Electron microscopy study of Formin conformations during actin filament assembly ; Etude par microscopie électronique des conformations structurales adoptées par la Formine durant l'assemblage de filaments d'actine
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Media type:
E-Book
Title:
Electron microscopy study of Formin conformations during actin filament assembly ; Etude par microscopie électronique des conformations structurales adoptées par la Formine durant l'assemblage de filaments d'actine
imprint:
[Erscheinungsort nicht ermittelbar]: HAL CCSD, 2021
Language:
French
Origination:
University thesis:
Dissertation, HAL CCSD, 2021
Footnote:
Description:
Formins are key regulators of the assembly of actin filaments in cells. Formins induce the nucleation and rapid elongation of actin filaments. The activity of formins thus participates in the generation of functional structures such as filopodia, stress fibers or the cytokinetic ring. Hence, formins are involved in fundamental cellular processes such as migration or cell division. At the molecular level, formins are homodimers capable of interacting with the barbed ends of elongating actin filaments via their FH2 domains. The conformational changes involved in tracking the barbed ends have never been directly observed. However, a set of biochemical, biophysical and structural biology studies evidence that the FH2 dimer moves stepwise at the barbed end, following an equilibrium between two states : closed" and "opened". In the closed state, no new actin subunit can be added to the barbed end, while it is allowed in the opened state. Several models, under discussion, describe the structural nature of these two states and how they alternate. One can then evoke the « stair-stepping » model and the « stepping second » model.My thesis aims at elucidating the debate about the conformational states adopted by formin at the barbed end, focusing on the mammalian formin mDia1. Our goal was undertaken using electron microscopy. So far, formins bound to the barbed ends of actin has not been described using electron microscopy. Indeed, to pursue single particle analysis by EM, a sufficient density of objects of interest randomly oriented have to be displayed within an image. Formins bound to actin rapidly generate long filaments, preventing the obtention of enough short filaments exhibiting formins at their barbed ends. Thus, a bottleneck had to be overcome to make the samples suitable for single particle structural analysis. A sonication approach was chosen to produce short actin filaments. A first 3D reconstruction of the FH2 dimer-actin complex was obtained from negative stain electron microscopy samples. This envelope ..."