scholarly journals Non-specificity of Pitstop 2 in clathrin-mediated endocytosis

2014 ◽  
Author(s):  
Anna K Willox ◽  
Yasmina M.E. Sahraoui ◽  
Stephen J Royle
Keyword(s):  
Heavy Chain ◽  
Membrane Trafficking ◽  
Specific Activity ◽  
Adaptor Proteins ◽  
Domain Interaction ◽  
Interaction Sites ◽  
Terminal Domain ◽  
Clathrin Heavy Chain ◽  
Potential Use ◽  
In Cells

Small molecule inhibitors of clathrin-mediated endocytosis are highly desired for the dissection of membrane trafficking pathways in the lab and for potential use as anti-infectives in the clinic. One inhibition strategy is to prevent clathrin from contacting adaptor proteins so that clathrin-mediated endocytosis cannot occur. "Pitstop" compounds have been developed which block only one of the four functional interaction sites on the N-terminal domain of clathrin heavy chain. Despite this limitation, Pitstop 2 causes profound inhibition of clathrin-mediated endocytosis. In this study, we probed for non-specific activity of Pitstop 2 by examining its action in cells expressing clathrin heavy chain harbouring mutations in the N-terminal domain interaction sites. We conclude that the inhibition observed with this compound is due to non-specificity, i.e. it causes inhibition away from its proposed mode of action. We recommend that these compounds be used with caution in cells and that they should not be used to conclude anything of the function of clathrin’s N-terminal domain.

Structure of clathrin cages and coats in ice

1986 ◽  
Vol 44 ◽  
pp. 94-97
Author(s):  
G.P.A. Vigers ◽  
R.A. Crowther ◽  
B.M.F. Pearse
Keyword(s):  
Electron Microscopy ◽  
Heavy Chain ◽  
Outer Part ◽  
Coated Vesicles ◽  
Eukaryotic Cells ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Clathrin Heavy Chain ◽  
Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

scholarly journals Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

FEBS Letters ◽  
2006 ◽  
Vol 581 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Min-Duk Seo ◽  
Sung Jean Park ◽  
Hyun-Jung Kim ◽  
Bong Jin Lee
Keyword(s):  
Domain Interaction ◽  
Ww Domain ◽  
Interaction Sites ◽  
Terminal Domain

scholarly journals Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

PLoS ONE ◽  
2010 ◽  
Vol 5 (8) ◽  
pp. e12017 ◽  
Author(s):  
Petra Neumann-Staubitz ◽  
Stephanie L. Hall ◽  
Joseph Kuo ◽  
Antony P. Jackson
Keyword(s):  
Heavy Chain ◽  
Temperature Sensitive ◽  
Clathrin Heavy Chain

scholarly journals Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

2020 ◽  
Author(s):  
Ridim D Mote ◽  
Jyoti Yadav ◽  
Surya Bansi Singh ◽  
Mahak Tiwari ◽  
Shivprasad Patil ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Young’S Modulus ◽  
Heavy Chain ◽  
Young's Modulus ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Actin Binding ◽  
Clathrin Heavy Chain

AbstractMouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Young’s modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young’s modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.

scholarly journals Clathrin heavy chain phosphorylated at T606 plays a role in proper cell division

Cell Cycle ◽  
2019 ◽  
Vol 18 (16) ◽  
pp. 1976-1994 ◽  
Author(s):  
Yusuke Yabuno ◽  
Toshihiro Uchihashi ◽  
Towa Sasakura ◽  
Hiroyuki Shimizu ◽  
Yoko Naito ◽  
...  
Keyword(s):  
Cell Division ◽  
Heavy Chain ◽  
Clathrin Heavy Chain
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