Caractérisation de l'intéraction de la stathmine avec les microtubules : une analyse par imagerie FRET dans la cellule.

.....

FRET and FRAP imaging: Approaches to characterise tau and stathmin interactions with microtubules in cells

International audienceMicrotubules (MTs) are involved in many crucial processes such as cell morphogenesis, mitosis and motility. These dynamic structures resulting from the complex assembly of tubulin are tightly regulated by stabilising MT-associated proteins (MAPs) such as tau and destabilising proteins, notably stathmin. Because of their key role, these MAPs and their interactions have been extensively studied using biochemical and biophysical approaches, particularly in vitro. Nevertheless, numerous questions remain unanswered and the mechanisms of interaction between MT and these proteins are still unclear in cells. Techniques coupling cell imaging and fluorescence methods, such as Förster resonance energy transfer and fluorescence recovery after photobleaching, are excellent tools to study these interactions in situ. After describing these methods, we will present emblematic data from the literature and unpublished experimental results from our laboratory concerning the interactions between MTs, tau and stathmin in cells

Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells: Stathmin binds to microtubules in cells

International audienceStathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies suggest strongly that stathmin could act by sequestering tubulin and/or by binding to the MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. Using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analysed the ability of stathmin and its phospho-forms (on Ser16, 25, 38 and 63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin-tubulin interactions at the MT plus-ends and in the cytosol. Interestingly, we also observed a novel pool of stathmin bound along the MT. The expression of truncated stathmin and the use of MT-stabilizing taxol further showed that the C-terminal domain of stathmin is the main contributor to this binding, and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus-end of a depolymerizing MT reaches the stathmin molecules

Molecular mechanisms of Tau binding to microtubules and its role in microtubule dynamics in live cells.

International audienceDespite extensive studies, the molecular mechanisms of Tau binding to microtubules (MTs) and its consequences on MT stability still remain unclear. It is especially true in cells where the spatiotemporal distribution of Tau-MT interactions is unknown. Using Förster resonance energy transfer (FRET), we showed that the Tau-MT interaction was distributed along MTs in periodic hotspots of high and low FRET intensities. Fluorescence recovery after photobleaching (FRAP) revealed a two-phase exchange of Tau with MTs as a rapid diffusion followed by a slower binding phase. A real-time FRET assay showed that high FRET occurred simultaneously with rescue and pause transitions at MT ends. To further explore the functional interaction of Tau with MTs, the binding of paclitaxel (PTX), tubulin acetylation induced by trichostatin A (TSA), and the expression of non-acetylatable tubulin were used. With PTX and TSA, FRAP curves best fitted a single phase with a long time constant, whereas with non-acetylatable α-tubulin, curves best fitted a two phase recovery. Upon incubation with PTX and TSA, the number of high and low FRET hotspots decreased by up to 50% and no hotspot was observed during rescue and pause transitions. In the presence of non-acetylatable α-tubulin, a 34% increase in low FRET hotspots occurred, and our real-time FRET assay revealed that low FRET hotspots appeared with MTs recovering growth. In conclusion, we have identified, by FRET and FRAP, a discrete Tau-MT interaction, in which Tau could induce conformational changes of MTs, favoring recovery of MT self-assembly