Nuclear import of cubitus interruptus is regulated by hedgehog via a mechanism distinct from Ci stabilization and Ci activation

Development ◽  
2000 ◽  
Vol 127 (14) ◽  
pp. 3131-3139 ◽  
Author(s):  
Q.T. Wang ◽  
R.A. Holmgren
Keyword(s):  
Nuclear Import ◽  
Full Length ◽  
Negative Regulators ◽  
Cell Fates ◽  
Cubitus Interruptus ◽  
Drosophila Wing ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
The Relationship

The Hedgehog (Hh) signal is transduced via Cubitus interruptus (Ci) to specify cell fates in the Drosophila wing. In the absence of Hh, the 155 kDa full-length form of Ci is cleaved into a 75 kDa repressor. Hh inhibits the proteolysis of full-length Ci and facilitates its conversion into an activator. Recently, it has been suggested that Hh promotes Ci nuclear import in tissue culture cells. We have studied the mechanism of Ci nuclear import in vivo and the relationship between nuclear import, stabilization and activation. We found that Ci rapidly translocates to the nucleus in cells close to the anteroposterior (AP) boundary and this rapid nuclear import requires Hh signaling. The nuclear import of Ci is regulated by Hh even under conditions in which Ci is fully stabilized. Furthermore, cells that exhibit Ci stabilization and rapid nuclear import do not necessarily exhibit maximal Ci activity. It has been previously shown that stabilization does not suffice for activation. Consistent with this finding, our results suggest that the mechanisms regulating nuclear import, stabilization and activation are distinct from each other. Finally, we show that cos2 and pka, two molecules that have been characterized primarily as negative regulators of Ci activity, also have positive roles in the activation of Ci in response to Hh.

scholarly journals Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

1997 ◽  
Vol 328 (3) ◽  
pp. 847-854 ◽  
Author(s):  
E. Margaret TOME ◽  
M. Steven FISER ◽  
M. Claire PAYNE ◽  
W. Eugene GERNER
Keyword(s):  
Ornithine Decarboxylase ◽  
Competitive Inhibitor ◽  
Initiation Factor ◽  
Protective Mechanism ◽  
Eukaryotic Initiation Factor ◽  
Post Translational Modification ◽  
Resistant Variant ◽  
Culture Cells ◽  
Tissue Culture Cells

DH23A cells, an α-difluoromethylornithine-resistant variant of the parental hepatoma tissue culture cells, express high levels of stable ornithine decarboxylase. Aberrantly high expression of ornithine decarboxylase results in a large accumulation of endogenous putrescine and increased apoptosis in DH23A cells when α-difluoromethylornithine is removed from the culture. Treatment of DH23A cells with exogenous putrescine in the presence of α-difluoromethylornithine mimics the effect of drug removal, suggesting that putrescine is a causative agent or trigger of apoptosis. Accumulation of excess intracellular putrescine inhibits the formation of hypusine in vivo, a reaction that proceeds by the transfer of the butylamine moiety of spermidine to a lysine residue in eukaryotic initiation factor 5A (eIF-5A). Treatment of DH23A cells with diaminoheptane, a competitive inhibitor of the post-translational modification of eIF-5A, causes both the suppression of eIF-5A modification in vivo and induction of apoptosis. These data support the hypothesis that rapid degradation of ornithine decarboxylase is a protective mechanism to avoid cell toxicity from putrescine accumulation. Further, these data suggest that suppression of modified eIF-5A formation is one mechanism by which cells may be induced to undergo apoptosis.

scholarly journals Bridging length scales to measure polymer assembly

2017 ◽  
Vol 28 (10) ◽  
pp. 1379-1388 ◽  
Author(s):  
Bryan Kaye ◽  
Tae Yeon Yoo ◽  
Peter J. Foster ◽  
Che-Hang Yu ◽  
Daniel J. Needleman
Keyword(s):  
Polymer Concentration ◽  
Microtubule Assembly ◽  
Acquisition Time ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Culture Cells ◽  
Cell Extracts ◽  
Egg Extracts ◽  
The Relationship

Time-resolvable quantitative measurements of polymer concentration are very useful to elucidate protein polymerization pathways. There are numerous techniques to measure polymer concentrations in purified protein solutions, but few are applicable in vivo. Here we develop a methodology combining microscopy and spectroscopy to overcome the limitations of both approaches for measuring polymer concentration in cells and cell extracts. This technique is based on quantifying the relationship between microscopy and spectroscopy measurements at many locations. We apply this methodology to measure microtubule assembly in tissue culture cells and Xenopus egg extracts using two-photon microscopy with FLIM measurements of FRET. We find that the relationship between FRET and two-photon intensity quantitatively agrees with predictions. Furthermore, FRET and intensity measurements change as expected with changes in acquisition time, labeling ratios, and polymer concentration. Taken together, these results demonstrate that this approach can quantitatively measure microtubule assembly in complex environments. This methodology should be broadly useful for studying microtubule nucleation and assembly pathways of other polymers.

scholarly journals Rap1A-Deficient T and B Cells Show Impaired Integrin-Mediated Cell Adhesion

2006 ◽  
Vol 26 (2) ◽  
pp. 643-653 ◽  
Author(s):  
Marlena Duchniewicz ◽  
Tomasz Zemojtel ◽  
Mateusz Kolanczyk ◽  
Steffen Grossmann ◽  
Jürgen S. Scheele ◽  
...  
Keyword(s):  
Functional Complementation ◽  
Wild Type ◽  
T And B Cells ◽  
Genetic Studies ◽  
Mild Phenotype ◽  
Cell Homing ◽  
Culture Cells ◽  
Cell Matrix ◽  
Tissue Culture Cells

ABSTRACT Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1−/− cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.

scholarly journals Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases

2008 ◽  
Vol 183 (4) ◽  
pp. 589-595 ◽  
Author(s):  
Chawon Yun ◽  
Yonggang Wang ◽  
Debaditya Mukhopadhyay ◽  
Peter Backlund ◽  
Nagamalleswari Kolli ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Ribosome Biogenesis ◽  
Mammalian Tissue ◽  
Hematopoietic Malignancies ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Egg Extracts ◽  
Protein B23 ◽  
Sumo Pathway

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.

scholarly journals Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo

2003 ◽  
Vol 14 (12) ◽  
pp. 5089-5097 ◽  
Author(s):  
Stephen J. King ◽  
Christa L. Brown ◽  
Kerstin C. Maier ◽  
Nicholas J. Quintyne ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Binding Assays ◽  
Microtubule Organization ◽  
Binding Domains ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Dynein Intermediate Chain ◽  
Transient Overexpression

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

In vivo association of lamins with nucleic acids in Drosophila melanogaster

1998 ◽  
Vol 111 (1) ◽  
pp. 121-129 ◽  
Author(s):  
R. Rzepecki ◽  
S.S. Bogachev ◽  
E. Kokoza ◽  
N. Stuurman ◽  
P.A. Fisher
Keyword(s):  
Nucleic Acids ◽  
Nuclear Organization ◽  
Eukaryotic Cell ◽  
Dna Interactions ◽  
Nuclear Lamins ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Nucleic Acid Interactions ◽  
Contrast Interaction

A 32P-labeling strategy was developed to study the interaction(s) in tissue culture cells between proteins and nucleic acids. Interphase and mitotic nuclear lamins were studied in Drosophila Kc cells. After bromodeoxyuridine incorporation and in vivo photo-crosslinking with 366 nm light, it was found that interphase lamins were associated with nucleic acid. Interactions with DNA as well as RNA were detected. In contrast, interaction of nucleic acids with mitotic lamin was not observed. Photo-crosslinking in the presence of antibiotics distamycin and/or chromomycin suggested that interphase lamins interacted with both A-T-rich DNA and G-C-rich DNA; interactions with G-C-rich DNA predominated. These results have implications for understanding the interphase organization of the higher eukaryotic cell nucleus as well as the transition of cells from interphase to mitosis. A model of nuclear organization, consistent with our results, is proposed.

scholarly journals Biochemical Characterization of the Drosophila Wingless Signaling Pathway Based on RNA Interference

2004 ◽  
Vol 24 (5) ◽  
pp. 2012-2024 ◽  
Author(s):  
Hiroko Matsubayashi ◽  
Sonoka Sese ◽  
Jong-Seo Lee ◽  
Tadaoki Shirakawa ◽  
Takeshi Iwatsubo ◽  
...  
Keyword(s):  
Rna Interference ◽  
Biochemical Characterization ◽  
The Other ◽  
Protein Levels ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Mediated Priming ◽  
Kinase A

ABSTRACT Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Iα (CKIα) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIα and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIα- or Zw3-mediated Arm phosphorylaytion in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIα and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIα-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIα-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIα-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphoylation is due to CKIα and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

scholarly journals Coordination of planar cell polarity pathways through Spiny-legs

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Abhijit A Ambegaonkar ◽  
Kenneth D Irvine
Keyword(s):  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Drosophila Wing ◽  
Direct Linkage ◽  
The Relationship

Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. We have characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, we define where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP, and provide a new understanding of the orientation of polarity and the basis of PCP phenotypes. Our results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals.

Further characterization of the antigen defined by the monoclonal antibody M27

1989 ◽  
Vol 94 (4) ◽  
pp. 725-731
Author(s):  
M.E. Bramwell ◽  
S.M. Humm
Keyword(s):  
Monoclonal Antibody ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Reducing Conditions ◽  
Rat Lymphocytes ◽  
Foetal Liver ◽  
Cultivation In Vitro

Using immunoblotting techniques, the antigen that binds the monoclonal antibody M27 has been clearly defined in terms of apparent molecular mass and distribution. In reducing conditions it has an apparent mass of 178K (K = 10(3) Mr) and is present in the cytoplasm and membranes of all mammalian tissue culture cells so far examined. It is absent from lines derived from avian, piscine and amphibian sources. It is also absent from foetal liver of both rat and mouse, but subsequently appears after cultivation in vitro. Similarly, it can be detected on rat lymphocytes only after mitogenic stimulation. However, it is found on both hepatoma and lymphoma cells in vitro, and on in vivo tumours from murine sources. It thus appears to be associated with cell proliferation.

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