7 research outputs found
FRET-based biosensors for in vivo measurements of kinases activities during cell cycle
Lâimplication et les rĂŽles de la PKA et de la MAPK/ERK lors de la division cellulaire, ont fait lâobjet de nombreuses Ă©tudes. Pourtant les profils spatio-temporels des activitĂ©s de ces kinases au cours des diffĂ©rentes Ă©tapes du cycle et notamment lors de la mitose sont controversĂ©s et restent Ă Ă©claircir. Le but de ce travail a Ă©tĂ© la dĂ©termination de ces profils grĂące Ă lâutilisation et au dĂ©veloppement dâoutils molĂ©culaires basĂ©s sur des propriĂ©tĂ©s de la fluorescence, capables de rapporter lâactivitĂ© kinase in vivo, qui sont appelĂ©s biosenseurs FRET. Nous avons mis en Ă©vidence que lâactivitĂ© de PKA augmente lors de la mitose pour ensuite chuter rapidement lors de la cytokinĂšse dans les cellules HeLa. Lors de la mĂ©taphase et de lâanaphase, l'activitĂ© de PKA est particuliĂšrement Ă©levĂ©e Ă proximitĂ© des chromosomes et ce, indĂ©pendamment dâune relocalisation de ses sous-unitĂ©s catalytiques. De plus, lâutilisation dâinhibiteur de PKA conduit Ă lâapparition de phĂ©notypes mitotiques aberrants, indiquant le rĂŽle essentiel de cette augmentation dâactivitĂ© dans le maintien de lâintĂ©gritĂ© du gĂ©nome. Ces phĂ©notypes sont similaires Ă ceux dĂ©crits pour des perturbations de lâactivitĂ© de MAPK/ERK. Le dĂ©veloppement dâun biosenseur FRET optimisĂ© pour les mesures dâactivitĂ© de MAPK/ERK nous a permis de dĂ©terminer que son activitĂ© globale ne varie pas lors de la mitose mais connait en revanche une diminution forte et trĂšs brĂšve lors de la cytokinĂšse. Lâinhibition de PKA induit une augmentation sensible de la phosphorylation de MAPK/ERK, ce qui pourrait suggĂ©rer ainsi un lien entre les activitĂ©s de ces deux protĂ©ines dans la rĂ©partition correcte du matĂ©riel gĂ©nĂ©tique lors de la mitose.Even if the roles and contribution of PKA and MAPK/ERK in cell cycle have been the topic of several studies, the spatio-temporal profiles of their activities are still controversial and remain to be clarified. The aim of my PhD was to highlight those activity profiles during the cell cycle in HeLa cells, by using or developing new molecular tools, based on fluorescence properties that are able to report kinase activity in vivo and named FRET-based biosensors.The use of these biosensors allowed us to reveal that PKA activity increased at the onset of mitosis and stayed high until the completion of cytokinesis. During metaphase and anaphase, this activity was especially high in the close vicinity of the condensed chromosomes, independently of any concomitant relocalization of PKA catalytic sub-units within the cell. Moreover inhibition of PKA activity during mitosis lead to improper mitotic phenotype (i.e. : misalignment of the DNA on the spindle, precocious segregation of part of the chromosomes), pointing out the essential role of the activity increase in genetic stability. Those observed phenotypes are similar to those described upon experimental modifications of the MAPK/Erk activity level. By means of the development of a new improved MAPK/Erk activity biosensor, we showed that its global activity does not change during mitosis, but goes through a brief and strong decrease during cytokinesis. As the inhibition of PKA induces a noticeable increase of MAPK/Erk phosphorylation, those results could suggest a link between those two kinases activities in the correct distribution of the DNA to daughter cells during mitosis
Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties
The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation
International audienceThe cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells
Methylation-dependent transcriptional regulation of crescentin gene (creS) by GcrA in Caulobacter crescentus
In Caulobacter crescentus the combined action of chromosome replication and the expression of DNA methylâtransferase CcrM at the end of Sâphase maintains a cyclic alternation between a fullâ to hemiâmethylated chromosome. This transition of the chromosomal methylation pattern affects the DNAâbinding properties of the transcription factor GcrA that controls the several key cell cycle functions. However, the molecular mechanism by which GcrA and methylation are linked to transcription is not fully elucidated yet. Using a combination of cell biology, genetics, and in vitro analysis, we deciphered how GcrA integrates the methylation pattern of several Sâphase expressed genes to their transcriptional output. We demonstrated in vitro that transcription of ctrA from the P1 promoter in its hemiâmethylated state is activated by GcrA, while in its fully methylated state GcrA had no effect. Further, GcrA and methylation together influence a peculiar distribution of creS transcripts, encoding for crescentin, the protein responsible for the characteristic shape of Caulobacter cells. This gene is duplicated at the onset of chromosome replication and the two hemiâmethylated copies are spatially segregated. Our results indicated that GcrA transcribed only the copy where coding strand is methylated. In vitro transcription assay further substantiated this finding. As several of the cell cycleâregulated genes are also under the influence of methylation and GcrAâdependent transcriptional regulation, this could be a mechanism responsible for maintaining the gene transcription dosage during the Sâphase
From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells
International audienceno abstrac
Research data supporting for "Shadow Technique Algorithm (STA) Sheds a New Light on Differential Interference Contrast (DIC) Microscopy"
<p>This folder contains data to support the paper entitled:</p>
<p>Shadow Technique Algorithm (STA) Sheds a New Light on Differential Interference Contrast (DIC) Microscopy<br>
Trinel D ,Vandame P, Hervieu M, Floquet E, Aumercier M, Biondi EG , Bodart JF and Spriet C*<br>
Analytical & Bioanalytical Techniques (2015)</p>
<p>Supplementary data and the STA macro for imageJ are at the folder root.<br>
Data used for the paper are available as test sample in the data folder.<br>
In each case, the data before and after STA are present.<br>
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