Spatio-temporal composition of the mitotic Chromosomal Passenger Complex detected using in situ proximity ligation assay.
Cell division is orchestrated by a complex protein network that aims to maintenance of genomic stability. Visualisation of mitotic protein-protein associations in space and time has been limited due to the lack of proper biochemical and easy-to-use imaging tools. Here we report adaptation of the in situ proximity ligation assay (is-PLA) to study mitotic protein interactions with spatio-temporal resolution. We examined the composition of the Chromosomal Passenger Complex (CPC) at various mitotic phases and after chemical treatments using is-PLA with antibodies against the core CPC subunits Aurora B, INCENP, Survivin and Borealin. Our results support the notion that the core CPC functions as a single structural unit at centromeres in early mitosis and at central spindle after the onset of anaphase. Treatment of cells with the Aurora B inhibitor ZM447439 diminished the is-PLA signals at centromeres suggesting that Aurora B activity contributes to structural maintenance and/or proper subcellular localization of the core CPC. Is-PLA-based analysis of interaction between INCENP and Polo-like kinase 1 (Plk1) proposes that the kinase co-travels with CPC during late mitosis. The data illustrates both the strengths and limitations of the is-PLA in the analysis of mitotic macromolecule associations at sub-organelle level.
VTT Technical Research Centre of Finland, Medical Biotechnology, Itäinen Pitkäkatu 4C, 20520 Turku, Finland.
Mariaana
M
Vuoriluoto
Leena J
LJ
Laine
Petri
P
Saviranta
Jeroen
J
Pouwels
Marko J
MJ
Kallio
D002877:Chromosomes, Human; D006801:Humans