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.

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

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239357
Author(s):  
Junior J. West ◽  
Tony J. C. Harris
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Genetic Interaction ◽  
Coiled Coil ◽  
Leading Edge ◽  
Cell Junctions ◽  
Dorsal Closure ◽  
Ph Domain ◽  
Dorsal Midline ◽  
Actin Networks

Cytohesin 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. Inversely, 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.

scholarly journals WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7

2021 ◽  
Vol 9 ◽  
Author(s):  
Sophie Sluysmans ◽  
Isabelle Méan ◽  
Lionel Jond ◽  
Sandra Citi
Keyword(s):  
Protein Interactions ◽  
Cultured Cells ◽  
Coiled Coil ◽  
Cell Junctions ◽  
Structural Basis ◽  
Chimeric Proteins ◽  
Ph Domain ◽  
Protein Protein Interactions ◽  
Ww Domains ◽  
Lateral Localization

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

scholarly journals The Drosophila Afadin and ZO-1 homologs Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling

2019 ◽  
Author(s):  
Lathiena A. Manning ◽  
Kia Z. Perez-Vale ◽  
Kristina N. Schaefer ◽  
Mycah T. Sewell ◽  
Mark Peifer
Keyword(s):  
Cultured Cells ◽  
Adherens Junction ◽  
Leading Edge ◽  
Cell Junctions ◽  
Dorsal Closure ◽  
Epithelial Integrity ◽  
Tissue Integrity ◽  
Cell Intercalation ◽  
Actomyosin Cytoskeleton ◽  
Cell Cell

AbstractDuring morphogenesis cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting force generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation. Canoe’s mammalian homolog Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We took these insights back into the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found Canoe helps cells maintain junctional-cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation and neuroblast invagination, or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homolog Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest Canoe and Polychaetoid stabilize Bazooka/Par3 at cell-cell junctions, helping maintain balanced apical contractility and tissue integrity.

scholarly journals The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

2014 ◽  
Vol 25 (20) ◽  
pp. 3147-3165 ◽  
Author(s):  
Stephanie H. Nowotarski ◽  
Natalie McKeon ◽  
Rachel J. Moser ◽  
Mark Peifer
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Leading Edge ◽  
Dorsal Closure ◽  
Primary Role ◽  
Loss Of Function ◽  
Timely Completion ◽  
And Migration ◽  
Different Tissues

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous’ role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure—migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells—as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative “fingerprint”—the protrusive profile—which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.

scholarly journals The AF-6 Homolog Canoe Acts as a Rap1 Effector During Dorsal Closure of the Drosophila Embryo

Genetics ◽  
2003 ◽  
Vol 165 (1) ◽  
pp. 159-169
Author(s):  
Benjamin Boettner ◽  
Phoebe Harjes ◽  
Satoshi Ishimaru ◽  
Michael Heke ◽  
Hong Qing Fan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Interaction ◽  
Critical Role ◽  
Adherens Junction ◽  
Small Gtpases ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Cell Sheets ◽  
Jnk Pathway

Abstract Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Here, we demonstrate that Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, acts as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. We further show that Rap1 acts upstream of Cno, but that Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

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).

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 Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium

2001 ◽  
Vol 153 (4) ◽  
pp. 795-810 ◽  
Author(s):  
Satoru Funamoto ◽  
Kristina Milan ◽  
Ruedi Meili ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Wild Type ◽  
Pi3k Inhibitor Ly294002 ◽  
Effector Pathways

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3′ kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain–containing proteins. We demonstrate that in response to chemoattractants three PH domain–containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

scholarly journals Bleb-driven chemotaxis of Dictyostelium cells

2014 ◽  
Vol 204 (6) ◽  
pp. 1027-1044 ◽  
Author(s):  
Evgeny Zatulovskiy ◽  
Richard Tyson ◽  
Till Bretschneider ◽  
Robert R. Kay
Keyword(s):  
Cyclic Amp ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Pi3 Kinase ◽  
Mechanical Resistance ◽  
Ph Domain ◽  
Cortical Function ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.

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