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New approaches to study the architecture and dynamics of the sealing zone in osteoclasts

Abstract : Bone is a complex biological environment, and a living tissue under constant renewal throughout life. Its remodeling process is orchestrated by three different cell types: osteoblasts, osteoclasts and osteocytes. While osteoblasts are in charge of the generation of new bone matrix, the main function of osteoclasts is to degrade it, and both activities are under the regulation of osteocytes. In order to efficiently dissolute both mineralized and organic bone components, osteoclasts compartmentalize their plasma membrane to create a resorption microenvironment. The ruffled border is the site of extensive vesicular trafficking, and the sealing zone confines the digestion site. The sealing zone has been identified in the late 80s as a dense actin structure, then in the late 2000s as composed of seemingly podosomal subunits. However, little is still known about the precise inner organization of this specific cytoskeletal arrangement. This study proposed the first quantitative nanoscale characterization of the sealing zone in human osteoclasts. It benefited from state-of-the-art super-resolution microscopy, compatible with observation of both fixed and live samples on bone. Hence, it also yielded the first assessment of the internal dynamics of single actin cores within the sealing zone. It provided the precise in-plane localization of major actin-binding proteins associated with the sealing zone. In addition, it evaluated the 3D distribution of the same proteins in the podosome belt, counterpart of the sealing zone on glass substrates. In contrast to podosomes, the mechanical properties of the sealing zone have been but poorly investigated. Thus, this work also aimed at developing a new traction force microscopy environment, allowing for 3D characterization of forces thanks to a 3D nanoscopy technique. The acute sensitivity of the observation method implied challenging technological requirements, the feasibility of which have been explored. Even if this project was not successfully achieved, it helped identifying key information in order to raise technological bolts in the future. In addition, micro- and nanotechnological resources have been applied to the development of new substrates to assess topography sensing at the nanoscale. Indeed, micron-sized lines presenting nanoscale heights were directly shaped on glass coverslips thanks to photolithography and chemical etching, and their characterization was carried out with atomic force microscopy. [...]
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https://hal.laas.fr/tel-02535262
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Submitted on : Monday, January 11, 2021 - 2:15:14 PM
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2019TOU30294a.pdf
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  • HAL Id : tel-02535262, version 2

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Marion Portes. New approaches to study the architecture and dynamics of the sealing zone in osteoclasts. Biochemistry, Molecular Biology. Université Paul Sabatier - Toulouse III, 2019. English. ⟨NNT : 2019TOU30294⟩. ⟨tel-02535262v2⟩

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