Miniaturization and validation of a sensitive multiparametric cell-based assay for the concomitant detection of microtubule-destabilizing and microtubule-stabilizing agents.
The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin alpha-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with double-immunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cell-based assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons.
Vassal
Emilie
E
Université Joseph Fourier, INSERM U366-CS/DRDC/CEA Grenoble, France.
Barette
Caroline
C
Fonrose
Xavier
X
Dupont
Raphaël
R
Sans-Soleilhac
Emmanuelle
E
Lafanechère
Laurence
L
D002452:Cell Count; D002463:Cell Membrane Permeability; D004121:Dimethyl Sulfoxide; D004353:Drug Evaluation, Preclinical / Q000379:methods* / Q000706:statistics & numerical data; D005404:Fixatives; D006367:HeLa Cells; D006801:Humans; D000432:Methanol; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008904:Miniaturization; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
D005404:Fixatives; D014404:Tubulin; D050257:Tubulin Modulators; D000432:Methanol; D004121:Dimethyl Sulfoxide