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The present work takes advantage of the intrinsic localisation of two-photon fluorescence excitation to develop two-photon fluorescence recovery after photobleaching -TP-FRAP - as a method to assess fluorophore dynamics with microscopic resolution. Numerical simulations are proposed to improve data interpretation beyond the usual frame of FRAP data analysis. This work was developed for measuring the dynamics of cytoskeleton proteins. The apical face of epithelial cells is covered with a dense set of microvilli, the main components of which have been identified and localized at the ultrastructural level, but their dynamic organisation remains largely unknown. To understand the apical morphogenesis and to assess the dynamics of cytoskeleton proteins that might underlie the steady-state morphology, GFP-fusion proteins were expressed. Using TP-FRAP, fluorophore dynamics could be resolved between the plasma membrane and the cytosol, and interpreted in terms of diffusive mobility or exchange rates within and between these two compartments. This is applied in particular to ezrin, a membrane-actin linker protein localized in the cytosol and at the plasma membrane, which plays a key role in coupling signal transduction to cortical morphogenesis. TP-FRAP experiments in conjunction with ezrin mutaganesis and numerical modelling strongly suggest a fast cyclic renewal dynamics with three sequential membrane binding states with distinct mobilities and biochemical reactivities. This paper presents a detailed account of the instrumental design and the numerical method developed to interpret recovery data. This approach should be more generally useful to locally assess the dynamics of protein turnover in submicroscopic structures, and resolve its molecular basis.
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