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© Sandrine Etienne-Manneville
Photo prise à l'avant (dans la protrusion) d'astrocytes primaires de rat en migration. Marquage par immunofluorescence montrant en rouge, p150 Glued, une protéine associée aux extrémités 'plus' des microtubules et en vert la tubuline des microtubules. La photographie montre l'accumulation de p150 Glued à l'avant des cellules en migration, où la protéine pourrait participer à l'ancrage des microtubules à la membrane plasmique. Pour essayer de corriger, les dérèglements observés lors de la migration des cellules d'astrocytes tumuraux ou gliomes on cherche à connaitre les mécanismes moléculaires fondamentaux qui controlent la polarisation et la migration cellulaires.
Publication : Methods in molecular biology (Clifton, N.J.)

A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway Cloning

Team: Biochemistry of Macromolecular Interactions
Member: Daniel Ladant
Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Methods in molecular biology (Clifton, N.J.) - 01 Jan 2018

Olson MG, Goldammer M, Gauliard E, Ladant D, Ouellette SP

Link to Pubmed [PMID] – 29855952

Methods Mol. Biol. 2018;1794:75-96

The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway recombinational cloning, BACTH. This chapter explains the principle of the BACTH, its Gateway modified system, and details of the general procedure.