scholarly journals Multiple Forces Contribute to Cell Sheet Morphogenesis for Dorsal Closure in Drosophila

2000 ◽  
Vol 149 (2) ◽  
pp. 471-490 ◽  
Author(s):  
Daniel P. Kiehart ◽  
Catherine G. Galbraith ◽  
Kevin A. Edwards ◽  
Wayne L. Rickoll ◽  
Ruth A. Montague
Keyword(s):  
Wound Healing ◽  
Cell Shape ◽  
Cell Sheet ◽  
Leading Edge ◽  
Drosophila Embryo ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Dorsal Closure ◽  
Purse String ◽  
Ultraviolet Laser

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29–41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602–R605).

scholarly journals Quantifying dorsal closure in three dimensions

2016 ◽  
Vol 27 (25) ◽  
pp. 3948-3955 ◽  
Author(s):  
Heng Lu ◽  
Adam Sokolow ◽  
Daniel P. Kiehart ◽  
Glenn S. Edwards
Keyword(s):  
Surface Tension ◽  
Wound Healing ◽  
Internal Pressure ◽  
Leading Edge ◽  
Dorsal Surface ◽  
Model System ◽  
Three Dimensions ◽  
Dorsal Closure ◽  
Purse String ◽  
Drosophila Embryogenesis

Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure, two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus (“corner” of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis (“lids” of the eye). Here we report that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help to shape the dorsal surface.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

scholarly journals A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.

1996 ◽  
Vol 16 (5) ◽  
pp. 1896-1908 ◽  
Author(s):  
N Harden ◽  
J Lee ◽  
H Y Loh ◽  
Y M Ong ◽  
I Tan ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Cell Shape ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Epidermal Cells ◽  
Dorsal Closure ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Amino Terminal ◽  
Drosophila Homolog

Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.

scholarly journals The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure

2020 ◽  
Author(s):  
Junior J. West ◽  
Tony J. C. Harris
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Genetic Interaction ◽  
Coiled Coil ◽  
Leading Edge ◽  
Cell Junctions ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Ph Domain ◽  
Actin Networks

AbstractCytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke’s previously reported regulation of myosin contractility during dorsal closure.

Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis

1999 ◽  
Vol 112 (3) ◽  
pp. 273-284 ◽  
Author(s):  
N. Harden ◽  
M. Ricos ◽  
Y.M. Ong ◽  
W. Chia ◽  
L. Lim
Keyword(s):  
Leading Edge ◽  
Cell Types ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Contractile Apparatus ◽  
Serine Threonine Kinase ◽  
Down Regulation ◽  
Cellular Events

The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signalling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. We have been studying the participation of the RHO subfamily in dorsal closure of the Drosophila embryo, a process involving morphogenesis of the epidermis. We have previously shown that Drac1, a Rho subfamily protein, is required for the presence of an actomyosin contractile apparatus believed to be driving the cell shape changes essential to dorsal closure. Expression of a dominant negative Drac1 transgene causes a loss of this contractile apparatus from the leading edge of the advancing epidermis and dorsal closure fails. We now show that two other Rho subfamily proteins, Dcdc42 and RhoA, as well as Ras1 are also required for dorsal closure. Dcdc42 appears to have conflicting roles during dorsal closure: establishment and/or maintenance of the leading edge cytoskeleton versus its down regulation. Down regulation of the leading edge cytoskeleton may be controlled by the serine/threonine kinase DPAK, a potential Drac1/Dcdc42 effector. RhoA is required for the integrity of the leading edge cytoskeleton specifically in cells flanking the segment borders. We have begun to characterize the interactions of the various small GTPases in regulating dorsal closure and find no evidence for the hierarchy of Rho subfamily activity described in some mammalian cell types. Rather, our results suggest that while all Ρ subfamily p21s tested are required for dorsal closure, they act largely in parallel.

scholarly journals CKA, a Novel Multidomain Protein, Regulates the JUN N-Terminal Kinase Signal Transduction Pathway in Drosophila

2002 ◽  
Vol 22 (6) ◽  
pp. 1792-1803 ◽  
Author(s):  
Hua-Wei Chen ◽  
Maria Julia Marinissen ◽  
Su-Wan Oh ◽  
Xiu Chen ◽  
Michael Melnick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Sheet ◽  
Signal Transduction Pathway ◽  
Leading Edge ◽  
Genetic Screen ◽  
Signal Transduction Pathways ◽  
Dorsal Closure ◽  
Transduction Pathway ◽  
Temporal Expression ◽  
Multidomain Protein

ABSTRACT The Drosophila melanogaster JUN N-terminal kinase (DJNK) and DPP (decapentaplegic) signal transduction pathways coordinately regulate epithelial cell sheet movement during the process of dorsal closure in the embryo. By a genetic screen of mutations affecting dorsal closure in Drosophila, we have now identified a multidomain protein, connector of kinase to AP-1 (cka), that functions in the DJNK pathway and controls the localized expression of dpp in the leading-edge cells. We have also investigated how CKA acts. This unique molecule forms a complex with HEP (DJNKK), BSK (DJNK), DJUN, and DFOS. Complex formation activates BSK kinase, which in turn phosphorylates and activates DJUN and DFOS. These data suggest that CKA represents a novel molecule regulating AP-1 activity by organizing a molecular complex of kinases and transcription factors, thus coordinating the spatial-temporal expression of AP-1-regulated genes.

scholarly journals Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Konrad Steinestel ◽  
Eva Wardelmann ◽  
Wolfgang Hartmann ◽  
Inga Grünewald
Keyword(s):  
Tumor Cell ◽  
Cell Invasion ◽  
Intracellular Signaling ◽  
Expression Patterns ◽  
Leading Edge ◽  
Tumor Cell Invasion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Surface Receptors ◽  
Cytoskeletal Dynamics

Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells.

scholarly journals Interplay between integrins and PI4P5K Sktl is crucial for cell polarization and reepithelialisation during Drosophila wound healing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Si-Hyoung Park ◽  
Chan-wool Lee ◽  
Kwang-Min Choe
Keyword(s):  
Wound Healing ◽  
Cell Adhesion ◽  
Cell Polarity ◽  
Myosin Ii ◽  
Cell Sheet ◽  
Cell Polarization ◽  
Actin Dynamics ◽  
Rear Side ◽  
Jnk Pathway ◽  
Pathway Activation

Abstract Phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2] regulates cell adhesion and actin dynamics during cell migration. PI(4,5)P2 binds various components of the cell adhesion machinery, but how these processes affect migration of the epithelial cell sheet is not well understood. Here, we report that PI(4,5)P2 and Sktl, the kinase that converts PI4P to PI(4,5)P2, are both localized to the rear side of cells during wound healing of the Drosophila larval epidermis. The Sktl localization requires JNK pathway activation and integrins, but not PVR. The sktl knockdown epidermis displays strong defects in would closure, reminiscent of the JNK-depleted epidermis, and shows severe disruption of cell polarity, as determined by myosin II localization. Sktl and βPS integrin colocalize at the rear side of cells forming the trailing edge during wound healing and the two are inter-dependent in that the absence of one severely disrupts the rear localization of the other. These results strongly suggest that the JNK pathway regulates the rear localization of Sktl and integrins and the interplay between Sktl and integrins sets up cell polarity, which is crucial for reepithelialisation during wound healing.

Cell-Level Finite Element Studies of Viscous Cells in Planar Aggregates

2000 ◽  
Vol 122 (4) ◽  
pp. 394-401 ◽  
Author(s):  
Helen H. Chen ◽  
G. Wayne Brodland
Keyword(s):  
Experimental Data ◽  
Wound Healing ◽  
Finite Element ◽  
Cell Shape ◽  
Cell Sheet ◽  
Element Formulation ◽  
Cell Level ◽  
Cell Sheets ◽  
Real Cell ◽  
Sheet Stretching

A new cell-level finite element formulation is presented and used to investigate how epithelia and other planar collections of viscous cells might deform during events such as embryo morphogenesis and wound healing. Forces arising from cytoskeletal components, cytoplasm viscosity, and cell-cell adhesions are included. Individual cells are modeled using multiple finite elements, and cell rearrangements can occur. Simulations of cell-sheet stretching indicate that the initial stages of sheet stretching are characterized by changes in cell shape, while subsequent stages are governed by cell rearrangement. Inferences can be made from the simulations about the forces that act in real cell sheets when suitable experimental data are available. [S0148-0731(00)01404-7]

scholarly journals N-terminal modification of actin by acetylation and arginylation determines the architecture and assembly rate of linear and branched actin networks

2020 ◽  
Author(s):  
Samantha M. Chin ◽  
Tomoyuki Hatano ◽  
Lavanya Sivashanmugam ◽  
Andrejus Suchenko ◽  
Anna S. Kashina ◽  
...  
Keyword(s):  
Cell Migration ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Network ◽  
Post Translational Modification ◽  
Actin Binding Proteins ◽  
N Terminus

AbstractThe great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, recent work implicates post-translational modification of the actin N-terminus by either acetylation or arginylation itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing, and modification of the N-terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in Cofilin-mediated severing. Taken together, these results suggest that cells can employ the differently modified actins to precisely regulate actin dynamics. In addition, unprocessed, or non-acetylated actin show very different effects on formin-mediated-elongation, Arp2/3-mediated nucleation, and severing by Cofilin. Altogether, this study shows that the nature of the N-terminus of actin can induce distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge.

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