FRET biosensors for spatio-temporal dynamic kinase activity by fast FLIM and fluorescence anisotropy microscopy – KinBioFRET

KinBioFRET will develop new technological and methodological approaches by using fastFLIM for quantitative heteroFRET measurement and by introducing homoFRET bioprobes by fluorescence anisotropy measurement.

the methodological approaches developed here will be directly used to investigate two biological questions: (i) the comparison of blastula and gastrula dividing cells to simultaneously follow spatio-temporal dynamics of MELK kinase activities and mechanical tension in Xenopus embryos, and (ii) the spatio-temporal regulation of Aurora A activity following symmetric and asymmetric cellular division in the context of Drosophila live pupae.

this proposal is novel and ambitious for several reasons: multidisciplinary approaches, development of quantitative fluorescence microscopy methods dedicated to FRET bioprobes, development of new Aurora A kinase FRET biosensors, development of homoFRET biosensors by anisotropy measurements, development of simultaneous spatio-temporal dynamics of FRET biosensors, novelty of expected biological results in the field of kinase regulation during cell division in developing living organism.

Not for the moment.

KinBioFRET proposal is based on an original combination of multidisciplinary partners: technological and methodological development team (Marc Tramier) directly concerned by the development of FRET biosensor methodologies and three biological partners (Jean-Pierre Tassan, Claude Prigent and Roland Le Borgne) directly interested into dynamics of kinase activities in different fields of biology. We propose to develop new FRET biosensors to measure the activity of two essential mitotic kinases, MELK and Aurora A, in the frame of spatio-temporal regulation of these kinases during cell division in living genetically tractable model organisms.
First, to be able to increase sensitivity, speed of time-lapse acquisition and then biological relevance of these bioprobes, KinBioFRET will develop new technological and methodological approaches by using fastFLIM for quantitative heteroFRET measurement and by introducing homoFRET bioprobes by fluorescence anisotropy measurement. Second, one of the methodological objectives is to be able to follow in the same time two (or more) FRET bioprobes using the homoFRET approach. This will be achieved by measuring MELK and Vinculin tension sensors using multicolor homoFRET biosensors simultaneously in Xenopus embryos. Third, we will engineer new FRET biosensors to study spatio-temporal regulation of the mitotic kinase Aurora A in Drosophila. The choice of Aurora A is intimately linked to the biological questions addressed by four independent groups of biologists at IGDR. In addition to the FRET change from C- and N-ter fluorescent protein tags within Aurora A, we propose to develop an Aurora A FRET biosensor constituted of a phospho-binding domain recognizing an Aurora A substrate peptide sequence and sandwiched between two fluorescent proteins. This latter FRET biosensor will contain various target sequences for specific subcellular localizations such as centromers, centrosomes, and plasma membrane.
Finally, the methodological approaches developed here will be directly used to investigate two biological questions: (i) the comparison of blastula and gastrula dividing cells to simultaneously follow spatio-temporal dynamics of MELK kinase activities and mechanical tension in Xenopus embryos, and (ii) the spatio-temporal regulation of Aurora A activity following symmetric and asymmetric cellular division in the context of Drosophila live pupae. We believe that this proposal is novel and ambitious for several reasons: multidisciplinary approaches, development of quantitative fluorescence microscopy methods dedicated to FRET bioprobes, development of new Aurora A kinase FRET biosensors, development of homoFRET biosensors by anisotropy measurements, development of simultaneous spatio-temporal dynamics of FRET biosensors, novelty of expected biological results in the field of kinase regulation during cell division in developing living organism.