Recruitment of Tube and Pelle to signaling sites at the surface of the Drosophila embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2443-2450 ◽  
Author(s):  
P. Towb ◽  
R.L. Galindo ◽  
S.A. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Immunofluorescence Microscopy ◽  
Nuclear Translocation ◽  
Signal Transduction Pathway ◽  
Drosophila Embryo ◽  
Transduction Pathway ◽  
Entire Surface ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence

A signaling pathway initiated by activation of the transmembrane receptor Toll defines dorsoventral polarity in the Drosophila embryo. Toll, which is present over the entire surface of the embryo, is activated ventrally by interaction with a spatially restricted, extracellular ligand. Tube and Pelle then transduce the signal from activated Toll to a complex of Dorsal and Cactus. Here we demonstrate by an mRNA microinjection assay that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy we also show that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle.

The protein kinase Pelle mediates feedback regulation in the Drosophila Toll signaling pathway

Development ◽  
2001 ◽  
Vol 128 (23) ◽  
pp. 4729-4736 ◽  
Author(s):  
Par Towb ◽  
Andreas Bergmann ◽  
Steven A. Wasserman
Keyword(s):  
Protein Kinase ◽  
Immunofluorescence Microscopy ◽  
Nuclear Translocation ◽  
Adaptor Protein ◽  
Feedback Regulation ◽  
Kinase Domain ◽  
Drosophila Embryo ◽  
Confocal Immunofluorescence Microscopy ◽  
Toll Signaling ◽  
Confocal Immunofluorescence

Dorsoventral polarity in the Drosophila embryo is established through a signal transduction cascade triggered in ventral and ventrolateral regions. Activation of a transmembrane receptor, Toll, leads to localized recruitment of the adaptor protein Tube and protein kinase Pelle. Signaling through these components directs degradation of the IκB-like inhibitor Cactus and nuclear translocation of the Rel protein Dorsal. Here we show through confocal immunofluorescence microscopy that Pelle functions to downregulate the signal-dependent relocalization of Tube. Inactivation of the Pelle kinase domain, or elimination of the Tube-Pelle interaction, dramatically increases Tube recruitment to the ventral plasma membrane in regions of active signaling. We also characterize a large collection of pelle alleles, identifying the molecular lesions in these alleles and their effects on Pelle autophosphorylation, Tube phosphorylation and Tube relocalization. Our results point to a mechanism operating to modulate the domain or duration of signaling downstream from Tube and Pelle.

scholarly journals Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis

2003 ◽  
Vol 77 (9) ◽  
pp. 5360-5369 ◽  
Author(s):  
Luc Snyers ◽  
Hannes Zwickl ◽  
Dieter Blaas
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Human Rhinovirus ◽  
Coated Pits ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Transient Overexpression

ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

Regulation of chemotaxis by the cytoplasmic domain of tissue factor

2005 ◽  
Vol 93 (01) ◽  
pp. 27-34 ◽  
Author(s):  
Matilda Johnell ◽  
Brit Sorensen ◽  
Lars Petersen ◽  
Carl-Henrik Heldin ◽  
Agneta Siegbahn
Keyword(s):  
Signal Transduction ◽  
Tissue Factor ◽  
Intracellular Signaling ◽  
Signal Transduction Pathway ◽  
Cytoplasmic Domain ◽  
Cellular Migration ◽  
Boyden Chamber ◽  
Transduction Pathway ◽  
Low Levels

SummaryWe previously demonstrated that FVIIa bound to tissue factor (TF) induces a hyperchemotactic response towards PDGF-BB. The aim of the present study was to investigate the role of the cytoplasmic domain of TF in cell migration. Porcine aortic endothelial (PAE) cells expressing human PDGF β -receptors (PAE/ PDGFR β ) were transfected for expression of human wildtype TF (PAE/PDGFRβ ,TF), a construct lacking the cytoplasmic domain (PAE/PDGFRβ ,TF ∆ cyto), a construct with alanine replacement of serine 258 (PAE/PDGFRβ ,TFS258A), or a construct with alanine replacement of serine 253, 258 and 263 in the cytoplasmic domain (PAE/PDGFRβ ,TF3SA). All stably transfected cell lines expressed functional TF. Chemotaxis was analyzed in a modified Boyden chamber assay. PAE/PDGFRβ ,TF cells stinulated with FVIIa migrated towards a 100-fold lower concentration of PDGF-BB than in the absence of FVIIa,however,hyperchemotaxis was not seen in PAE/PDGFRβ ,TF ∆ cyto cells. PAE/ PDGFR β /TFS258A and PAE/PDGFRβ ,TF3SA cells responded to low levels of PDGF-BB, but migrated a significantly shorter distance than PAE/PDGFRβ ,TF cells. Thus, hyperchemotaxis towards PDGF-BB is likely to depend in part on phosphorylation of the cytoplasmic domain of TF.We conclude that the cytoplasmic domain of TF plays a pivotal role in modulating cellular migration response.Our results suggest that the FVIIa/TF complex mediates intracellular signaling by alternative signal transduction pathway(s).

scholarly journals Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

1995 ◽  
Vol 6 (5) ◽  
pp. 587-596 ◽  
Author(s):  
K A Winans ◽  
C Hashimoto
Keyword(s):  
Cell Fate ◽  
Transmembrane Protein ◽  
Signal Transduction Pathway ◽  
Extracellular Domain ◽  
Drosophila Embryo ◽  
Transduction Pathway ◽  
Wild Type ◽  
Truncated Form ◽  
Biochemical Analyses ◽  
Leucine Rich Repeats

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.

scholarly journals Adipocytes support cAMP-dependent translocation of aquaporin-2 from intracellular sites distinct from the insulin-responsive GLUT4 storage compartment

2006 ◽  
Vol 290 (5) ◽  
pp. F985-F994 ◽  
Author(s):  
Giuseppe Procino ◽  
Donne Bennett Caces ◽  
Giovanna Valenti ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Aquaporin 2 ◽  
Confocal Immunofluorescence Microscopy ◽  
Dominant Negative Form ◽  
Confocal Immunofluorescence ◽  
Hormone Sensitive ◽  
Insulin Responsiveness

Aquaporin-2 (AQP2), when expressed in fully differentiated 3T3-L1 adipocytes, displays cAMP-dependent plasma membrane translocation in a manner similar to its behavior in renal epithelial cells. The translocation of AQP2 required phosphorylation at serine 256, as the expression of AQP2/S256D was constitutively plasma membrane localized, whereas AQP2/S256A was refractory to forskolin stimulation. Unlike GLUT4, this property is not inhibited by depolymerization of cortical actin. In addition, coexpression with the dominant negative form of TC10 (TC10/T31N) or inhibition of phosphatidylinositol 3-kinase did not abrogate the cAMP-mediated response. Under basal conditions, AQP2 is localized in both the perinuclear region and in punctate vesicles scattered within the periphery of the cell. Two- and three-dimensional confocal immunofluorescence microscopy demonstrated that the adipocyte AQP2 cAMP-responsive compartment was distinct from the GLUT4 insulin-responsive compartment. Consistent with this conclusion, insulin was an effective stimulator of GLUT4 translocation but had no effect on AQP2. Conversely, forskolin induced AQP2 translocation but not GLUT4. Colocalization studies with the early endosomal marker EEA1 and transferrin receptor suggested that the AQP2 compartment is mostly distinct from endosomal vesicles. Interestingly, however, the peripheral AQP2 vesicles significantly overlapped vesicle-associated membrane protein-2, underscoring the role of the latter in hormone-regulated exocytosis. To acquire insulin responsiveness following biosynthesis, GLUT4 undergoes a slow sorting step that requires 6–9 h. In contrast, AQP2 rapidly acquires forskolin responsiveness (3 h following biosynthesis) and directly enters the cAMP-regulated compartment without transiting the plasma membrane. Together, these data demonstrate that adipocytes display two different intracellular sorting mechanisms that direct distinct hormone-sensitive partitioning of GLUT4 and AQP2.

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