Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos

Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. Here we show that the Drosophila myosin XV homolog, Sisyphus, is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. We have identified several putative Sisyphus cargos, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. Our data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure.     

Wound healing and inflammation: embryos reveal the way to perfect repair

Tissue repair in embryos is rapid, efficient and perfect and does not leave a scar, an ability that is lost as development proceeds. Whereas adult wound keratinocytes crawl forwards over the exposed substratum to close the gap, a wound in the embryonic epidermis is closed by contraction of a rapidly assembled actin purse string. Blocking assembly of this cable in chick and mouse embryos, by drugs or by inactivation of the small GTPase Rho, severely hinders the re-epithelialization process. Live studies of epithelial repair in GFP-actin-expressing Drosophila embryos reveal actin-rich filopodia associated with the cable, and although these protrusions from leading edge cells appear to play little role in epithelial migration, they are essential for final zippering of the wound edges together-inactivation of Cdc42 prevents their assembly and blocks the final adhesion step. This wound re-epithelialization machinery appears to recapitulate that used during naturally occurring morphogenetic episodes as typified by Drosophila dorsal closure. One key difference between embryonic and adult repair, which may explain why one heals perfectly and the other scars, is the presence of an inflammatory response at sites of adult repair where there is none in the embryo. Our studies of repair in the PU. 1 null mouse, which is genetically incapable of raising an inflammatory response, show that inflammation may indeed be partly responsible for scarring, and our genetic studies of inflammation in zebrafish (Danio rerio) larvae suggest routes to identifying gene targets for therapeutically modulating the recruitment of inflammatory cells and thus improving adult healing.     

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.     

Dynamic analysis of actin cable function during Drosophila dorsal closure

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. Here, we test the function of this cable by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. We show that the cable serves a dual role during dorsal closure. It is contractile and thus can operate as a "purse string," but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila

Studies in cultured cells and in vitro have identified many actin regulators and begun to define their mechanisms of action. Among these are Enabled (Ena)/VASP proteins, anti-Capping proteins that influence fibroblast migration, growth cone motility, and keratinocyte cell adhesion in vitro. However, partially redundant family members in mammals and maternal Ena contribution in Drosophila previously prevented assessment of the roles of Ena/VASP proteins in embryonic morphogenesis in flies or mammals. We used several approaches to remove maternal and zygotic Ena function, allowing us to address this question. We found that inactivating Ena does not disrupt cell adhesion or epithelial organization, suggesting its role in these processes is cell type-specific. However, Ena plays an important role in many morphogenetic events, including germband retraction, segmental groove retraction and head involution, whereas it is dispensable for other morphogenetic movements. We focused on dorsal closure, analyzing mechanisms by which Ena acts. Ena modulates filopodial number and length, thus influencing the speed of epithelial zippering and the ability of cells to match with correct neighbors. We also explored filopodial regulation in cultured Drosophila cells and embryos. These data provide new insights into developmental and mechanistic roles of this important actin regulator.     

A functional genomic screen combined with time-lapse microscopy uncovers a novel set of genes involved in dorsal closure of Drosophila embryos

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes.     

Desmoplakin: an unexpected regulator of microtubule organization in the epidermis

Despite their importance in cell shape and polarity generation, the organization of microtubules in differentiated cells and tissues remains relatively unexplored in mammals. We generated transgenic mice in which the epidermis expresses a fluorescently labeled microtubule-binding protein and show that in epidermis and in cultured keratinocytes, microtubules stereotypically reorganize as they differentiate. In basal cells, microtubules form a cytoplasmic network emanating from an apical centrosome. In suprabasal cells, microtubules concentrate at cell-cell junctions. The centrosome retains its ability to nucleate microtubules in differentiated cells, but no longer anchors them. During epidermal differentiation, ninein, which is a centrosomal protein required for microtubule anchoring (Dammermann, A., and A. Merdes. 2002. J. Cell Biol. 159:255-266; Delgehyr, N., J. Sillibourne, and M. Bornens. 2005. J. Cell Sci. 118:1565-1575; Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. J. Cell Sci. 113:3013-3023), is lost from the centrosome and is recruited to desmosomes by desmoplakin (DP). Loss of DP prevents accumulation of cortical microtubules in vivo and in vitro. Our work uncovers a differentiation-specific rearrangement of the microtubule cytoskeleton in epidermis, and defines an essential role for DP in the process.     

Dynamic imaging of mammalian neural tube closure

Neurulation, the process of neural tube formation, is a complex morphogenetic event. In the mammalian embryo, an understanding of the dynamic nature of neurulation has been hampered due to its in utero development. Here we use laser point scanning confocal microscopy of a membrane expressed fluorescent protein to visualize the dynamic cell behaviors comprising neural tube closure in the cultured mouse embryo. In particular, we have focused on the final step wherein the neural folds approach one another and seal to form the closed neural tube. Our unexpected findings reveal a mechanism of closure in the midbrain different from the zipper-like process thought to occur more generally. Individual non-neural ectoderm cells on opposing sides of the neural folds undergo a dramatic change in shape to protrude from the epithelial layer and then form intermediate closure points to “button-up” the folds. Cells from the juxtaposed neural folds extend long and short flexible extensions and form bridges across the physical gap of the closing folds. Thus, the combination of live embryo culture with dynamic imaging provides intriguing insight into the cell biological processes that mold embryonic tissues in mammals.     

Noncentrosomal microtubules and type II myosins potentiate epidermal cell adhesion and barrier formation

During differentiation, many cells reorganize their microtubule cytoskeleton into noncentrosomal arrays. Although these microtubules are likely organized to meet the physiological roles of their tissues, their functions in most cell types remain unexplored. In the epidermis, differentiation induces the reorganization of microtubules to cell–cell junctions in a desmosome-dependent manner. Here, we recapitulate the reorganization of microtubules in cultured epidermal cells. Using this reorganization assay, we show that cortical microtubules recruit myosin II to the cell cortex in order to engage adherens junctions, resulting in an increase in mechanical integrity of the cell sheets. Cortical microtubules and engaged adherens junctions, in turn, increase tight junction function. In vivo, disruption of microtubules or loss of myosin IIA and B resulted in loss of tight junction–mediated barrier activity. We propose that noncentrosomal microtubules act through myosin II recruitment to potentiate cell adhesion in the differentiating epidermis, thus forming a robust mechanical and chemical barrier against the external environment.     

Microtubules and Microfilaments in Amphibian Neurulation

In the salamander embryo, the morphogenetic movements of neurulationare correlated with two cell shape changes in the neural epithelium:elongation and apical constriction of the columnar neural platecells. Cells first elongate to form the flat open neural plateand then constrict apically as the plate rolls up to form theneural tube. Evidence is presented that these cell shape changesare intrinsic to the cells themselves and that they play a causalrole in the morphogenetic movements. Neural plate cells containnumerous microtubules oriented parallel to the axis of elongation.These microtubules are critical to the elongation process. Possiblemechanisms for microtubule function in cell elongation are considered.During apical constriction the cells contain bundles of microfilamentswhich encircle the cell apex in purse-string fashion. Evidenceis presented which suggests that microfilament bundles playan active role in apical constriction, and that this localizedcontraction is produced by filament sliding.     

Spermiogenesis in Xenopus laevis: from late spermatids to spermatozoa

Spermatogenesis is a complex morphogenetic process in which microfilaments and microtubules have been shown to play an important role. The last steps of Xenopus spermatogenesis, i.e., the corkscrew shaping of the sperm head, have been followed to study actin and microtubule distribution by conventional and immunoelectron microscopy. During sperm head morphogenesis, actin is absent in the elongating spermatids, but it is present in the Sertoli cells where results localized at the periphery of their cytoplasm that surrounds the developing germ cells. Sertoli cell actin and microtubules may assist the elongation and the shaping of the spermatids and function in maintaining the Sertoli-spermatid association.     

Mutational analysis of the zippering reaction during flavivirus membrane fusion

The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra- and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.     

Epithelial morphogenesis in embryos: asymmetries, motors and brakes

Epithelial cells play a central role in many embryonic morphogenetic processes, during which they undergo highly coordinated cell shape changes. Here, we review some common principles that have recently emerged through genetic and cellular analyses performed mainly with invertebrate genetic models, focusing on morphogenetic processes involving epithelial sheets. All available data argue that myosin II is the main motor that induces cell shape changes during morphogenesis. We discuss the control of myosin II activity during epithelial morphogenesis, as well as the recently described involvement of microtubules in this process. Finally, we examine how forces unleashed by myosin II can be measured, how embryos use specific brakes to control molecular motors and the potential input of mechano-sensation in morphogenesis.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.     

Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes

We have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome have increased and vimentin is organized as an aggregate located near the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearrangements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.     

Stabilization of post-translational modification of microtubules during cellular morphogenesis

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.     

Selection of initial conditions for recursive production of multicellular organisms

The development of a multicellular organism is a dynamic process. Starting from one or a few cells, the organism becomes a set of cells with different types that form well-determined patterns. It is rather surprising that differentiation in cell types and formation of controlled patterns are compatible, because the former gives morphogenetic diversification whereas the latter implies recursive production of a cell ensemble, reducing individual differences. We studied this problem by taking a simple cell model with intracellular reaction dynamics of chemical concentrations, cell-cell interactions, and increase in cell numbers. We observed successive differentiation from a cell type with diverse chemicals and chaotic concentration dynamics to cell types with oscillatory or fixed-point dynamics, leading to morphogenetic diversity in a spatial pattern. We further show that, by starting from an initial object consisting of both the former cell type with diverse chemicals and the latter differentiated cell type, the recursive production of a multicellular organism with morphogenetic diversity is possible. By relating the former type to a cell in the vegetal pole and the latter to one in the animal pole, classic experimental results with separation of blastomeres in sea urchin eggs are coherently explained, while some predictions are made for in vitro morphogenesis from embryonic stem cells.