Étude multiéchelle du transport intracellulaire passif de macromolécules à l'aide d'une modélisation milieu poreux et d'expériences de fluorescence sur cellules vivantes

Intracellular macromolecule transport is essential to most biological processes. Unlike active transport mechanisms, diffusion has the advantage that it does not require energy to transport macromolecules through the intracellular medium. However, the intracellular medium features complex structures that are likely to impact macromolecule diffusion. Though cytoplasmic diffusion is known to be heterogeneous, hindered compared to diffusion in pure water, and often anomalous, the physical mechanisms behind these observations are still unclear.In this thesis, we propose to study the effect of the cytoplasmic structure on passive diffusion using a transverse approach inspired by the porous media theory and applied to intracellular biophysics.First, we use live-cell fluorescence experiments to study MDCK living cells expressing free-GFP. We use confocal imaging to conduct Z-stack experiments to explore the structure of the cytoplasm. We also develop a FRAP and a FCS protocols to measure the cytoplasmic diffusivity of free-GFP in different cytoplasmic regions. The Z-stack and FRAP experiments are applied to cells submitted to different medium osmolarities and to different cytoskeleton depolymerizing drugs.Then, using the theoretical framework of transport in porous media and of solutal diffusion in hydrogels, we develop a multiscale model for free-GFP diffusion in the cytoplasm. We thus derive a macroscopic transport equation for free-GFP in the cytoplasm. This equation features an effective diffusivity that depends on the cytoplasm local structure. The cytoplasm structure affects the free-GFP effective diffusivity through two physical mechanisms: the tortuous hindrance (TH) and the hydrodynamic diffusional hindrance (HDH). These two sources of diffusional hindrance are quantified by numerically solving the obtained closure problems on simplified cytoplasmic structure geometries using the finite element method.Finally, using supplementary developments, the cytoplasmic solid fractions and the effect of the cytoplasmic structure on free-GFP diffusivity are inferred from our experimental results. The model predictions and the experimental results obtained for the different osmotic conditions are then compared.The results highlight the impact of the cytoplasm structure on cytoplasmic diffusion and the importance of taking HDH into account to correctly predict the effective cytoplasmic diffusivity. Altogether, this work lays the foundations for an original intracellular macromolecule transport description based on porous media modeling and offers a framework to study intracellular diffusion processes in various biological contexts, such as caspase transport during apoptosis.