BIOLOGICAL OVERVIEW

Myosins are a functionally divergent group of molecular motors that are involved in various non-muscle cell motile activities. Zipper, hence referred to as Myosin II, or non-muscle myosin, is crucial to the viability of the Drosophila embryo throughout its development. The clearest example of nonmuscle-driven shape change is cytokinesis, in which an actin and myosin-rich contractile ring cleaves the cell during mitosis. Null alleles of zipper coding for the non-muscle heavy chain cause failure of dorsal closure, during which the lateral epidermal cells elongate to cover the dorsal surface of the embryo after germ band retraction. Axon pathfinding and head involution defects also contribute to the embryonic lethality of zipper mutants. Early dorsal closure is normal in these mutants, suggesting that maternal coded Zipper protein is sufficient for early stages of closure. By late closure however, nonmuscle myosin is either improperly localized in or absent from the leading edge of the lateral epithelium. By the time zip mutant embryos arrest during late dorsal closure stages, the leading edge is dramatically disorganized, the lateral epidermis is not fused along the dorsal midline, and the amnioserosa remains exposed. In normal flies, dorsal closure is accompanied by a dramatic cell shape change as the entire epidermal cell sheet thins out to spread over the amnioserosa. As dorsal closure proceeds, more and more cells throughout the epidermis are elongated. This change in epidermal cell shape, driven for the most part by maternal myosin II, is sufficient to account for the spreading of the lateral epithelium over the region occupied by the amnioserosa (Young, 1993).

Nonmuscle myosin II is also required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos. Developing oocytes containing germline mutant spaghetti squash(sqh) clones fail to attain full suze due to a defect in 'dumping,' the rapid phase of cytoplasmic transport in nurse cells. Spaghetti squash is the regulatory light chain of nonmuscle myosin II; along with the Essential light chain, it acts to regulate the function of Myosin II. Two subunits of the regulatory light chain, along with two subunits of the essential light chain, each combine with two subunits of Myosin II to form the functional Myosin II hexamer. spaghetti squash mutant egg chambers show no evidence of ring canal obstruction, and no obvious alteration in the actin network. However, the distribution of myosin II is abnormal. It is thought that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively by nurse cell myosin II. Regulation of myosin activity is one means by which cytoplasmic transport is controlled during oocyte development. Fertilized eggs developed from spaghetti squash maternal mutant clones begin development by exhibiting an early defect in axial migration of cleavage nuclei toward the posterior pole of the embryo. This is similar to development seen in early cleavage zygotes, in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration of the zygotic nuclei require maternally supplied myosin II (Wheatley, 1995).

In order to study the role of myosin II during imaginal disc development, spaghetti squash was put under control of a heat shock promoter and induced at various times in sqh mutants. sqh mutants can be rescued to adulthood by daily induction of sqh from a hsp 70 promoter. When SQH is transiently depleted in larvae, the resulting adult phenotypes demonstrate that SQH is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When SQH is depleted in adult females, oogenesis is reversibly disrupted. Without SQH induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping) (Edwards, 1996).

Normally at stage 9 of oogenesis, approximately eight so-called border cells form at the anterior tip of the egg chamber, delaminate, and migrate past the phalanx of nurse cells until they reach the anterior tip of the oocyte. This migration of border cells between and through the nurse cells is abnormal in sqh mutants. Normal border cell myosin II staining is much brighter than that of the surrounding nurse cells, indicating a presence of myosin II in border cells. Centripetal cells, a second morphologically distinct type of follicle cell, is derived from the most anterior ring of oocyte follicle cells. Centripetal cells normally elongate and plunge inward toward the border cells, which have come to rest at the anterior face of the oocyte. These centripetal cells will eventually cover the anterior of the oocyte and build several specialized structures of the anterior chorion. Each centripetal cell specifically accumulates a bright bar of myosin II staining at the edge of the apical (inner) surface that leads the penetration between the nurse cells and the oocyte (Edwards, 1996).

sqh mutant ovaries also display major defects in migration of centripetal cells. After three days without SQH, centripetal cells fail to elongate. Follicle cells that construct the dorsal appendages also fail to migrate properly as the supply of SQH dwindles and eventually is depleted. A shortage of SQH in migrating cells appears to be the primary cause of the shortened dorsal appendage phenotype found in sqh mutants (Edwards, 1996).

In sqh mutant tissues, Myosin II heavy chain is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail binding protein p127. p127 is coded for by the gene lethal (2) giant larvae tumor suppressor gene. In follicle cells, p127 is normally concentrated at the lateral membranes. In sqh mutant tissue, p127 shows the same pattern, with no punctate cytoplasmic staining. Likewise F-actin is not mislocalized in sqh mutants. Thus sqh mutations cause mislocalization of myosin, but not the other major cytoskeletal proteins (Edwards, 1996).

Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila

Diverse types of epithelial morphogenesis drive development. Similar cytoskeletal and cell adhesion machinery orchestrate these changes, but it is unclear how distinct tissue types are produced. Thus, it is important to define and compare different types of morphogenesis. Cell flattening and elongation were investigated in the amnioserosa, a squamous epithelium formed at Drosophila gastrulation. Amnioserosa cells are initially columnar. Remarkably, they flatten and elongate autonomously by perpendicularly rotating the microtubule cytoskeleton - this is called 'rotary cell elongation'. Apical microtubule protrusion appears to initiate the rotation and microtubule inhibition perturbs the process. F-actin restrains and helps orient the microtubule protrusions. As amnioserosa cells elongate, they maintain their original cell-cell contacts and develop planar polarity. Myosin II localizes to anterior-posterior contacts, while the polarity protein Bazooka (PAR-3) localizes to dorsoventral contacts. Genetic analysis revealed that Myosin II and Bazooka cooperate to properly position adherens junctions. These results identify a specific cellular mechanism of squamous tissue morphogenesis and molecular interactions involved (Pope, 2008).

Amnioserosa tissue morphogenesis involves dramatic cell shape change. Before amnioserosa morphogenesis, cells are columnar with lateral MT bundles in a basket-like array along the apicobasal axis. With amnioserosa morphogenesis, the cells elongate and flatten. Amnioserosa cells could change shape by symmetrically re-positioning cellular contents (full cytoskeleton and/or membrane reorganization) or by perpendicularly rotating cellular components to reorient the long axis of the cell into the plane of tissue extension. To distinguish these possibilities, 3D cell organization was studied over time; it was discovered that the MT array plus the nucleus, centrosomes and ER rotate, apparently as a unit, into the plane of tissue extension. More symmetric adherens junction (AJ) and cortical reorganizations appear to accompany the rotation. MT arrays also reorient to polarize cells during chemotaxis and tissue migration, and to reposition cell contents as occurs during cortical rotation in early Xenopus embryos. These results reveal rotation of the MT cytoskeleton linked to cell shape change and amnioserosa morphogenesis. Similar mechanisms may underlie the development of other squamous epithelial monolayers (Pope, 2008).

Regulated apical MT protrusion appears to initiate amnioserosa rotary cell elongation. MT inhibition perturbs initial elongation, and the process normally begins with MTs protruding into the apical domain, bending perpendicularly and then extending in the axis of cell elongation. Pre-existing lateral MT bundles appear to protrude across the apical domain - they are mainly non-centrosomal and contain older (acetylated) MTs. However, EB1-GFP imaging also revealed bi-directional MT growth across the apical domain. This indicates that the bundles are dynamic, but argues against MT bundle protrusion through polarized individual MT polymerization. Instead, MT bundle protrusion may involve greater net renewal of bundles apically versus basally, motors sliding MTs past MTs in the bundles and/or motors moving bundles along the cell cortex. Distinguishing these models requires further study. As MTs extend apically the actin cytoskeleton appears to inhibit them, as weakening actin leads to excessively long and randomly oriented MT-based protrusions. Actin is normally found around the full apical circumference as amnioserosa cells elongate. By contrast, Myosin II becomes enriched at AP contacts and is gradually lost from the full cell cortex. Thus, different pools of actin may regulate MT protrusion. It is speculated that the gradual overall loss of cortical actin-myosin complexes permits, and may help orient, regulated MT protrusion. Actin also antagonizes cortical MTs in other systems. MT-based primary axons form where cortical actin is weakest. Actin inhibits cortical MT protrusion in neutrophils and Myosin IIA inhibits cortical MTs in mammalian cells. Actin might physically block MT protrusion, but direct or indirect molecular interactions may also be involved (Pope, 2008).

In Drosophila embryos, MT-actin interactions also affect germband cells. At stage 7-8, actin disruption enhances AJ planar polarity at DV contacts. MT disruption suppresses this, suggesting that actin inhibits MT-based AJ positioning in these cells - however, germband cells show minimal shape change with actin disruption at this stage. Remarkably, the same actin disruption causes stage 9-10 germband cells to rotate analogously to early amnioserosa cells. Their apical domains elongate and their lateral regions rotate perpendicularly, becoming exposed to the embryo surface. Implicating MTs in this change, lateral MT bundles run into the extended apical domains and simultaneous MT disruption suppresses the cell shape change. Thus, actin may inhibit apical MTs to regulate tissue structure in many parts of the embryo. This MT inhibition may also involve coordination with AJs, as disrupted germband cells in arm^m/z mutants also display MTs protruding into extended apical domains (Pope, 2008).

How do MTs elongate the apical domain and how is this linked to the rotation of the full MT cytoskeleton in the amnioserosa? It is proposed that rotary cell elongation occurs in two phases. In phase one, the MT imaging and inhibitor studies indicate that regulated MT protrusion elongates the apical domain. This may involve a combination of physical force, membrane delivery and/or relaxation of actin-myosin contractility. The AJ clustering observed at abnormal apical MT protrusions formed with actin inhibition in both early amnioserosa cells and the later germband suggests that MTs may apply force to AJs. Consistent with this idea, MT inhibition affected both initial amnioserosa cell elongation and later amnioserosa cell-cell interactions. However, amnioserosa cell elongation in arm^m/z mutants suggests that MTs may not necessarily engage AJs directly. Since Baz localizes apically in early arm^m/z mutants, and is enriched at DV amnioserosa cell contacts to which MTs rotate in wild type, it is a strong candidate for coordinating these interactions. However, severe early defects in baz^m/z mutants would confound analysis of amnioserosa development - this may require conditional mutants. Phase two of rotary cell elongation requires full perpendicular rotation of the MT array, apical and basal membrane growth, and lateral membrane removal. Although it is unclear how full rotation occurs, MT rotation and cortical remodeling may occur in concert. For example, membrane remodeling may explain how amnioserosa cells remain elongated with MT disruption during later development (Pope, 2008).

For rotary cell elongation to translate into tissue extension, cell contacts and AJs must be remodeled. Remarkably, amnioserosa cells maintain their neighbor relationships as they elongate, and two contact types develop; highly elongated AP contacts and lesser elongated DV contacts. Each appears to involve unique AJ remodeling. Intriguingly, Myosin II and Baz localize to AP and DV contacts, respectively -- the same reciprocal planar polarized relationship displayed in the germband. In the amnioserosa, Myosin II and Baz synergize to control overall AJ positioning, a regulatory interaction that has not been shown elsewhere (Pope, 2008).

Myosin II and Baz may regulate specific AJ remodeling events occurring at AP and DV contacts, respectively. Amnioserosa cells increase their apical circumference 10-fold, initially doubling the length of their AP cell contacts every 5-10 minutes. Remarkably, AJs localize around the full circumference as this occurs. This contrasts elongating Drosophila follicle cells, which lose AJ continuity, suggesting specific mechanisms for maintaining AJ continuity during amnioserosa morphogenesis. amnioserosa AJs do lose continuity with actin disruption, suggesting a role for actin. More specifically, AJ fragmentation in baz zip double mutants suggests a role for Myosin II. In the neighboring ventral furrow and germband, actin-myosin contractility is coupled to AJs during apical constriction and cell intercalation, respectively. The actin-myosin complexes enriched along amnioserosa AP contacts may also be contractile, but here they may counterbalance MT protrusion. Slowing apical elongation may indirectly allow AJ remodeling. However, Myosin II may also have direct affects on AJs (Pope, 2008).

Baz may regulate distinct AJ re-modeling at DV contacts. It is hypothesized that MT protrusion applies force to the DV contact at the cell 'front', and that cell elongation may also pull the 'rear' contact. Either force could detach AJs and necessitate AJ remodeling. Dynamic looping of DE-CadGFP and ArmCFP was observed at D-V contacts, and BazGFP partly colocalized with these loops. Although further experiments (e.g., photobleaching) are needed to understand this and other amnioserosa AJ remodeling, Baz localization at DV contacts and abnormal AJ aggregation at DV contacts in baz zip double mutants suggests a role for Baz in AJ remodeling at these sites. Baz appears to interact with MTs and Dynein to initially position AJs during Drosophila cellularization, and Baz might re-position AJs at DV amnioserosa contacts in a similar way (Pope, 2008).

In four different cases, cells were observed elongating towards potential sources of pulling forces. First, wild-type amnioserosa cells elongate along the DV axis towards the germband (potential source of DV pulling forces during convergent extension) and the ventral furrow (potential source of DV pulling forces during invagination). Second, amnioserosa cells elongate along the DV axis of bcd nos tsl mutants, in which germband extension fails, but ventral furrow formation occurs. Third, in dl mutants in which the ventral furrow does not form and the amnioserosa forms a ring around the DV axis, amnioserosa cells reoriented along the AP axis towards ectopic contractile furrows. Fourth, in the stage 9-11 wild-type germband, cells artificially induced to flatten and elongate did so in coordinated groups oriented towards contractile regions of the germband. Thus, polarized pulling forces across a tissue may orient rotary cell elongation. In wild-type embryos, these forces may come from germband extension and/or ventral furrow formation. However, Zen must first trigger the amnioserosa cell shape change, while AP patterning may specifically regulate AJ remodeling (Pope, 2008).

GENE STRUCTURE

The nonmuscle myosin heavy chain from Drosophila contains an alternatively spliced exon at the 5' end which generates two distinct heavy-chain transcripts: the longer transcript inserts an additional start codon upstream of the primary translation start site and encodes a myosin heavy chain with a 45-residue extension at its amino terminus. The shorter transcript is 3.5 times more abundant than the longer one (Ketchum, 1990).

A second alternative exon (40aa) is close to the nucleotide binding pocket. The position, size and sequence of this exon is conserved in D. simulans and putative alternative exons of different size (7 to 16 aa), but identical positions have been reported for other myosins in many phyla. The functional significance of either alternative splice is not clear (Mansfield, 1996)

Exons - 14

Bases in 3' UTR - 188

PROTEIN STRUCTURE

Amino Acids - 2057

Structural Domains

The coding sequence of zipper reveals extensive homology with other conventional myosins, especially metazoan nonmuscle and smooth muscle myosin isoforms. Comparisons among available myosin heavy-chain sequences establish that characteristic differences in sequence throughout the length of both the globular myosin head and extended rod-like tail readily distinguish nonmuscle and smooth muscle myosins from striated muscle isoform,s and predict a basis for their functional diversity. The ATP binding region is in the first 200 amino acids. The actin binding region is within 30 amino acids centered on amino acid 670. The myosin tail includes the 1085 residues from leucine-843 to leucine-1927 and is characterized by a strong heptad repeat, common to conventional myosin tails and other alpha-helical coiled-coil proteins. These regions dimerize and fold into the extended rod-like tail of the native Drosophila myosin molecule. The hinge region is in the center of the tail sequence. The terminal 47 amino acids consist of the globular tail region. The tail region contains putative casein kinase II and protein kinase C phosphorylation sites (Ketchum, 1990 and Mansfield, 1996).

date revised:  22 Dec 96

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