FRET-based biosensors for in vivo measurements of kinases activities during cell cycle

L’implication et les rôles de la PKA et de la MAPK/ERK lors de la division cellulaire, ont fait l’objet de nombreuses études. Pourtant les profils spatio-temporels des activités de ces kinases au cours des différentes étapes du cycle et notamment lors de la mitose sont controversés et restent à éclaircir. Le but de ce travail a été la détermination de ces profils grâce à l’utilisation et au développement d’outils moléculaires basés sur des propriétés de la fluorescence, capables de rapporter l’activité kinase in vivo, qui sont appelés biosenseurs FRET. Nous avons mis en évidence que l’activité de PKA augmente lors de la mitose pour ensuite chuter rapidement lors de la cytokinèse dans les cellules HeLa. Lors de la métaphase et de l’anaphase, l'activité de PKA est particulièrement élevée à proximité des chromosomes et ce, indépendamment d’une relocalisation de ses sous-unités catalytiques. De plus, l’utilisation d’inhibiteur de PKA conduit à l’apparition de phénotypes mitotiques aberrants, indiquant le rôle essentiel de cette augmentation d’activité dans le maintien de l’intégrité du génome. Ces phénotypes sont similaires à ceux décrits pour des perturbations de l’activité de MAPK/ERK. Le développement d’un biosenseur FRET optimisé pour les mesures d’activité de MAPK/ERK nous a permis de déterminer que son activité globale ne varie pas lors de la mitose mais connait en revanche une diminution forte et très brève lors de la cytokinèse. L’inhibition de PKA induit une augmentation sensible de la phosphorylation de MAPK/ERK, ce qui pourrait suggérer ainsi un lien entre les activités de ces deux protéines dans la répartition correcte du matériel génétique 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 methyl‐transferase CcrM at the end of S‐phase maintains a cyclic alternation between a full‐ to hemi‐methylated chromosome. This transition of the chromosomal methylation pattern affects the DNA‐binding 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 S‐phase expressed genes to their transcriptional output. We demonstrated in vitro that transcription of ctrA from the P1 promoter in its hemi‐methylated 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 hemi‐methylated 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 cycle‐regulated genes are also under the influence of methylation and GcrA‐dependent transcriptional regulation, this could be a mechanism responsible for maintaining the gene transcription dosage during the S‐phase

From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells

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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>  </p