scholarly journals Zipper Is Necessary for Branching Morphogenesis of the Terminal Cells in the Drosophila melanogaster’s Tracheal System

Biology ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 729
Author(s):  
Jong-Hyeon Shin ◽  
Chan-Woo Jeong
Keyword(s):  
Developmental Stages ◽  
Cell Sheet ◽  
Leading Edge ◽  
Branching Morphogenesis ◽  
Confocal Imaging ◽  
Specific Role ◽  
Actin Binding ◽  
Loss Of Function ◽  
Tracheal System

Branching morphogenesis and seamless tube formation in Drosophila melanogaster are essential for the development of vascular and tracheal systems, and instructive in studying complex branched structures such as human organs. Zipper is a myosin II’s actin-binding heavy chain; hence, it is important for contracting actin, cell proliferation, and cell sheet adhesion for branching of the tracheal system in post-larval development of the D. melanogaster. Nevertheless, the specific role of Zipper in the larva is still in question. This paper intended to investigate the specific role of Zipper in branching morphogenesis and lumenogenesis in early developmental stages. It did so by checking the localization of the protein in the cytoplasm of the terminal cells and also by analyzing the morphology of zipper RNAi loss-of-function mutants in regard to branching and lumen formation in the terminal cells. A rescue experiment of RNAi mutants was also performed to check the sufficiency of Zipper in branching morphogenesis. Confocal imaging showed the localization of Zipper in the cytoplasm of the terminal cells, and respective quantitative analyses demonstrated that zipper RNAi terminal cells develop significantly fewer branches. Such a result hinted that Zipper is required for the regulation of branching in the terminal cells of D. melanogaster. Nevertheless, Zipper is not significantly involved in the formation of seamless tubes. One hypothesis is that Zipper’s contractility at the lateral epidermis’ leading edge allows cell sheet movement and respective elongation; as a result of such an elongation, further branching may occur in the elongated region of the cell, hence defining branching morphogenesis in the terminal cells of the tracheal system.

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).

The dual-targeted prolyl aminopeptidase PAP1 is involved in proline accumulation in response to stress and during pollen development

2021 ◽  
Author(s):  
Abi S Ghifari ◽  
Pedro F Teixeira ◽  
Beata Kmiec ◽  
Neha Singh ◽  
Elzbieta Glaser ◽  
...  
Keyword(s):  
Pollen Development ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Physiological Role ◽  
Loss Of Function ◽  
Prolyl Aminopeptidase ◽  
Cellular Localisation ◽  
Alternative Transcription ◽  
Response To Stress

Abstract Plant endosymbiotic organelles such as mitochondria and chloroplasts harbour a wide array of biochemical reactions. As a part of protein homeostasis to maintain organellar activity and stability, unwanted proteins and peptides need to be completely degraded in a stepwise mechanism termed the processing pathway, where at the last stage single amino acids are released by aminopeptidases. Here, we determined the molecular and physiological functions of a prolyl aminopeptidase homologue PAP1 (At2g14260) that is able to release N-terminal proline. Transcript analyses demonstrate that an alternative transcription start site (TSS) gives rise to two alternate transcripts, generating two in-frame proteins PAP1.1 and PAP1.2. Sub-cellular localisation studies revealed that the longer isoform PAP1.1, which contains a 51-residue N-terminal extension is exclusively targeted to chloroplasts, while the truncated isoform PAP1.2 is located in the cytosol. Distinct expression patterns in different tissues and developmental stages were observed. Investigations into the physiological role of PAP1 using loss-of-function mutants revealed that PAP1 activity may be involved in proline homeostasis and accumulation, required for pollen development and tolerance to osmotic stress. Enzymatic activity, sub-cellular location, and expression patterns of PAP1 suggest a role in the chloroplastic peptide processing pathway and proline homeostasis.

scholarly journals Borrelia burgdorferi ftsZ Plays a Role in Cell Division

2006 ◽  
Vol 188 (5) ◽  
pp. 1969-1978 ◽  
Author(s):  
Lydia Dubytska ◽  
Henry P. Godfrey ◽  
Felipe C. Cabello
Keyword(s):  
Cell Division ◽  
Borrelia Burgdorferi ◽  
Leading Edge ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Lethal Mutant ◽  
E Coli ◽  
Its Gene ◽  
Conditional Lethal Mutant

ABSTRACT ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZBbu ). Its gene product (FtsZBbu) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZBbu might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZBbu antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZBbu could interfere with the function of E. coli FtsZ, ftsZBbu was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZBbu generated a filamentous phenotype. This suggested interference of ftsZBbu with E. coli FtsZ function and confirmed the role of ftsZBbu in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470 ◽  
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

Abstract This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

scholarly journals CYFIP2 containing WAVE complexes inhibit cell migration

2020 ◽  
Author(s):  
Anna Polesskaya ◽  
Arthur Boutillon ◽  
Yanan Wang ◽  
Marc Lavielle ◽  
Sophie Vacher ◽  
...  
Keyword(s):  
Cell Migration ◽  
Mammary Epithelial Cells ◽  
Leading Edge ◽  
Good Prognosis ◽  
Mammary Epithelial ◽  
Breast Cancer Patients ◽  
Loss Of Function ◽  
A Cell ◽  
Wave Complex

ABSTRACTBranched actin networks polymerized by the Arp2/3 complex are critical for cell migration. The WAVE complex is the major Arp2/3 activator at the leading edge of migrating cells. However, multiple distinct WAVE complexes can be assembled in a cell, due to the combinatorial complexity of paralogous subunits. When systematically analyzing the contribution of each WAVE complex subunit to the metastasis-free survival of breast cancer patients, we found that overexpression of the CYFIP2 subunit was surprisingly associated with good prognosis. Gain and loss of function experiments in transformed and untransformed mammary epithelial cells revealed that cell migration was always inversely related to CYFIP2 levels. The role of CYFIP2 was systematically opposite to the role of the paralogous subunit CYFIP1 or of the NCKAP1 subunit. The specific CYFIP2 function in inhibiting cell migration was related to its unique ability to down-regulate classical pro-migratory WAVE complexes. The anti-migratory function of CYFIP2 was also revealed in migration of prechordal plate cells during gastrulation of the zebrafish embryo, indicating that the unique function of CYFIP2 is critically important in both physiological and pathophysiological migrations.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

Thorax closure in Drosophila: involvement of Fos and the JNK pathway

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3947-3956 ◽  
Author(s):  
J. Zeitlinger ◽  
D. Bohmann
Keyword(s):  
Functional Module ◽  
Cell Sheet ◽  
Dorsal Closure ◽  
Loss Of Function ◽  
Jnk Pathway ◽  
Insect Metamorphosis ◽  
Wing Imaginal Discs ◽  
The One ◽  
Jnk Activation

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, we studied another cell sheet movement that we term thorax closure, the joining of the parts of the wing imaginal discs which give rise to the adult thorax during metamorphosis. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibres during midline attachment. Genetic data indicate a requirement of D-Fos and the JNK pathway for thorax closure, and a negative regulatory role of Puc. Furthermore, puc expression co-localises with elevated levels of D-Fos, is reduced in a JNK or D-Fos loss-of-function background and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well.

scholarly journals Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.

1993 ◽  
Vol 120 (1) ◽  
pp. 163-173 ◽  
Author(s):  
E L de Hostos ◽  
C Rehfuess ◽  
B Bradtke ◽  
D R Waddell ◽  
R Albrecht ◽  
...  
Keyword(s):  
Leading Edge ◽  
Gene Replacement ◽  
Actin Binding ◽  
Beta Subunits ◽  
Wild Type ◽  
Daughter Cells ◽  
Immunofluorescence Labeling ◽  
Mutant Cells

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.

Analytical Techniques for Determining the Role of Domain of Unknown Function 579 Proteins in the Synthesis of O-Methylated Plant Polysaccharides

2020 ◽  
Vol 25 (4) ◽  
pp. 345-355 ◽  
Author(s):  
Peter J. Smith ◽  
Malcolm A. O’Neill ◽  
Jason Backe ◽  
William S. York ◽  
Maria J Peña ◽  
...  
Keyword(s):  
Unknown Function ◽  
Developmental Stages ◽  
Family Members ◽  
Analytical Techniques ◽  
Arabinogalactan Protein ◽  
Loss Of Function ◽  
Domain Of Unknown Function ◽  
Matrix Glycans

Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4- O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4- O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.

scholarly journals Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

2013 ◽  
Vol 202 (1) ◽  
pp. 163-177 ◽  
Author(s):  
Ingo Thievessen ◽  
Peter M. Thompson ◽  
Sylvain Berlemont ◽  
Karen M. Plevock ◽  
Sergey V. Plotnikov ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Leading Edge ◽  
Flow Speed ◽  
Actin Binding ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Primary Fibroblasts

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.

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