Drac1 and Crumbs participate in amnioserosa morphogenesis during dorsal closure in Drosophila

2002 ◽  
Vol 115 (10) ◽  
pp. 2119-2129 ◽  
Author(s):  
Nicholas Harden ◽  
Michael Ricos ◽  
Kelly Yee ◽  
Justina Sanny ◽  
Caillin Langmann ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Shape Change ◽  
Apical Cell ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Epithelial Morphogenesis ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Contractile Apparatus ◽  
Constitutively Active

Dorsal closure of the Drosophila embryo involves morphological changes in two epithelia, the epidermis and the amnioserosa, and is a popular system for studying the regulation of epithelial morphogenesis. We previously implicated the small GTPase Drac1 in the assembly of an actomyosin contractile apparatus, contributing to cell shape change in the epidermis during dorsal closure. We now present evidence that Drac1 and Crumbs, a determinant of epithelial polarity, are involved in setting up an actomyosin contractile apparatus that drives amnioserosa morphogenesis by inducing apical cell constriction. Expression of constitutively active Drac1 causes excessive constriction of amnioserosa cells and contraction of the tissue, whereas expression of dominant-negative Drac1 impairs amnioserosa morphogenesis. These Drac1 transgenes may be acting through their effects on the amnioserosa cytoskeleton, as constitutively active Drac1 causes increased staining for F-actin and myosin, whereas dominant-negative Drac1 reduces F-actin levels. Overexpression of Crumbs causes premature cell constriction in the amnioserosa, and dorsal closure defects are seen in embryos homozygous for hypomorphic crumbs alleles. The ability of constitutively active Drac1 to cause contraction of the amnioserosa is impaired in a crumbsmutant background. We propose that amnioserosa morphogenesis is a useful system for studying the regulation of epithelial morphogenesis by Drac1.

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 Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells

2000 ◽  
Vol 20 (19) ◽  
pp. 7378-7387 ◽  
Author(s):  
Hironori Katoh ◽  
Hidekazu Yasui ◽  
Yoshiaki Yamaguchi ◽  
Junko Aoki ◽  
Hirotada Fujita ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Morphological Changes ◽  
Cell Types ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Wild Type ◽  
Rho Family ◽  
Constitutively Active

ABSTRACT The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

scholarly journals ACK Family Tyrosine Kinase Activity Is a Component of Dcdc42 Signaling during Dorsal Closure in Drosophila melanogaster

2002 ◽  
Vol 22 (11) ◽  
pp. 3685-3697 ◽  
Author(s):  
Kai Ping Sem ◽  
Baharak Zahedi ◽  
Ivan Tan ◽  
Maria Deak ◽  
Louis Lim ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Leading Edge ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dorsal Closure ◽  
Tyrosine Kinase Activity ◽  
Amino Terminal

ABSTRACT We have characterized Drosophila melanogaster ACK (DACK), one of two members of the ACK family of nonreceptor tyrosine kinases in Drosophila. The ACKs are likely effectors for the small GTPase Cdc42, but signaling by these proteins remains poorly defined. ACK family tyrosine kinase activity functions downstream of Drosophila Cdc42 during dorsal closure of the embryo, as overexpression of DACK can rescue the dorsal closure defects caused by dominant-negative Dcdc42. Similar to known participants in dorsal closure, DACK is enriched in the leading edge cells of the advancing epidermis, but it does not signal through activation of the Jun amino-terminal kinase cascade operating in these cells. Transcription of DACK is responsive to changes in Dcdc42 signaling specifically at the leading edge and in the amnioserosa, two tissues involved in dorsal closure. Unlike other members of the ACK family, DACK does not contain a conserved Cdc42-binding motif, and transcriptional regulation may be one route by which Dcdc42 can affect DACK function. Expression of wild-type and kinase-dead DACK transgenes in embryos, and in the developing wing and eye, reveals that ACK family tyrosine kinase activity is involved in a range of developmental events similar to that of Dcdc42.

scholarly journals RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

2021 ◽  
Author(s):  
Futaba Osaki ◽  
Takahide Matsui ◽  
Shu Hiragi ◽  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Proteins ◽  
Neurite Outgrowth ◽  
Membrane Trafficking ◽  
Specific Binding ◽  
Binding Specificity ◽  
Small Gtpase ◽  
Binding Domain ◽  
Dominant Negative ◽  
Epithelial Morphogenesis ◽  
Phenotype Characteristic

ABSTRACTThe small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

Drosophila RhoA regulates the cytoskeleton and cell-cell adhesion in the developing epidermis

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3173-3183 ◽  
Author(s):  
James W. Bloor ◽  
Daniel P. Kiehart
Keyword(s):  
Cell Adhesion ◽  
Leading Edge ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Cell Physiology ◽  
Dorsal Closure ◽  
Adhesive Properties ◽  
Actin Organization ◽  
On Line ◽  
Cell Cell

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. We have investigated its role in the developing Drosophila embryonic epidermis during the process of dorsal closure. By expressing the dominant negative DRhoAN19 construct in stripes of epidermal cells, we confirm that Rho function is required for dorsal closure and demonstrate that it is necessary to maintain the integrity of the ventral epidermis. We show that defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoAN19 expression. Furthermore, we demonstrate that these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoAN19-expressing cells. Movies available on-line

scholarly journals Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

2011 ◽  
Vol 301 (2) ◽  
pp. C507-C521 ◽  
Author(s):  
Lilian Chiang ◽  
Julie Ngo ◽  
Joel E. Schechter ◽  
Serhan Karvar ◽  
Tanya Tolmachova ◽  
...  
Keyword(s):  
Lacrimal Gland ◽  
Morphological Changes ◽  
Acinar Cells ◽  
Secretory Vesicle ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Apical Surface ◽  
Tear Proteins ◽  
Vesicle Exocytosis ◽  
Constitutively Active

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b −/− and Rab27 ash/ash /Rab27b −/− mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

scholarly journals RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11

2021 ◽  
pp. jcs.257311
Author(s):  
Futaba Osaki ◽  
Takahide Matsui ◽  
Shu Hiragi ◽  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Proteins ◽  
Neurite Outgrowth ◽  
Membrane Trafficking ◽  
Specific Binding ◽  
Binding Specificity ◽  
Small Gtpase ◽  
Binding Domain ◽  
Dominant Negative ◽  
Epithelial Morphogenesis ◽  
Phenotype Characteristic

The small GTPase Rab11 plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth, and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain of one of its effectors, e.g., the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, e.g., to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 visualized endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking: tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

Drosophila puckered regulates Fos/Jun levels during follicle cell morphogenesis

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1845-1856 ◽  
Author(s):  
L.L. Dobens ◽  
E. Martin-Blanco ◽  
A. Martinez-Arias ◽  
F.C. Kafatos ◽  
L.A. Raftery
Keyword(s):  
Nurse Cell ◽  
Ectopic Expression ◽  
Follicle Cell ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Drosophila Embryo ◽  
Follicle Cells ◽  
Cell Morphogenesis ◽  
Jnk Pathway ◽  
Egg Chambers

puckered (puc) encodes a VH1-like phosphatase that down-regulates Jun kinase (JNK) activity during dorsal closure of the Drosophila embryo. We report a role for puc in follicle cell morphogenesis during oogenesis. puc mRNA accumulates preferentially in the centripetally migrating follicle cells and cells of the elongating dorsal appendages. Proper levels of Puc activity in the follicle cells are critical for the production of a normal egg: either reduced or increased Puc activity result in incomplete nurse cell dumping and aberrant dorsal appendages. Phenotypes associated with puc mutant follicle cells include altered DE-cadherin expression in the follicle cells and a failure of nurse cell dumping to coordinate with dorsal appendage elongation, leading to the formation of cup-shaped egg chambers. The JNK pathway target A251-lacZ showed cell-type-specific differences in its regulation by puc and by the small GTPase DRac1. puc mutant cells displayed region-specific ectopic expression of the A251-lacZ enhancer trap whereas overexpression of a transgene encoding Puc was sufficient to suppress lacZ expression in a cell autonomous fashion. Strikingly, decreased or increased puc function leads to a corresponding increase or decrease, respectively, of Fos and Jun protein levels. Taken together, these data indicate that puc modulates gene expression responses by antagonizing a Ρ GTPase signal transduction pathway that stabilizes the AP-1 transcription factor. Consistent with this, overexpression of a dominant negative DRac1 resulted in lower levels of Fos/Jun.

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 ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

2002 ◽  
Vol 157 (5) ◽  
pp. 819-830 ◽  
Author(s):  
Takahiro Tsuji ◽  
Toshimasa Ishizaki ◽  
Muneo Okamoto ◽  
Chiharu Higashida ◽  
Kazuhiro Kimura ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Induced Membrane ◽  
3T3 Fibroblasts ◽  
C3 Exoenzyme ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts ◽  
Membrane Ruffle

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

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