scholarly journals The Myopic-Ubpy-Hrs nexus enables endosomal recycling of Frizzled

2015 ◽  
Vol 26 (18) ◽  
pp. 3329-3342 ◽  
Author(s):  
Tirthadipa Pradhan-Sundd ◽  
Esther M. Verheyen
Keyword(s):  
Wnt Signaling ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Proximity Ligation Assay ◽  
Endosomal Trafficking ◽  
Proximity Ligation ◽  
Drosophila Wing ◽  
Wing Disk ◽  
Molecular Events ◽  
Endosomal Recycling

Endosomal trafficking of signaling proteins plays an essential role in cellular homeostasis. The seven-pass transmembrane protein Frizzled (Fz) is a critical component of Wnt signaling. Although Wnt signaling is proposed to be regulated by endosomal trafficking of Fz, the molecular events that enable this regulation are not completely understood. Here we show that the endosomal protein Myopic (Mop) regulates Fz trafficking in the Drosophila wing disk by inhibiting the ubiquitination and degradation of Hrs. Deletion of Mop or Hrs results in endosomal accumulation of Fz and therefore reduced Wnt signaling. The in situ proximity ligation assay revealed a strong association between Mop and Hrs in the Drosophila wing disk. Overexpression of Hrs rescues the trafficking defect caused by mop knockdown. Mop aids in the maintenance of Ubpy, which deubiquitinates (and thus stabilizes) Hrs. In the absence of the ubiquitin ligase Cbl, Mop is dispensable. These findings support a previously unknown role for Mop in endosomal trafficking of Fz in Wnt-receiving cells.

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (10) ◽  
Author(s):  
Valentin Derangère ◽  
Mélanie Bruchard ◽  
Frédérique Végran ◽  
François Ghiringhelli
Keyword(s):  
T Cells ◽  
Proximity Ligation Assay ◽  
Proximity Ligation

scholarly journals AHR:IKAROS Interaction Promotes Platelet Biogenesis in Response to SR1

Reports ◽  
2021 ◽  
Vol 4 (1) ◽  
pp. 7
Author(s):  
Lea Mallo ◽  
Valentin Do Sacramento ◽  
Christian Gachet ◽  
Susan Chan ◽  
Philippe Kastner ◽  
...  
Keyword(s):  
High Capacity ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Short Report ◽  
Production Engineering ◽  
Platelet Production ◽  
Proximity Ligation ◽  
Aryl Hydrocarbon ◽  
Dependent Pathway

In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved IKAROS. The downregulation/inhibition of IKAROS (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and IKAROS. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and IKAROS. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ IKAROS -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- IKAROS dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.

Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (17) ◽  
Author(s):  
Matthew Keeney ◽  
Eric Hoffman ◽  
J. Greenamyre ◽  
Roberto Di Maio
Keyword(s):  
Kinase Activity ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Lrrk2 Kinase ◽  
Lrrk2 Kinase Activity
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