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Régulation biochimique et mécanique de l'assemblage de filaments d'actine par la formine

Abstract : Actin filament assembly plays a pivotal role in cellular processes such as cell motility, morphogenis or division. Elucidating how the actin cytoskeleton is globally controlled remains a complex challenge. We know that it is orchestrated both by actin regulatory proteins and mechanical constraints.The formin protein is an essential actin regulator. Anchored to the cell membrane, it is responsible for the assembly (nucleation and elongation) of actin filaments found in linear and unbranched architectures. It is notably involved in the generation of filopodia protrusions at the leading edge of a motile cell. One important feature is that it processively tracks the barbed end of an actin filament, while stimulating its polymerization in the presence of profilin.Formin processivity and its regulation is not yet completely understood. As an important factor determining the length of the resulting filament, it must be investigated further.A perfect assay to look at formin processivity in vitro is an innovative microfuidics assay coupled to TIRF microscopy, pioneered by the team, to simultaneously track tens of individual filaments. In a designed chamber,filaments are anchored to the surface by one end, and aligned with the solution flow. We can precisely control the biochemical environment of the filaments. Moreover, we can exert and modulate forces on filaments, due to the viscous drag of flowing solutions. By varying chemical conditions during formin action at the barbed end, I investigated how others proteins or the elongation rate can modulate formin processivity, by looking at the detachment rate of formins.Moreover, we can mimic the membrane anchoring in the cell by specifically attaching formins at the surface. In our chamber, through the filament they elongate, we can apply force to formins.In complement to biochemical studies, we then investigate the effect oftension on their processivity.I first investigated the impact of a capping protein on formin action at the barbed end. Their barbed end binding is thought to be mutually exclusive.We measured that the affinity of one protein is reduced by the presence of the other. However we observed they both can bind simultaneously the barbed end, in a transient complex, which block barbed end elongation.Competition of formin and CP would regulate barbed end dynamics in a cell situation where length is tightly controlled.I next studied formin processivity dependence on various parameters. We show that processivity is sensitive to salt and labelling fraction used in our solutions. We also looked at how processivity is affected by the elongation rate, which can either be varied by actin or profilin concentration. On one hand, actin concentration reduces processivity, at any given concentrationsof profilin. On the other hand, raising the concentration of profilin increasesprocessivity, regardless of the elongation rate. This indicates that theincorporation of actin monomers decreases processivity while in contrast,the presence of the profilin at the barbed end increases it.Moreover, tension exerted on formin was observed to largely favor its detachment. In a quantitative matter, the effect of tension prevails over anyothers biochemical factor on processivity : only a few piconewtons decreaseit by several orders of magnitude. This important effect helps us build amore complete model of processive elongation. These results indicate thatmechanical stress is likely to play an important role in a cellular context.In conclusion, this project brings insights into the molecular properties offormin and helps to decipher the mechanism of processive elongation and its regulation.
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Submitted on : Friday, January 19, 2018 - 4:45:07 PM
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Mikaël Kerleau. Régulation biochimique et mécanique de l'assemblage de filaments d'actine par la formine. Biochimie [q-bio.BM]. Université Paris-Saclay, 2017. Français. ⟨NNT : 2017SACLS583⟩. ⟨tel-01688756⟩

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