Plasma membrane mechanosensing upon stretch-induced topography remodelling

Five years ago, I started walking this path that now seems like an entire life. Although everyone around tried to explain how this would feel, none of their explanations could have match what it has been in the end. And this is exactly how living systems are, at all levels. The harder the scientists try to feed our curiosity taking closer looks to them, inspecting the question from a different angle, and despite all the previous knowledge that we could gather; the more they surprise us and reveal new ways of sensing, reacting and adapting to the environment. And I think this is exactly what drove me here. I wanted to understand how this is done. I wanted to “see” it. How is it possible that a cell “understands” what is going on around? Which are the parameters that they sense? Biochemistry alone does not answer the question. In a crowded environment, such as it is our body, cells are exposed to thousands of mechanical stimuli too. And those can be also harnessed and transformed into chemical responses as a way of signalling. While the classical biochemical inputs have long been studied, loads of questions remain open about how cells interpret those physical stimuli around them, and microscopy comes as a powerful technique to try to answer these queries. In that sense, this thesis represents a small approach in trying to unravel how the plasma membrane, the first boundary between the cell and the extracellular media, can receive mechanical stimuli and convert them into biochemical signals amenable for the cell. To try to answer this question, this work starts with an introduction to the structure and physicochemical characteristics of the plasma membrane. An overview of the cortical component of the cytoskeleton, intrinsically interconnected to this structure, is also provided. Next, a summary of the literature available on how the plasma membrane can perceive mechanical stimuli and which are the associated biochemical responses triggered by them is included as well. This part is based on a review article published by my colleague and co-supervisor Dr. Le Roux and myself at Philosophical Transactions B as part of the 2019 issue “Forces in cancer” [1]. After the introduction, chapter 2 describes the objectives of this study, which can be summarised as trying to unravel the way in which cells couple mechanical signals at their plasma membrane to biochemical cascades that mediate a response to those. Following, chapters 3 and 4 compose the main body of this thesis, including the methods and the experimental results coming from this research work. Both sections constitute a scientific article that has been recently submitted for publication. In chapter 3 the simplified model chosen to study the question of how cells sense and integrate mechanical stimuli at their plasma membrane is described. This consisted in submitting fibroblast to a controlled stretch-release cycle, forcing them to quickly adapt their shape, mimmicking a highly-relevant scenario in physiology. Chapter 4 recapitulates the way in which plasma membrane reacted to this mechanical perturbation. In the first place, the structure reacted by passively forming evaginations on the nanometric scale of homogeneous size and shape. These evaginations are next recognised by the I- BAR protein IRSp53, which subsequently organizes a node of actin polymerisation dependent on Rac1 and Arp2/3 that mediates the flattening of the structures. Absence of IRSp53 results, thus, in an impaired recovery of homeostasis after stretch. To reinforce the obtained experimental results, theoretical framework to model the mechanics of the system was generated in collaboration with the group of Dr. Arroyo at the Centre Internacional de Mètodes Numèrics en Enginyeria (CIMNE). The aim of this model was to describe how a network generated by the Arp2/3 complex, until now described to push, is able to generate in-plane forces that mediate the ironing of the evagination. Chapter 5 includes a discussion about the limitations of the technique, the novelty of the presented findings, the possible physiological scenarios where the described mechanochemical pathway can be of relevance and, finally, some exciting and unexplored questions that remained open after this work. Additionally, other scientific production obtained during this thesis consisting in unexplored results or work belonging to other publications, has been added at the end of this manuscript in four appendixes. On the first one, I describe all the efforts made during the first 1 year and a half of the PhD to improve immunostaining technique for plasma membrane bound proteins in order to try to stain endogenous BAR proteins. The second appendix gathers the findings obtained from the silencing assay of BAR candidates likely to recognise the curved shape of the stretch-release generated evaginations. The third appendix contains experimental results part of a different publication from Le Roux et al. now under review in Nature Communications [2]. Here, I studied the response of the N-BAR protein Amphiphysin after mechanical stimulation in cells. A fourth appendix including scanning electron microscopy representative images of stretch-release generated evaginations in other cell lines is also added. Finally, I included two more appendix containing the sequencing of all the IRSp53 plasmids used for the body of the work of this thesis and the MATLAB code used for analysis of evaginations flattening dynamics after stretch