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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Gene Expr Patterns. Author manuscript; available in PMC 2012 January 1.
Published in final edited form as:
PMCID: PMC3025304
NIHMSID: NIHMS243199

Distinct tissue distributions and subcellular localizations of differently phosphorylated forms of the myosin regulatory light chain in Drosophila

Abstract

Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRCL, encoded by spaghetti squash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila.

Keywords: Drosophila, myosin, myosin regulatory light chain, spaghetti squash, dorsal closure, morphogenesis

1. Results and Discussion

Nonmuscle myosin II (referred to as myosin hereafter) is a motor protein that binds reversibly to actin filaments and generates contractile forces necessary for a variety of cellular processes including cell shape changes, cell movements, cytokinesis, maintenance of cell morphology and polarity, and intracellular vesicle transport (reviewed in Conti and Adelstein, 2008; Vicente-Manzanares et al., 2009). Consistent with these roles, loss-of-function mutations in myosin in various organisms result in severe developmental defects and are inevitably lethal. For example, in Dictyostelium, cells that lack either myosin heavy chain or the regulatory light chain undergo cytokinesis with greatly decreased efficiency, have dramatic reduction of cell polarity, show greatly reduced motility, cease development after aggregation and fail to form fruiting bodies (reviewed in Bosgraaf and van Haastert, 2006). In Drosophila, strong loss-of-function mutations in the myosin heavy chain (encoded by zipper or zip) are embryonic lethal with defects in axon pathfinding, dorsal closure and head involution (Young et al., 1993). Hypomorphic zip alleles produce malformed adult legs due to defects in cell shape changes during imaginal disc morphogenesis (Gotwals and Fristrom, 1991; Halsell and Kiehart, 1998). Similarly, mutations in the myosin regulatory light chain (encoded by spaghetti squash or sqh) result in disrupted cytokinesis, defects in imaginal disc morphogenesis including eversion of the legs and wings, eye morphogenesis, and oogenesis (Edwards and Kiehart, 1996; Karess et al., 1991).

Myosin molecules are hexamers composed of a pair of heavy chains (MHC), a pair of essential light chains, and a pair of regulatory light chains (MRLC). The activity of myosin is mainly controlled by phosphorylation and dephosphorylation of MRLC (reviewed in Tan et al., 1992). Phosphorylation of Ser19 on MRLC (which corresponds to Ser21 in Drosophila Sqh) can greatly increase the actin-dependent myosin ATPase activity and thus the motor activity of myosin (Ikebe et al., 1988). Subsequent phosphorylation of Thr18 (Thr20 in Sqh) can further increase myosin activity (Ikebe and Hartshorne, 1985; Ikebe et al., 1986; Ikebe et al., 1988). Protein kinases including myosin light chain kinase (MLCK), Rho-associated protein kinase (ROK), Rho-dependent Citron kinase, AMP-activated kinase, leucine zipper interacting kinase (ZIPK) and myotonic dystrophy kinase-related CDC42-binding kinase can phosphorylate and activate MRLC (Kosako et al., 2000; Lee et al., 2007; Tan et al., 1992; Ueda et al., 2002; Vicente-Manzanares et al., 2009; Yamashiro et al., 2003). Myosin phosphatase acts antagonistically, dephosphorylating MRLC, thereby leading to inactivation of myosin (Hartshorne, 1998; Vereshchagina et al., 2004).

1.1. Antibodies specific for phosphorylated forms of Sqh

To identify spatial and temporal patterns of myosin activation during Drosophila development, we generated two site-specific antibodies against the phosphorylated forms of Sqh: one directed against the monophosphorylated form (with phospho-Ser21; referred to hereafter as Sqh1P), and the other directed against the diphosphorylated form (with phospho-Thr20 and phospho-Ser21; referred to as Sqh2P). We also generated an antibody against full-length Sqh protein to use as a control (initially described in Wang and Ward, 2010). To test the specificity of these antibodies, we conducted western blotting of lysates from wild type imaginal discs. All three antibodies recognized a single band of ~21-kD on these blots, suggesting that all three antibodies recognize endogenous Sqh in this tissue (Fig. 1A). To confirm this notion, we used the antibodies on blots containing protein samples extracted from yw cv sqhAx3; P{sqh-GFP} animals. sqhAx3 is an amorphic allele that produces no Sqh protein (Jordan and Karess, 1997), whereas sqh-GFP encodes a full-length Sqh protein tagged with Green Fluorescent Protein (GFP). The recombinant protein has a predicted molecular mass of ~48-kD. All three antibodies recognize only a single band of ~48-kD from these lysates, consistent with the antibodies specifically recognizing the recombinant Sqh-GFP (Fig. 1A).

Fig. 1
Antibodies against Sqh1P and Sqh2P are specific

We next performed Urea-glycerol-PAGE followed by immunoblotting to further test the specificity of the antibodies. Urea-glycerol-PAGE has been used to separate the phosphorylated forms of MRLC based on their native charges instead of molecular weight (Perrie and Perry, 1970). When a urea-glycerol-PAGE blot of lysate from wild type imaginal discs is probed with the polyclonal antibody against Sqh, three bands are clearly visible (Fig. 1B). From top to bottom, these bands represent nonphosphorylated, monophosphorylated and diphosphorylated forms of Sqh, respectively. In contrast, when these blots are probed with anti-Sqh1P or anti-Sqh2P antibodies, only a single band is revealed (Fig. 1B). To confirm that the single band represents either Sqh1P or Sqh2P specifically, we re-probed these blots with the anti-Sqh antibody and visualized the presence of the other two bands at the expected positions (data not shown). These blots also revealed that less than 20% of total Sqh proteins are phosphorylated in late larval imaginal discs.

To further demonstrate the specificity of the antibodies against the phosphorylated forms of Sqh, we treated protein samples from wild type imaginal discs with protein phosphatase 1 (PP1) and analyzed the samples by western blotting. PP1 is a general phosphatase that dephosphorylates phospho-Ser and phospho-Thr residues (Cohen, 1989), and thus is predicted to be able to dephosphorylate phospho-Thr20 and phospho-Ser21 on Sqh. On western blots, we observed that the signal of both Sqh1P and Sqh2P decreased significantly after PP1 treatment, whereas the expression of Coracle, a cytoplasmic protein used as a control for protein stability was unaffected during the time course (Fig. 1C, and data not shown).

To demonstrate the specificity of these phospho-specific antibodies to recognize Sqh in Drosophila tissues, we generated mitotic clones of the null sqh allele sqhAX3 in the ovary and immunostained them with antibodies against Sqh1P and Sqh2P. sqhAX3 homozygous mutant clones in the ovarian follicular epithelium showed clearly decreased levels of both Sqh1P and Sqh2P in the mutant cells compared to the neighboring heterozygous cells (Fig. 2). Taken together, these immunoblot and immunostaining experiments clearly demonstrate that the antibodies directed against Sqh1P and Sqh2P are highly specific for the phosphorylated forms of Sqh in Drosophila tissues.

Fig. 2
Sqh1P and Sqh2P levels are strongly reduced in follicle cell clones of sqhAX3

1.2. Sqh1P and Sqh2P show distinct tissue distributions in wild type embryos

We next used these antibodies to investigate myosin activation on staged wild type Drosophila embryos, and discovered that the different phosphorylated forms of Sqh show distinct tissue distributions and subcellular localizations. Sqh1P is fairly ubiquitously expressed and has a pattern that is virtually indistinguishable from total Sqh and Zip (Fig. 3A–C; Young et al., 1991; Young et al., 1993). Specifically, Sqh1P can first be detected during cellularization, where it localizes to the leading edge of the cleavage furrows (Fig. 3A). During gastrulation, Sqh1P and Sqh are predominantly found in the cytoplasm, but there is a transient enrichment near the basal plasma membrane (arrows in Fig. 3B) that first disappears in the invaginating cells of the ventral furrow. This pattern of expression is similar to that reported for Zip (myosin heavy chain; (Dawes-Hoang et al., 2005). In the ventral furrow cells, Zip relocalizes to the apical plasma membrane (Dawes-Hoang et al., 2005). Consistent with Sqh (and Sqh1P) working in a complex with Zip in these cells, we occasionally detected an enrichment of Sqh and Sqh1P near the apical plasma membrane of ventral furrow cells (arrowhead in Fig. 3B). It is possible that we could not detect more frequent or robust apical enrichment of Sqh and Sqh1P in ventral furrow cells due to interference from the cytoplasmic expression that persisted in embryos fixed by paraformaldehyde (please see notes on fixation in Experimental Procedures). In subsequent stages, Sqh1P is expressed in nearly all embryonic tissues including the epidermis, foregut, hindgut, midgut, the central nervous system, and the extraembryonic amnioserosa (Fig. 3C–G, Fig. 5A–C and data not shown). It is noteworthy that there are some tissues that show substantially higher levels of Sqh1P expression than the surrounding tissues, and that this difference is not reflected in the total Sqh staining. This is most evident in the brain and ventral nerve cord (Fig. 3D and E). In addition, there is an unusual distribution of Sqh1P in the dorsal most row of epidermal (DME) cells during dorsal closure (described in more detail below), but this expression is reflected in the total Sqh pattern as well.

Fig. 3
The expression pattern of Sqh1P in staged embryos
Fig. 5
Sqh1P and Sqh2P have distinct distributions in the DME cells during dorsal closure

Subcellularly, Sqh1P is cytoplasmic but appears to be enriched at cell membranes, thereby producing a cell-outlining pattern (for example Fig. 3C). Sqh1P is enriched apically in the epidermis, salivary gland, foregut and hindgut (Fig. 3C, F, and G), but it is also present along basolateral membranes in these tissues and is noticeably enriched on the basal surface of the hindgut (arrow in Fig. 3G).

Sqh2P, in contrast, is expressed in a limited pattern during embryogenesis. Sqh2P is not detected at all during cellularization or gastrulation (data not shown), and is first noticeably expressed at stage 6 in the cephalic furrow (data not shown), and then later in the invaginating proctodeal and stomodeal openings at stage 9 and 10 (Fig. 4A). It is also strongly expressed in tracheal placodes as they begin to invaginate in stage 11 and continuing throughout tracheal development (Fig. 4B–D and data not shown). We also observe strong Sqh2P expression on the apical surface of the invaginating salivary glands starting at stage 11, although we did not detect strong expression in the salivary gland placode prior to invagination (data not shown). During dorsal closure we noted strong Sqh2P expression in the DME cells, but in a pattern distinct from that of Sqh1P (described in more detail below). Sqh2P is also strongly expressed in the apically constricted cells found at segmental boundaries (Fig. 4D). During head involution, Sqh2P is strongly expressed in the pharynx and foregut (data not shown), as well as in a structure known as the dorsal pouch, which becomes apparent in stage 14 embryos (Fig. 4E). Finally, Sqh2P is expressed in puncta in the epidermis starting at stage 15 in a pattern that presages the epidermal hairs, as well as in other actin rich structures including the posterior spiracles (data not shown). It should be noted that starting in stage 14 embryos, the most obviously staining structures appear to be in the lumen of the tracheal system and the fore- and hindgut. These patterns are non-specific, however, as evidenced by costaining with apical plasma membrane markers such as Crumbs (Crb; Tepass et al., 1990) and Uninflatable (Uif; Zhang and Ward, 2009) that show that the anti-Sqh2P serum predominantly recognizes a luminal component instead of the epidermis (Supplemental Fig. 1).

Fig. 4
The expression pattern of Sqh2P in staged embryos

Subcellularly, Sqh2P is found on the apical membrane. This can be demonstrated most clearly by counterstaining these embryos with antibodies against apically localized proteins. Sqh2P colocalizes with Uif and Crb on the apical surface of the tracheal system and in the dorsal pouch (Figs. 4B–E and data not shown). These co-staining experiments also demonstrate that Sqh2P is upregulated in the segmental boundary cells relative to the rest of the epidermis. Specifically, Uif is expressed at relatively uniform levels at the apical surface of all the epidermal cells including cells of the segmental boundaries where the apical surfaces of adjacent cells are in close proximity (and thus Uif appears to be more strongly expressed here), and at the surface of the intersegmental cells that can be visualized on the ventral surface of the embryo (bracket in Fig. 4D”). Sqh2P, in contrast, is barely detectable in these intersegmental cells while maintaining strong expression in the cells at the segmental boundary (Fig. 4D’).

The expression differences between Sqh1P and Sqh2P are most evident in the DME cells during dorsal closure. Midway through embryogenesis, after the germband has retracted, the epidermis covers the ventral and lateral regions of the embryo, leaving a large dorsal hole covered only by a layer of squamous extraembryonic cells known as the amnioserosa. During dorsal closure, the lateral epidermal sheets on both sides of the embryo begin an orchestrated migration toward the midline, where they eventually meet and fuse and thereby close the dorsal surface of the embryo (reviewed in Harden, 2002). During this processes, the DME cells have a special role in organizing these morphogenetic events. Early in dorsal closure the DME cells undergo planer polarization in the dorsal-ventral axis resulting in a reorganization of the actin cytoskeleton (Kaltschmidt et al., 2002). These cells subsequently accumulate actin and myosin at the leading edge of these cells, eventually producing an actinomyosin cable through all the DME cells that helps coordinate the migration of the epidermal sheets towards the dorsal midline (Franke et al., 2005; Young et al., 1993). Sqh1P and Sqh2P are both upregulated in the DME cells during dorsal closure, but show a mostly non-overlapping pattern. Starting at stage 13, Sqh1P is strongly expressed in the leading edge of the DME cells in a “bars-on-a-string” distribution (Fig. 5A). This pattern persists during dorsal closure and grows more intense as dorsal closure proceeds (Fig. 5B and C). Similar patterns of expression have been described for both Sqh and Zip (Franke et al., 2005; Wang and Ward, 2010; Young et al., 1993). In contrast to the specific localization of Sqh1P along the actin cable, Sqh2P is strongly upregulated throughout the DME cells during the initiation phase of dorsal closure (stage 13; Fig. 5D), and localizes along the membrane. This expression appears to start in the cells near the anterior end of the row of DME cells and often remains strongest there. We also frequently observe large puncta of Sqh2P expression in these cells (Figs. 5D and E). Beginning in stage 13 embryos, Sqh2P can be found decorating large filopodia that emanate from the dorsal side of the DME cells (Fig. 5D). These filopodia become more numerous during the lateral spreading phase of dorsal closure (stage 14; Fig. 5E), and during the fusion or suturing phase (stage 15; Fig. 5F). It should be noted that Sqh2P also decorates the leading edge of the DME cells, but does so in a more continuous manner than Sqh1P, and does not appear to be enriched in the “bars on a string” pattern found by staining with Sqh1P.

1.3. Sqh1P and Sqh2P have distinct subcellular localization in imaginal discs

Sqh1P and Sqh2P are also expressed at high levels in the larval imaginal discs where they also display differential subcellular localizations, as well as slight differences in their expression pattern across the disc epithelium. Confocal microscopy shows that whereas Sqh1P appears to be expressed in all imaginal disc cells (Fig. 6A’), Sqh2P appears to strongly localize to regions of epithelial folds (Fig. 6A”). The epithelial cells of imaginal discs are highly polarized and are interconnected by a junctional complex on the lateral membrane of these cells. From apical to basal, the junction consists of a marginal zone (MZ), adherens junction (AJ) and septate junction (SJ) (Fig. 6B) (reviewed in Badouel and McNeill, 2009; Tepass et al., 2001). The center of a fold in the epithelium represents the juxtaposition of two apical surfaces, with lateral junctions spreading basally on both sides of the fold (schematic drawing in Fig. 6C). Thus, the high expression of Sqh2P in the folds likely reflects a strong apical localization in this tissue. Sqh1P then, appears to have a more basal localization in these cells, but does appear to localize to the apical region of the lateral membrane. In addition, due to the folding of the wing disc, the basal surfaces of imaginal disc cells can be observed in other folds (asterisk in Fig. 6A). In this fold, Sqh1P is expressed at high levels on the basal surface, whereas Sqh2P is barely detectable (Fig. 6A). Note the similarity of the apical and basal expression of Sqh1P in this tissue to that observed in the embryonic epithelia (Fig. 3E and F).

Fig. 6
Sqh1P and Sqh2P have distinct subcellular localizations in wing imaginal discs

To confirm the differences in subcellular localizations between Sqh1P and Sqh2P on the apical surface of these cells, we examined a confocal Z-series through the depth of an imaginal disc. An XZ projection representing a sagittal cross section through the disc (schematic drawing shown in Fig. 6E) confirms that Sqh2P is more apically localized than Sqh1P. In addition, this sagittal cross section reveals that Sqh2P is expressed in all cells in the imaginal epithelium, although it is more highly expressed in the apically constricted cells lining the fold (arrows in Fig. 6D”). Note the similarity in the expression of Sqh2P in the imaginal disc folds to that of Sqh2P in the cells of the segmental boundary during embryogenesis (Fig. 4D).

To more firmly define the subcellular localization of Sqh1P and Sqh2P in imaginal discs, we co-stained wing imaginal discs with Sqh1P and Sqh2P and different cellular markers of the lateral membrane. For these experiments we used the septate junction marker Coracle (Cor; Fehon et al., 1994), the adherens junction marker Armadillo (Arm; Peifer and Wieschaus, 1990), and marginal zone maker Crb (Tepass et al., 1990). We found that Sqh1P localizes more apical than Cor, mainly co-localizes with Arm and partially co-localizes with Crb (Fig. 7A–C). In contrast, Sqh2P localizes more apically than both Cor and Arm (Fig. 8B–C). The distribution of Sqh2P appears to primarily co-localize with Crb, although Crb has a punctate expression in the folds consistent with a marginal zone localization, whereas Sqh2P shows a continuous distribution at the apical ends of these cells, indicating that it is even more apically localized than Crb (Fig. 8A). Thus, Sqh1P mainly localizes to adherens junction with some expression in the marginal zone (Fig. 7D), whereas Sqh2P mainly localizes in the marginal zone and the apical plasma membrane (Fig. 8D).

Fig. 7
Sqh1P localizes primarily to the adherens junction in wing imaginal disc cells
Fig. 8
Sqh2P localizes primarily to the apical surface of wing imaginal disc cells

One of the most interesting observations made with these antibodies is that the different phosphorylated forms of Sqh show distinct subcellular localizations in Drosophila embryos and imaginal discs. Studies in cultured cells have described similar differential subcellular localizations of the monophosphorylated and diphosphorylated forms of MRLC. In resting rat aortic smooth muscle cells, diphosphorylated MRLC was found only on the peripheral regions of the cells, whereas monophosphorylated MRLC was more strongly expressed along fibrillary structures that ran along the long axis of these cells (Sakurada et al., 1998). In motile HeLa cells, the monophosphorylated form of MRLC was found in the cell periphery as well as at the leading edge of the motile cells, whereas the diphosphorylated form was concentrated at the base of the leading edge in a region that was sprouting filopodia, likely associated with focal adhesion complexes (Uchimura et al., 2002). Here we show for the first time that the monophosphorylated and diphosphorylated forms of Sqh also have distinct subcellular localizations in Drosophila cells in an intact organism. In all the Drosophila tissues we observed, both total Sqh and Sqh1P predominantly localizes to the adherens junction, consistent with a role for Sqh in regulating myosin on the cortical actin cytoskeleton. These observations suggest that there is a pool of Sqh in these junctions that is in an equilibrium between nonphosphorylated and monophosphorylated. Likely, cycles of phosphorylation and dephosphorylation occur in situ at the adherens junction to maintain this equilibrium. Sqh1P also localizes to the basal surface of epithelial cells, again consistent with a localization with filamentous actin, and suggesting a pool of Sqh that might be involved in focal adhesion. In contrast, Sqh2P specifically localizes to the very apical surface and in filopodia of imaginal disc cells and a subset of ectodermally-derived epithelial cells in the embryo.

What could account for the different subcellular distribution of Sqh1P and Sqh2P in these tissues? There are at least two, non exclusive possibilities. First, this distribution could be controlled primarily by localized phosphatases. In vitro, phosphorylation of Ser19 on MRLC (Ser21 in Sqh) by myosin light chain kinase (MLCK) occurs 500-fold more readily than phosphorylation on Thr18 (Thr20 in Sqh) (Ikebe and Hartshorne, 1985; Ikebe et al., 1986), suggesting that the equilibrium point in the cell is likely between nonphosphorylated and monophosphorylated, consistent with our observations on western blots from total protein extracts from imaginal discs (Fig. 1). Sakurada et al. (1998) reported that treatment with the protein phosphatase inhibitor, calyculin A, strongly induced diphosphorylation of MRLC in cultured SM-3 smooth muscle cells, arguing that the equilibrium in these cells was being largely regulated by phosphatase activity. Thus an adherens junction localized phosphatase could easily shift the equilibrium point away from diphosphorylated Sqh in the adherens junction, leaving Sqh2P to be found only in places that lack concentration of this phosphatase. In support of this idea is work showing that the myosin binding subunit of both vertebrate and Drosophila myosin phosphatase (MYPT) localizes to the adherens junction (Inagaki et al., 1997; Mitonaka et al., 2007). An alternative hypothesis, however, argues that there are unique kinases that show specific subcellular localizations and have a high activity on Sqh that can thereby phosphorylate Sqh mainly to Sqh2P in these locations (Ikebe and Hartshorne, 1985; Ikebe et al., 1986; Ueda et al., 2002). In support of this idea, Sakurada et al. (1998) observed that diphosphorylation of MRLC in rabbit aortic smooth muscle cells is more sensitive to certain protein kinase inhibitors than others, suggesting that different kinases can have differential activities on the phosphorylation state of MRLC (and by extension, Sqh). In addition, MLCK has been shown to have actin binding activity in vertebrate cell, which may account for the higher concentration of diphosphorylated MRLC in actin rich surface features such as lamellipodia and filopodia (Uchimura et al., 2002). There are more than a dozen kinases that have been reported to phosphorylate MRLC (Vicente-Manzanares et al., 2009), but at this point there are no studies showing specific localization of kinases able to phosphorylate Sqh at the apical plasma membrane in Drosophila. Additional studies will be required to address these ideas. For example, if this equilibrium is predominantly maintained by the activity of myosin phosphatase in the adherens junction, then imaginal disc clones of cells mutant for myosin phosphatase should have an upregulation of Sqh2P at the adherens junction.

A potentially more interesting question is what are the functional differences of myosin complexes containing predominately Sqh1P or Sqh2P. As part of the nonmuscle myosin II complex, Sqh serves as the regulatory light chain, affecting the actin-dependent ATPase activity of the complex. Through this activity, myosin can exert motile force along actin filaments. It is well described that phosphorylation of Ser19 on MRLC (Ser21 in Sqh) can greatly increase the actin-dependent ATPase activity of myosin (Ikebe et al., 1988), and that subsequent phosphorylation of Thr18 (Thr20 in Sqh) can further increase this activity (Ikebe and Hartshorne, 1985; Ikebe et al., 1986; Ikebe et al., 1988). The localization of Sqh1P primarily to the adherens junction, a region where filamentous actin bands encircle the cells, suggests that Sqh1P exerts a role primarily in force generation. This activity would be necessary for simple cell shape changes, including apical cell constrictions. The localization of Sqh1P at the leading edge of DME cells during dorsal closure is an excellent example of how this function might work. Previous studies have shown that F-actin forms a continuous cable at the leading edge of the DME cells (Franke et al., 2005; Jacinto et al., 2000; Jacinto et al., 2002a; Jacinto et al., 2002b). The myosin heavy chain (Zip) localizes in a “bars-on-a-string” distribution along this actin cable (Franke et al., 2005; Young et al., 1993), identical to what we observed with Sqh1P (Fig. 5). The colocalization of Sqh1P, Zip and actin would then be capable of generating coordinated contractile forces that would close the epidermis around the dorsal hole analogous to the closure of a purse string, and would additionally contribute to the maintenance of a uniform migration front as dorsal closure proceeds (Jacinto et al., 2002a; Kiehart et al., 2000).

What then might be the function of Sqh2P in Drosophila cells? The tissue distribution of Sqh2P may shed some light on this question. During embryogenesis, Sqh2P is strongly expressed primarily in cells that are actively moving, including a number of invaginating tissues such as the salivary glands, the tracheal system, the stomodeum and proctodeum, as well as tissues that coordinate the migration of epithelial sheets including the DME cells during dorsal closure and the dorsal pouch during head involution. The dorsal pouch originates from the most dorsal anterior region of the procephalon and is among the first tissues to involute during head involution. In subsequent stages, the remaining head segments are drawn into the embryo under the dorsal pouch while the dorsal epidermis, led by the dorsal ridge, migrate over the dorsal pouch (Nassif et al., 1998). It is likely that all of these cells that express higher levels of Sqh2P are not typical polarized epithelia, but rather have taken on a pseudo-mesenchymal behavior. As such, the plasma membrane, and in particular the actin cytoskeleton, is more dynamic, producing extensive filopodia and lamellipodia. Jacinto et al. (2000) reported that these filopodial protrusions are remarkably dynamic during dorsal closure, and may play an important role in scanning the opposing epithelial cells and for dragging the contralateral cell sheets together. Similarly, it has been shown that the apical regions of imaginal disc cells have long filopodial extensions, and that the disc proper cells are in contact with the peripodial epithelium through these actin-rich membrane extensions (Demontis and Dahmann, 2007; Gibson and Schubiger, 2000). Since these structures are actin-rich surface protrusions, Sqh2P may act as part of a myosin motor carrying cargo to and from the tips of these structures, and as such may play important roles in building these structures and for signaling events occurring in them. There is precedence for myosin activity in filopodial structures. Tokuo et al. (2007) showed that myosin X is crucial for actin reorganization leading to formation of filopodia in African green monkey kidney COS7 cells and NIH3T3 fibroblasts. Myosin X also plays an important role in elongation of filopodia (Berg and Cheney, 2002; Tokuo and Ikebe, 2004). There is no homolog of myosin X in Drosophila (Berg and Cheney, 2002), however. The function of myosin X might therefore be carried out by nonmuscle myosin II in conjunction with Sqh2P. Finally, it is also noteworthy that Sqh2P is first upregulated in the DME cells during the initiation phase of dorsal closure, before migration begins. It is possible that the expression of a Sqh2P-containing myosin complex is a necessary early step in this transition to a pseudo-mesenchymal migratory tissue. The early expression of Sqh2P in the tracheal placodes prior to invagination supports this idea. Finally, although Sqh2P containing myosin complexes may be important for proper control of these cellular processes and developmental events, it appears that it is not strictly essential. Jordan and Karess (1997) used a sqh transgene coding for a nonphosphorylatable Ala in place of Thr20 and found that it could rescue the sqhAX3 null allele to viability. Perhaps there was an increase in the amount of Sqh1P that could compensate for the loss of Sqh2P. Further experiments will need to determine if dorsal closure, tracheal morphogenesis and head involution are less well coordinated or fail in a certain percentage of animals that cannot expresses Sqh2P.

With these antibodies we now have the tools that will enable us to explore a host of cell biological and developmental questions. An antibody directed against the monophosphorylated form of MRLC cross-reacts with Sqh (Matsumura et al., 1998), and has been used to examine myosin activation in a number of developmental systems including planer cell polarity in imaginal discs and the formation and movement of the morphogenetic furrow during adult eye development (Corrigall et al., 2007; Escudero et al., 2007; Winter et al., 2001). With these antibodies, we now have better matched antibodies against both phosphorylated forms of Sqh and thus can investigate a wide range of morphogenetic processes that ultimately require cell shape changes mediated through myosin activation. It will be particularly interesting to dissect the Rho signaling pathway during embryogenesis, as zygotic loss of Rho1 specifically affect processes involving tissues that normally express high levels of Sqh2P including dorsal closure and head involution (Magie et al., 1999).

2. Experimental Procedures

2.1. Drosophila strains

yw cv sqhAX3; P{sqh-GFP} and yw, sqhAX3, FRT101/FM7C fly lines were generous gifts from Roger Karess (CNRS, France; Jordan and Karess, 1997). Other stocks were obtained from Bloomington Drosophila Stock Center (Bloomington, IN). w1118 was used as the wild type strain for the experiments conducted in this study. All Drosophila stocks were maintained on media consisting of corn meal, sugar, yeast, and agar in incubators maintained at a constant temperature of 21°C or in a room that typically fluctuated between 21°C and 22.5°C. Genetic experiments were conducted in incubators controlled at a constant temperature of 25°C.

2.2. Generation of antibodies

To generate phosphorylation site-specific antibodies, two peptides were commercially synthesized corresponding to regions of the Sqh protein with phosphorylated derivatives at Ser21 or Thr20/Ser21. The sequences of the peptides used were Sqh1P: A-Q-R-A-T-S*-N-V-F-A-M-C and Sqh2P: A-Q-R-A-T*-S*-N-V-F-A-M-C (* indicates phosphorylated amino acid). The cysteine at the C-terminus of each peptide was added for the conjugation of these peptides to Keyhole limpet hemocyanin (KLH). After conjugation to KLH, the peptides were mixed with Freund’s complete adjuvant and used to immunize animals. The Sqh1P peptide was synthesized at Pocono Rabbit Farm and Laboratory Inc. (PRF&L, Canadensis, PA) and used to immunize guinea pigs. The Sqh2P peptide was synthesized at the Immunological Resource Center at the University of Illinois and used to immunize rats. The animals were boosted according to standard protocols until strong immunoreactivity was observed on immunoblots and on tissue stains. The guinea pig anti-Sqh1P serum was subsequently affinity purified at PRF&L. Antibodies specific for full-length Sqh are described in Wang and Ward (2010). It should be noted that a commercially available antibody to the monophosphorylated version of vertebrate MRLC differs slightly from the peptide we used for Sqh1P (vertebrate sequence: R-P-Q-R-A-T-S*-N-V-F-A-C) (Matsumura et al., 1998). We have compared this antibody to the one we generated and found essentially no differences (data not shown).

2.3. Gel electrophoreses and immunoblotting

SDS PAGE was performed according to standard procedures (Laemmli, 1970). ~25 imaginal discs from wandering 3rd instar larvae were hand dissected in cold fresh PBS. The discs were collected in 1.5ml centrifuge tubes and homogenized in 1× SDS-PAGE sample buffer (~1 wing disc per µl buffer) and boiled for 5 min. 20µl samples were loaded in each lane of 12% polyacrylamide gels and eletrophoresed at 120 V. The proteins on the gel were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA) overnight at 30V or for 1 h at 100V at 4°C in transfer buffer (25mM Tris, 192mM Glycine and 20% methanol) followed by immunoblotting as described below.

Phosphatase treatment of protein samples was done by homogenizing imaginal discs in 1×PBS with protease inhibitors (0.1mM PMSF, 10µM leupeptin) (~1.5wing discs/µl PBS), followed by treatment of the disc lysates with 0.025unit/µl protein phosphatase 1(PP1) (New England Biolabs, Ipswich, MA) for the indicated times.

Urea-glycerol-PAGE and immunoblotting was adapted from Seto et al. (Seto et al., 1990). Imaginal discs from wondering 3rd instar larvae were hand dissected in fresh PBS. ~20 wing discs plus associated leg and halter discs were homogenized in 20µl urea sample buffer (20mM Tris pH 8.6, 22 mM glycine, 10mM DTT, 8M urea, 0.1% bromophenol blue). The glycerol-polyacrylamide gels contained 20mM Tris pH 8.6, 23mM glycine, 40% glycerol, 15% acrylamide and 0.75% Bis. The reservoir buffer consisted of 20mM Tris pH 8.6, 23mM glycine, 1mM sodium thioglycolate, and 1mM DTT. The gels were pre-electrophoresed for 1 h at 400V in 4°C. The disc samples were then loaded onto the gel and eletrophoresed for 5 h at 400V in 4°C. The proteins on the gel were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA) overnight at 30V (4°C) in transfer buffer containing 20mM Tris (pH 7.5) and 20% methanol followed by immunoblotting as described below.

Immunoblotting of all blots were blocked in 1XTBS (0.1M NaCl, 0.02M Tris, pH7.5~7.6) plus 0.1% Tween20 and 5% nonfat dry milk (blocking buffer) for 1 h in room temperature, then incubated with primary antibodies in blocking buffer overnight at 4°C. After three quick rinses and three 15-min washes with 1XTBS plus 0.1% Tween20, the blots were incubated with HRP-conjugated secondary antibodies in blocking buffer for 2–3 h at room temperature. After three quick rinses and three 15-min washes with 1XTBS plus 0.1% Tween20, the blots were then developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL) for 1 min. Chemiluminescent signals were detected on a KODAC imaging station or exposed to X-ray film, developed and scanned.

The following antibodies were used at indicated dilutions: mouse anti-Sqh (1:5000); affinity purified guinea pig anti-Sqh1P (1:1000); rat anti-Sqh1P (1:3000); mouse anti-Coracle (1:1000) (Fehon et al., 1994). HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) were used at 1:5000.

2.4. Immunohistochemistry

Imaginal discs were hand dissected from wandering 3rd instar larvae or prepupae in fresh PBS, and fixed immediately in 4% paraformaldehyde for 20 min. For embryos, we tested three different fixation methods, as well as combinations of the three. We found that 4% paraformaldehyde fixation for 20 min at room temperature (Fehon et al., 1994) preserved the tissue morphology and gave the best staining for all three antibodies. 10% ice-cold trichloroacetic acid fixation for 20 minutes appeared to preserve dynamic membrane structures well, and preserved the epitope for Sqh2P, possibly owing to its ability to inhibit phosphatases (Hayashi et al., 1999), but did not permit efficient staining of Sqh or Sqh1P. Similarly, neither Sqh, Sqh1P nor Sqh2P stained well in embryos fixed using a heat-methanol procedure (attempted for staining gastrulating embryos; Muller and Wieschaus, 1996). Therefore, all of the embryos shown here were fixed using 4% paraformaldehyde for 20 minutes. The following antibodies were used at the given dilutions: mouse anti-Sqh 1:3000; guinea pig anti-Sqh1P 1:1000 (or affinity purified guinea pig anti-Sqh1P 1:500); rat anti-Sqh2P 1:2000; mouse anti-Crumbs 1:100 (clone Cq4 from Developmental Studies Hybridoma Bank at the University of Iowa); mouse anti-Armadillo 1:100 (clone N27A1, DSHB); mouse anti-Coracle 1:400 (gift from Richard Fehon, University of Chicago; Fehon et al., 1994). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and used at 1:400. Confocal images were acquired on a Zeiss LSM510 Meta Laser Scanning Confocal Microscope (Carl Zeiss Inc, Thornwood NY). Photomicrographs were adjusted for brightness and contrast with the Adobe Photoshop (version CS3, San Jose, CA), and Figures were complied in Adobe Illustrator (version CS3, San Jose, CA).

2.5. Mosaic clones

The FLP/FRT system (Xu and Rubin, 1993) was used to generate sqhAX3 homozygous mutant clones in the ovary. To generate sqh clones, yw, Ubi-GFP, FRT101/Y; hsFLP/+ males were crossed to yw, sqhAX3, FRT101/FM7C virgin females. Then, yw, ubiGFP, FRT101/yw, sqhAX3, FRT101; hsPLF/+ 2–3 day old females were selected and heat shocked at 37°C for 1 h. Ovaries were dissected 24 h after heat shock, and fixed and stained as described above. Homozygous sqh mutant cells are marked by the absence of ubiquitous GFP.

Supplementary Material

01

Supplemental Fig. 1 Sqh2P antibody shows nonspecific staining in the lumen of late stage embryos:

(A) Confocal optical section of a stage 16 wild type embryo stained with antibodies 36 against Sqh2P (red and as single channel in A’) and Uif (green and as single channel in A”). Note that the Sqh2P antibody recognizes an epitope in the lumen of the salivary gland (asterisk), but is barely detectable on the apical surface of the tracheal cells (arrows). Sqh2P is found in punctate structures in the tracheal cells (arrowheads).

Acknowledgments

We thank Carl Thummel (University of Utah), in whose lab antibodies against Sqh2P were generated. We thank Michelle Wickersheim and Shannon Stewart for technical help with the testing of the Sqh and Sqh1P sera, and Courtney Bone for technical help with fixing and mounting gastrulating embryos. We thank the developmental studies hybridoma bank for antibodies used in this study. We thank Roger Karess for the sqhAX3 strains used in these studies. We thank Brooke McCartney and Shi Mulinari for insightful discussions about the study. The project was supported by NIH Grant Number P20 RR15563 and NIH Grant Number R01HD047570 from the National Center for Research Resources.

Footnotes

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