detectable levels of F-actin and formed specifically at the plasma membrane and preferentially at the equator, although subsequent lateral movement often led to a more random distribution. Thus, anillin responds to spatiotemporal cytokinetic cues even after major disruption of the F-actin cytoskeleton. A substantial (albeit incomplete) reacquisition of cortical phalloidin staining was observed in cells fixed after washing out the drug for a few minutes. In live cells, LatA washout immediately after formation of the anillin structures allowed the preformed structures to migrate from a broad to a compact equatorial zone as the cells attempted to complete cytokinesis. This movement indicates that an F-actin-dependent process can contribute to the equatorial focusing of anillin (Hickson, 2008).

The influence of RhoGEF^Pbl on anillin behavior was tested in LatA. After RNAi of RhoGEF^Pbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEF^Pbl and Rho1 are required for anaphase anillin behavior, whether the cortex is intact or disrupted by LatA treatment (Hickson, 2008).

Tests were performed to see whether myosin II impacts anillin-GFP localization. Compared with controls, RNAi of the gene encoding MRLC spaghetti squash (MRLC^Sqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEF^Pbl RNAi, equatorial accumulation of anillin-GFP was not altogether blocked. It was still recruited but in a broad zone. Furthermore, in the presence of LatA, MRLC^Sqh RNAi did not affect the formation of the anillin-GFP structures. It is concluded that myosin II contributes to the equatorial focusing of anillin when the F-actin cortex is unperturbed but that myosin II is dispensable for anillin behavior in LatA (Hickson, 2008).

Collectively, these data suggest that multiple RhoGEF^Pbl-dependent inputs control anillin localization. The slowed equatorial accumulation of anillin when myosin II function was impaired indicates a myosin II-dependent input. That reassembly of the cortical F-actin network (after washout of LatA) allowed preformed anillin structures to move toward the cell equator indicates an F-actin-dependent input. This is consistent with the concerted actions of myosin II and F-actin driving cortical flow, as observed in other cells, and is reminiscent of the coalescence of cortical nodes during contractile ring assembly in Schizosaccharomyces pombe. However, the F-actin- and myosin II-independent behavior of anillin in LatA indicates an additional RhoGEF^Pbl-dependent input. Thus, RhoGEF^Pbl can control anillin behavior in anaphase via a previously unrecognized route. Immunofluorescence analysis revealed extensive colocalization between endogenous Rho1 and anillin-GFP in LatA, indicating that Rho1 was itself a component of these structures. These findings are consistent with the idea that Rho1 and anillin directly interact (Hickson, 2008).

Myosin II localization was studied, since it can bind anillin and is controlled by RhoGEF^Pbl. MRLC^Sqh-GFP is able to localize to the equatorial membrane independently of F-actin, and in doing so forms filamentous structures resembling those observed with anillin-GFP. Indeed, anillin and MRLC^Sqh (detected as either MRLC^Sqh-GFP or with an antibody to serine 21-phosphorylated pMRLC^Sqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLC^Sqh-GFP localization were tested. MRLC^Sqh-GFP recruitment and furrow initiation appeared normal, but within a few minutes of initiation, furrows became laterally unstable and oscillated back and forth across the cell cortex, parallel to the spindle axis, in repeated cycles, each lasting ~1-2 min and eventually subsiding to yield binucleate cells after ~20 min. The phenotype was very similar to that reported for anillin RNAi in HeLa cells and represents a requirement for anillin at an earlier stage than previously noted in Drosophila. Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008).

In LatA, anillin RNAi did not prevent equatorial MRLC^Sqh-GFP recruitment, but instead of appearing as persistent linear structures distorting the cell surface, a more reticular and dynamic structure lacking cell surface protrusions was observed. Thus myosin II can localize independently of both anillin and F-actin but the filamentous appearance of myosin II in the presence of LatA requires anillin, indicating that anillin can influence myosin II behavior in the absence of F-actin, whereas myosin II appeared capable of influencing anillin behavior only in the presence of F-actin (Hickson, 2008).

Septins are multimeric filament-forming proteins that can bind anillin in vitro and function with anillin in vivo. Using an antibody to the septin Peanut, it was found that in nontransfected S2 cells, septin^Pnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septin^Pnut antibody also strongly labeled bundles of cytoplasmic ordered cylindrical structures, each ~0.6 µm in diameter and of variable length (up to several micrometers). These staining patterns could be greatly reduced by septin^Pnut RNAi and were thus specific. The cylindrical structures did not appear to be cell cycle regulated, as they were apparent in interphase, mitotic, and postmitotic cells. They also did not colocalize with anillin, nor did their stability rely on anillin. Incubation with 1 µg/ml LatA before fixation inevitably led to disassembly of most of these large structures; however, the resulting distribution of septin^Pnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septin^Pnut formed cytoplasmic rings, ~0.6 µm in diameter, which are similar to the Septin2 rings seen in interphase mammalian cells treated with F-actin drugs or in the cell body of unperturbed ruffling cells (Kinoshita, 2002; Schmidt, 2004). In LatA-treated mitotic cells, septin^Pnut was diffusely cytoplasmic (or barely detectable) in early mitosis, whereas in anaphase/telophase, it localized to the same plasma membrane-associated anillin-containing filamentous structures (Hickson, 2008).

Anillin behavior was analyzed after septin^Pnut RNAi. Although unable to fully deplete septin^Pnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septin^Pnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septin^Pnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septin^Pnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septin^Pnut. However, in this case the plasma membrane to which anillin-GFP localized subsequently exhibited unusual behavior. It was internalized in large vesicular structures, apparently in association with midzone MTs. Although this phenomenon is not understood, it may be related to events induced by point mutations in the septin-interacting region of anillin that give rise to abnormal vesicularized plasma membranes during Drosophila cellularization (Hickson, 2008).

The effects were tested of anillin RNAi on the localization of septin^Pnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septin^Pnut. In LatA-treated cells, anillin RNAi did not affect the formation of septin^Pnut rings in interphase cells, but it greatly reduced the formation of septin^Pnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septin^Pnut and for the formation of septin^Pnut-containing structures in 1 µg/ml LatA. In contrast, Dia could still localize to the equatorial plasma membrane after combined anillin RNAi and LatA treatment, indicating that it can localize independently of both anillin and F-actin. Thus, although Dia partially colocalized with anillin in LatA, this likely reflected independent targeting to the same location rather than an association between anillin and Dia (Hickson, 2008).

These data argue that Rho1, anillin, septins, and the plasma membrane participate independently of F-actin in the formation of a complex. However, anillin, septins and F-actin can also form a different complex in vitro, independently of Rho (Kinoshita, 2002). Perhaps two such complexes dynamically interchange in vivo (Hickson, 2008).

The involvement of MTs in anillin behavior was tested in LatA. Overnight incubation with 25 µM colchicine effectively depolymerized all MTs in mitotic cells and promoted mitotic arrest, as expected. Using Mad2 RNAi to bypass the arrest, anillin-GFP was observed during mitotic exit in the absence of MTs and in the presence of LatA. Under such conditions, anillin-GFP formed filamentous structures very similar to those formed when MTs were present, indicating that MTs were dispensable for their formation. However, the structures appeared uniformly around the plasma membrane rather than restricted to the equatorial region, which is consistent with the role MTs play in the spatial control of Rho activation (Hickson, 2008).

The LatA-induced anillin structures localize to the ends of nonoverlapping astral MTs directed toward the equator. Live imaging of cells coexpressing cherry-tubulin and anillin-GFP revealed bundles of MTs associating with the filamentous anillin-GFP structures as they formed. Colocalization between anillin-GFP structures and MT ends persisted over many minutes, even after considerable lateral movement at the membrane. Thus, although the anillin structures formed independently of MTs, they stably associated with MTs. These findings support prior biochemical evidence for interactions of MTs with both anillin and septins (Sisson, 2000) and reveal a potential positive-feedback loop in which MTs directed where Rho1-anillin-septin formed linear structures at the plasma membrane, whereas the structures in turn associated with the MT ends. An MT plus end-binding ability of anillin-septin could explain the furrow instability phenotype elicited by anillin RNAi. Accordingly, anillin may physically link Rho1 to MT plus ends during furrow ingression, thereby promoting the focusing and retention of active Rho1, thus stabilizing the furrow at the equator (Hickson, 2008).

These live-cell analyses highlight an unusual behavior of the Rho-dependent anillin-containing structures at the plasma membrane. Initially forming beneath and parallel to the plasma membrane, the structures then often lifted on one side to appear perpendicular to the cell surface while remaining anchored at their base by MTs. This reorientation is interpreted as reflecting avid binding to and subsequent envelopment by the plasma membrane. Although intrinsically stable, the structures exhibited dynamic movement within the plane of the plasma membrane and were capable of sticking to one another, via their ends, giving rise to branched structures that were also capable of breaking apart. Anillin has a pleckstrin homology domain within its septin-interacting region and a membrane-anchoring role of anillin has long been postulated (Field, 1995). These data support such a role and suggest that it is controlled by Rho (Hickson, 2008).

The data highlight the complexity of RhoGEF^Pbl signaling and lead to a model in which multiple Rho-dependent inputs synergize to control anillin behavior during cytokinesis (Hickson, 2008). At the appropriate time and location of normal cytokinesis, several proteins, including Rho1, MRLC^Sqh, Dia, anillin, and Septin^Pnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and Septin^Pnut were uniquely and specifically required for the formation of the linear filamentous structures describe in this study. The behaviors of these structures are consistent with prior studies of ordered assemblies of septins and anillin and of interaction between anillin and MTs. Although such structures are not normally seen in furrowing cells, anillin localizes to remarkably similar filamentous structures in the cleavage furrows of HeLa cells arrested with the myosin II inhibitor blebbistatin (Hickson, 2008).

It seems unlikely that LatA induces the described structures through nonspecific aggregation of proteins. Rather, it is proposed that LatA blocks a normally dynamic disassembly of Rho1-anillin-septin complexes (by blocking an F-actin-dependent process required for the event) and that continued assembly promotes formation of the linear structures. Because blebbistatin slows F-actin turnover, blebbistatin and LatA may have induced filamentous anillin-containing structures via a common mechanism. A dynamic assembly/disassembly cycle involving anillin could promote transient associations between the plasma membrane and elements of the contractile ring and MTs, properties that could contribute to furrow stability and plasticity. Finally, because local loss of F-actin accompanies and may indeed trigger midbody formation, LatA treatment may have stabilized events in a manner analogous to midbody biogenesis and could therefore be useful in understanding this enigmatic process (Hickson, 2008).

Energy-dependent regulation of cell structure by AMP-activated protein kinase

AMP-activated protein kinase (AMPK, also known as SNF1A: see Drosophila Alicorn) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Using the Drosophila model system, animals with no AMPK activity were genrated and physiological functions of the kinase investigated. Surprisingly, AMPK-null mutants are lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restores many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC, Spaghetti-squash; also known as MLC2 was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, these results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures (Lee, 2007).

The catalytic subunit of Drosophila AMPK is a single orthologue of its human and yeast counterparts, and is activated by LKB1 on energy deprivation. By imprecise excision of the EP-element (enhancer- and promoter-containing P-element) from the AMPK^G505 line, AMPK-null mutant lines, AMPK^D1 and AMPK^D2 were generated. Interestingly, all AMPK-null mutant flies are lethal before the mid-pupal stage and fail to enter adulthood, even in the presence of sufficient nutrients. Although transgenic expression of wild-type AMPK (AMPK^WT) allowed AMPK-null mutants to successfully develop into adults, the expression of kinase-dead AMPK (AMPK^KR) failed to rescue the lethality, demonstrating that the phosphotransferase activity of AMPK is crucial for its function. In summary, AMPK was found to be essential for normal development of Drosophila (Lee, 2007).

The developmental role of AMPK was further investigated by generating AMPK-null germ-line clone (AMPK-GLC) embryos, which are completely deprived of both the maternal and zygotic AMPK proteins. Surprisingly, AMPK-GLC embryos never developed into larvae, showing the requirement of AMPK during embryogenesis. In AMPK-GLC embryos, cuticle structures were severely deformed, and ventral denticle belts were missing. Furthermore, the surface of AMPK-GLC embryos was roughened and the columnar structure of the epidermis was disorganized, implicating defects in underlying epithelial structures (Lee, 2007).

To examine the embryonic epithelial structures, AMPK-GLC epithelia were examined with various polarity markers. Bazooka (Baz, apical complex marker) and β-catenin (Arm, adherens junction marker) lost their apical localization and were found in various locations around the basolateral cell surfaces. The Discs-large (Dlg, basolateral marker was also irregularly distributed throughout the epithelium in AMPK-GLC embryos. Moreover, actin staining demonstrated that the AMPK-GLC epithelium contains many unpolarized round cells that had lost contact with the underlying tissue. This disorganization of epithelial structures was not a result of cell death, because it could not be restored by overexpression of apoptosis inhibitor p35. In addition, wing discs of AMPK-null mutants also showed defective epithelial organization with ectopic actin structures in the basolateral region. These results indicate that AMPK is indispensable for epithelial integrity (Lee, 2007).

In addition, abnormally enlarged nuclei were found in some cells of AMPK-GLC embryos. Mitotic chromosome staining with anti-phospho-histone H3 (PH3) antibody demonstrated that AMPK-GLC embryos frequently contained defective mitotic cells with lagging or polyploid chromosomes. Consistently, aceto-orcein staining of squashed AMPK-null larval brains revealed polyploidy in ~30% of mitotic cells, and anti-PH3 staining showed a highly increased amount of chromosome content in some of the neuroblasts. These results indicate that AMPK is also required for the maintenance of genomic integrity (Lee, 2007).

Recently, it has been proposed that LKB1, a kinase upstream of AMPK, is involved in the regulation of epithelial polarity and mitotic cell division. Indeed, the abnormal polarity and mitosis phenotypes of lkb1-null mutants were highly similar to those of AMPK-null mutants. To test whether AMPK mediates the polarity- and mitosis-controlling functions of LKB1, constitutively active AMPK (AMPK^TD), which is catalytically active even without phosphorylation by LKB1, was expressed in lkb1-null mutants. Remarkably, AMPK^TD suppresses the epithelial polarity defects and the genomic instability of lkb1-null mutants, suggesting that AMPK is a critical downstream mediator of LKB1, controlling mitosis and cell polarity (Lee, 2007).

To understand the molecular mechanism underlying the AMPK-dependent control of mitosis and cell polarity, attempts were made to identify the downstream targets of AMPK. Intriguingly, MRLC, a critical molecule for the execution of mitosis and cell polarity establishment, contains a peptide sequence that can be phosphorylated by AMPK. Therefore, various experiments were performed to evaluate the ability of AMPK to phosphorylate MRLC. AMPK holoenzyme purified from rat liver strongly phosphorylated full-length MRLC, which was further enhanced by the addition of AMP. The phosphorylation of MRLC was more efficient than that of acetyl-CoA carboxylase 2 (ACC2), a representative substrate of AMPK, indicating that MRLC is a good in vitro substrate of AMPK. It was deduced that this phosphorylation is specifically performed by AMPK because Compound C, a specific inhibitor of AMPK, inhibited the phosphorylation, whereas ML-7, an inhibitor of another MRLC-phosphorylating kinase (MLCK), did not. A mutant form of MRLC, whose regulatory phosphorylation site (corresponding to Thr 21/Ser 22 in Drosophila and Thr 18/Ser 19 in human) was mutated into non-phosphorylatable alanines, was not phosphorylated by AMPK, suggesting that MRLC is exclusively phosphorylated at the regulatory phosphorylation site. Both the human and Drosophila forms of AMPK were able to phosphorylate MRLC from each of the respective species, which further demonstrates that the AMPK phosphorylation of MRLC is highly conserved between species (Lee, 2007).

Moreover, it was found that MRLC phosphorylation is indeed regulated by AMPK in vivo. The phosphorylation of MRLC was dramatically reduced in AMPK- and lkb1-GLC epithelia when compared with the wild-type epithelia, although the protein level of MRLC was unaffected. The reduced phosphorylation of MRLC in the AMPK-GLC epithelia was completely restored by transgenic expression of AMPK but not by overexpression of LKB1. Furthermore, in Drosophila S2 cells, energy deprivation induced by 2-deoxyglucose (2DG) enhanced MRLC phosphorylation, which was suppressed by double-strand-RNA-mediated silencing of lkb1 or AMPK. Collectively, these data strongly suggest that MRLC is specifically phosphorylated by AMPK both in vitro and in vivo (Lee, 2007).

To find out whether the phosphorylation of MRLC is critical for the physiological functions of AMPK, an active form of MRLC (MRLC^EE), whose regulatory phosphorylation site was mutated into phosphomimetic glutamates, was expressed in AMPK-GLC embryos. Strikingly, MRLC^EE rescued the epithelial polarity defects caused by the loss of AMPK, and increased the percentage of cuticle-forming embryos from ~10% to ~30. MRLC^EE also restored the epithelial polarity defects of lkb1-null wing imaginal discs. Furthermore, the genomic polyploidy of AMPK- and lkb1-null larval brain neuroblasts was suppressed by the expression of MRLC^EE. Therefore, it is concluded that MRLC is a critical downstream target of AMPK controlling cell polarity and mitosis (Lee, 2007).

Notably, the larval brains of MRLC loss-of-function mutants (spaghetti-squash¹) showed extensive polyploidy (~40% of mitotic neuroblasts), and their imaginal discs showed severe disorganization in epithelial structure, similar to those of lkb1- and AMPK-null mutants. These phenotypic similarities further support the conclusion that MRLC is an important functional mediator of LKB1 and AMPK (Lee, 2007).

Finally, it was asked whether AMPK is critical for directing cell polarity in mammalian cells as well. To assess this, it was asked whether the activation of AMPK by 2DG treatment could induce polarization of unpolarized epithelial cells such as LS174T, which can be polarized by the activation of LKB1, in a cell-autonomous manner. Surprisingly, on 2DG treatment, LS174T cells undergo a dramatic change in cell shape to have polarized actin cytoskeleton with a brush-border-like structure. Moreover, although brush border marker villin, apical marker CD66/CEA, and basal marker CD71/transferrin were distributed throughout untreated cells, they became dramatically polarized on 2DG treatment, supporting that the activation of AMPK by energy deprivation is sufficient to induce complete polarization of LS174T cells (Lee, 2007).

It was also found that the phosphorylation of MRLC by AMPK is involved in the energy-dependent polarization of LS174T cells. Phosphorylated MRLC was colocalized with the 2DG-induced polarized actin structures, and this phosphorylation, as well as the actin polarization, was suppressed by the AMPK-specific inhibitor Compound C. Overexpression of dominant-negative AMPK (AMPK^DN) and short interfering (si)RNA-mediated inhibition of MRLC (siMRLC) also blocked the polarization, although inhibition of Par-1, another downstream kinase of LKB1, by Par-1 siRNA (siPar-1) or overexpression of dominant-negative Par-1 (Par-1^DN) failed to cause a block. More strikingly, human MRLC^EE itself was sufficient to polarize LS174T cells, even without energy deprivation, showing that phosphorylation of MRLC is critical for the AMPK-dependent polarization (Lee, 2007).

Until now, the importance of AMPK has been limited to its role as a regulator of metabolism. However, by generating the first animal model with no AMPK activity, additional functions of AMPK were characterized: AMPK regulates mitotic cell division and epithelial polarity downstream of LKB1 by controlling the activity of MRLC through direct phosphorylation. The findings revealed a link between energy status and cell structures, providing a new perspective to the diverse molecular function of AMPK. Further studies are needed on the cell-structure-controlling function of AMPK with respect to the various metabolic and physiological contexts, which may also help to understand AMPK-related diseases such as cancer and diabetes (Lee, 2007).

Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity

The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. This study shows that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. These results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change (Foronda, 2012).

The elimination of a part of an animal body is a major change occurring during morphogenesis and evolution. This study has analyzed the mechanisms required for one such change, the absence of the male seventh abdominal segment. The study shows that the suppression of this segment involves the interplay between Hox and the sex determining genes, which regulate targets implementing the morphological change. The reduction or suppression of this segment is also a sexually dimorphic feature characteristic of higher Diptera, so the mechanisms shown here may be relevant for the evolution of morphology (Foronda, 2012).

In early pupa, during the second phase of cell division, there is a reduction in the number of A7 histoblasts, both in males and females, but stronger in males perhaps because wg is not expressed in the male A7 histoblasts. It has been shown that fewer histoblasts result in a smaller adult segment. Therefore, the reduced number of A7 histoblasts may account in part for the reduced size of the A7 segment in females. The control of the second phase of cell division involves the EGFR pathway, and Abd-B was found to reduce the number of histoblasts in the A7 through down-regulation of EGFR activity. If this activity is eliminated in the male A7, an increase is observed the number of histoblasts, that many of these cells remain at the surface at the time of extrusion and that a small A7 forms in the adult. It was also previously reported that a small A7 is observed in the male adult when expressing vein, an EGFR ligand. It is possible that the high number of histoblasts obtained when over-expressing elements of the EGFR pathway makes many of them unable to be extruded by a 'titration' effect, that is, there may be 'too many' histoblasts for the invagination mechanism to extrude them at the correct time. However, the EGFR pathway may also hinder extrusion since lower levels are seen of emc-GFP and also many histoblasts remain at the surface after high EGFR activation (Foronda, 2012).

At later pupal stages (around 35-40 h APF) there is the extrusion of the male A7 histoblasts. It was observed, however, that a few histoblasts also invaginate in the female A7, suggesting the male intensifies a mechanism present in both sexes. The extrusion requires the activity of emc, and correlates with higher emc expression in the male A7 histoblasts at about the time of extrusion. The invagination of histoblasts superficially resembles that of larval cells, and it also requires myosin activity. This would suggest that, due to the higher levels of Abd-B and DsxM, male A7 histoblasts may have adopted a mechanism similar to that used by cuticular larval epidermal cells (LECs) for their elimination. Recent reports, however, suggest an alternative mechanism. It has been demonstrated that an excess of proliferation in the epithelium leads to cell death-independent cell extrusion. Since this study has observed that prevention of cell death in the male A7 does not cause the development of an A7 (although delamination is delayed), the mechanism driving extrusion may be more similar to that of an overproliferating epithelium than to that taking place in larval cells (Foronda, 2012).

The data are consistent with emc increasing the expression of spaghetti-squash to accomplish apical constriction and extrusion. However, high expression of emc may not be sufficient to effectively induce histoblast extrusion, suggesting other genes are required. Besides, a strong reduction of emc leads to a very small and poor differentiated male A7 segment, reflecting that this gene is required for several cellular functions, among them cell survival. Perhaps significantly, emc is also expressed in embryonic tissues preceding invagination of different structures in the embryo, suggesting a common requirement for invagination at different developmental stages. It is thought that emc forms part of complex networks that have, among other cellular functions, that of contributing to the extrusion of A7 histoblasts (Foronda, 2012).

Although regulation of the EGFR pathway and emc are two key events in controlling male A7 development, previous experiments have also shown the contribution of the wingless gene, absent in male A7 but present in male A6 and female A7, in the development of this segment. These results have been confirmed and it was also shown that a reduction in wg expression can partially suppress the Abd-B mutant phenotype. Absence of wg is probably required to reduce cell proliferation in the male A7 but the data suggest wg may also be needed to maintain high emc levels. Apart from the role of wg, it was also shown that some A7a cells are transformed into A6p cells, thus reducing the number of A7 cells that might contribute to the adult segment. Finally, the expression of bric-a-brac must also be down-regulated in male A7 histoblasts to eliminate this metamere. Thus, this suppression is a complex process using different genes and mechanisms (Foronda, 2012).

The suppression of the male A7 depends ultimately on the levels of Abd-B expression. The role of this Hox gene is probably mediated in part by dsx, since Abd-B regulates dsx transcription and dsx governs, in turn, the expression of genes required for cell proliferation and extrusion. That Hox genes regulate dsx expression has also been demonstrated in the male foreleg, suggesting that Hox genes specify the different parts of the body where sexual dimorphism may evolve. The different dsx isoforms (DsxF and DsxM) determine the outcome of this regulation. A significant difference between the activities of these two proteins in the A7 is the regulation of emc levels. In the female, emc expression is similar in the A7 and the A6 and, accordingly, histoblast extrusion in females is small and confined to the central dorsal region, a domain virtually absent in the adult tergite. By contrast, the DsxM isoform increases Emc expression to drive large extrusion of A7 cells and elimination of the segment (Foronda, 2012).

Only the male A7, but not anterior abdominal segments, is eliminated. Therefore, the increase in emc expression, and subsequent events observed in the A7, depends on the higher Abd-B expression in the A7 in relation to the A6. Several Hox loci, like Sex combs reduced, Ultrabithorax or Abd-B are haplo-insufficient, and relatively small differences in the amount of some of these Hox proteins can drive major phenotypic changes, suggesting some downstream genes can sense these slight differences and implement major changes in morphology (Foronda, 2012).

Previous studies have shown the cooperation of Abd-B and the sex determination pathway in controlling the pigmentation of the posterior abdomen. It is thought that Abd-B plays a dual role in regulating the morphology of the posterior abdomen. First, it regulates dsx expression, thus allowing the possibility to develop sexually dimorphic characters; second, it cooperates with Dsx proteins in establishing pattern. Part of the effect implemented by Abd-B may be mediated by the levels of expression of dsx (distinguishing male A6 from male A7), and from the nature of the Dsx proteins (male and female ones). Although there is no conclusive evidence that the different levels of dsx in the A6 and A7 play a role in development, it is noted that this difference correlates with that of Abd-B (and depends on it), that high levels of DsxM are sufficient to increase emc-GFP in the A7 of females and eliminate this segment, and that these same high levels similarly increase emc-GFP and partially rescue the Abd-B mutant phenotype in males. Hox genes, therefore, may provide a spatial cue along the anteroposterior axis to activate dsx transcription and allow the formation of sexually dimorphic characters, but they may also cooperate with Dsx proteins to determine different morphologies. This double control by Hox genes may apply to all the sexually dimorphic characters and be also a major force in evolution (Foronda, 2012).

Search PubMed for articles about Drosophila spaghetti squash

Billuart, P., Winter, C. G., Maresh, A., Zhao, X. and Luo, L. (2001). Regulating axon branch stability. the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell 107(2): 195-207. 11672527

Chougule, A.B., Hastert, M.C. and Thomas, J.H. (2016). Drak is required for actomyosin organization during Drosophila cellularization. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26818071.

Corrigall, D., Walther, R. F., Rodriguez, L., Fichelson, P., Pichaud, F. (2007). Hedgehog signaling is a principal inducer of Myosin-II-driven cell ingression in Drosophila epithelia. Dev Cell 13: 730-742. PubMed ID: 17981140

Edwards, K. A. and Kiehart, D. P. (1996). Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis. Development 122: 1499-1511. PubMed ID: 8625837

Folker, E. S., Schulman, V. K. and Baylies, M. K. (2012). Muscle length and myonuclear position are independently regulated by distinct Dynein pathways. Development 139(20): 3827-37. PubMed ID: 22951643

Foronda, D., Martín, P., Sánnchez-Herrero, E. (2012). Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity. PLoS Genet. 8(8): e1002874. PubMed ID: 22912593

Hickson, G. R. and O'Farrell, P. H. (2008). Rho-dependent control of anillin behavior during cytokinesis. J. Cell Biol. 180(2): 285-94. PubMed ID: 18209105

Jordan, P. and Karess, R. (1997). Myosin light chain-activating phosphorylation sites are required for oogenesis in Drosophila. J. Cell Biol. 139: 1805-1819. PubMed ID: 9412474

Karess, R. E., Chang, X.-j., Edwards, K. A., Kulkarni, S., Aguilera, I. and Kiehart, D. P. (1991). The regulatory light chain of nonmuscle myosin is encoded by spaghetti squash, a gene required for cytokinesis in Drosophila. Cell 65: 1177-1189. PubMed ID: 1905980

Kasza, K. E., Farrell, D. L. and Zallen, J. A. (2014). Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation. Proc Natl Acad Sci U S A 111(32):11732-7. PubMed ID: 25071215

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): 1741-9. PubMed ID: 17277363

Lee, A. and Treisman, J. E. (2004). Excessive myosin activity in Mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium. Mol. Biol. Cell. 15: 3285-3295. 15075368

Lee, J. H., et al. (2007). Energy-dependent regulation of cell structure by AMP-activated protein kinase. Nature 447(7147): 1017-20. PubMed ID: 17486097

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

Neubueser, D. and Hipfner, D. R. (2010). Overlapping roles of Drosophila Drak and Rok kinases in epithelial tissue morphogenesis. Mol Biol Cell 21: 2869-2879. PubMed ID: 20573980

Robertson, F., Pinal, N., Fichelson, P. and Pichaud, F. (2012). Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis. Development 139: 3432-3441. PubMed ID: 22874916

Simoes Sde, M., Blankenship, J. T., Weitz, O., Farrell, D. L., Tamada, M., Fernandez-Gonzalez, R. and Zallen, J. A. (2010). Rho-kinase directs Bazooka/Par-3 planar polarity during Drosophila axis elongation. Dev Cell 19: 377-388. PubMed ID: 20833361

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

Tang, H. W., Wang, Y. B., Wang, S. L., Wu, M. H., Lin, S. Y. and Chen, G. C. (2011). Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy. EMBO J 30: 636-651. PubMed ID: 21169990

Vasquez, C. G., Tworoger, M., Martin, A. C. (2014). Dynamic myosin phosphorylation regulates contractile pulses and tissue integrity during epithelial morphogenesis. J Cell Biol 206: 435-450. PubMed ID: 25092658

Vereshchagina, N., et al. (2004). The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin, Mol. Biol. Cell 15: 4395-4405. PubMed ID: 15269282

Wang, Y. and Riechmann, V. (2007). The role of the actomyosin cytoskeleton in coordination of tissue growth during Drosophila oogenesis. Curr Biol 17(15): 1349-55. PubMed ID: 17656094

Wheatley, S., Kulkarni, S. and Karess, R. (1995). Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos. Development 121: 1937-1946. PubMed ID: 7601006

Widmann, T. J. and Dahmann, C. (2009). Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1. J. Cell Sci. 122(Pt 9): 1362-73. PubMed ID: 19366729

Winter, C. G., et al. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105: 81-91. 11301004

date revised: 23 October 2014 Biological Overview

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