Thesis defense – Violeta Milanovic

Wednesday 1 October / 09:30

Venue : CARF

Videoconference link: https://u-bordeaux-fr.zoom.us/j/83185319457

Defense in English

Violeta Milanovic

Team : Mechano-biology of motile and neuronal structures
IINS

Title

Studying cellular mechanotransduction in living cells using single molecule fluorescence polarization microscopy

Abstract

Mechanotransduction, the process by which mechanical forces are converted into biochemical signals, is fundamental to cellular adhesion, migration, and the spatial organization and integrity of tissues. Integrin adhesion sites (IAS) serve as the mechanical interface between the extracellular matrix and the cytoskeleton, and host a complex network of force-sensitive proteins such as talin. Key protein in integrin adhesive structure, talin establishes the physical linkage that transmits cell mechanical forces to its surroundings. Far from being a rigid structural element, talin is a large multimodular protein carrying multiple domains with binding sites which are exposed when talin is stretched by force. This complex mechanical response led to propose the interesting hypothesis of a talin code in which both the shape of talin scaffolds and the set of partners bound to talin could serve as a readout for the mechanical information encoded by cells.
While the force-induced conformational changes of talin have been extensively studied in vitro, how talin conformation is changing within the native architecture of living cells still remain poorly resolved, particularly in terms of spatial orientation.
This thesis focuses primarily on talin molecular orientation in focal adhesions (FAs) using single-molecule fluorescence polarization microscopy. To probe distinct mechanical segments along talin, we engineered a panel of talin constructs fused to mEos3.2 at specific domains – neck, R3, R12, and the C-terminus. Using an optimized live-cell imaging pipeline, we successfully extracted mEos3.2 in-plane (ρ), out-of-plane (η), and wobbling (δ) angles used as proxies for talin domain orientation. The orientation distributions of talin domains were consistent with their known mechanical context: domains closer to integrins, such as the neck and R3, exhibited strong directional alignment along the focal adhesion axis, while R12 and C-terminal tags showed more isotropic behavior. These results in living cells recapitulated those we obtained in fixed samples, confirming the robustness and reproducibility of the approach under basal contractile conditions.
This thesis demonstrates that molecular orientation serves as a powerful and previously underused live-cell readout of mechanical state. Beyond talin, the molecular tools, methods and insights developed here provide a framework to study how proteins such as integrins, vinculin, or actin reorganize under force in their native environment. With continued technological development, orientation-resolved single-molecule microscopy holds strong potential to decode the mechanical logic of adhesion and signaling in both physiological and pathological contexts.

Keywords

mechanotransduction, cell adhesion, single protein tracking, fluorescence polarization

Publication

Carla Silva Martins, Cyntia Taveneau, Gerard Castro-Linares, Mikhail Baibakov, Nicolas Buzhinsky, Mar Eroles, Violeta Milanović, Shizue Omi, Jean-Denis Pedelacq, Francois Iv, Léa Bouillard, Alexander Llewellyn, Maxime Gomes, Mayssa Belhabib, Mira Kuzmić, Pascal Verdier-Pinard, Stacey Lee, Ali Badache, Sanjay Kumar, Cristel Chandre, Sophie Brasselet, Felix Rico, Olivier Rossier, Gijsje H. Koenderink, Jerome Wenger, Stéphanie Cabantous, Manos Mavrakis; Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells. J Cell Biol 6 March 2023; 222 (3): e202203016. doi: https://doi.org/10.1083/jcb.202203016.