Relevance of formins in the wound healing and actomysin cortex dynamics of cardiac fibroblasts
by Aina Gallemi Perez
Date of Examination:2023-04-25
Date of issue:2023-06-12
Advisor:Dr. Marco Tarantola
Referee:Dr. Marco Tarantola
Referee:Prof. Dr. Claudia Steinem
Persistent Address:
http://dx.doi.org/10.53846/goediss-9927
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Abstract
English
Upon myocardial infarction, the natural healing response triggers the fibroblast-to-myofibroblast transition (FMT). Through this process, fibroblasts acquire a myofibroblast phenotype by which they present increased mobility, contractility, and capacity to produce components of the extracellular matrix. These factors contribute to their migration towards the injured site and their effective participation in scar formation. However, the FMT involves a stringent regulation of the mechanics of the cellular cytoskeleton and, therefore, of the dynamics of the actomyosin cortex upon directed cell migration. As actin nucleators, formins are therefore of central relevance in the study of the interplay between collective wound healing dynamics and cortical adaptations. In this thesis, the role of formins is quantified by comparing the behavior and properties of wild type NIH 3T3 fibroblasts against those of mutant lines with deletions of the formin isoforms mDia1, mDia3, and of their combination. Atomic force microscopy (AFM) is employed to retrieve, in accordance to the Evans model, the prestress, area compressibility modulus and fluidity of single cells presenting different degrees of cell adhesion, hence mimicking different stages of the initial chemotactic migrating process. In parallel, the Electric Cell-Substrate Impedance Sensing (ECIS) device, in combination with fluorescence microscopy, is used to determine the wound closure dynamics of the different cell lines. Recovery kinetics are analysed in terms of the parameters derived from the passive electrical properties of the cell layer, namely: surface coverage, cell membrane integrity, cell-cell and cell-substrate morphology. Changes in the biological activity linked to formin modulations are further quantified via variance-, micromotion- and detrended fluctuation analysis of height oscillations in the cellular ventral membranes. Finally, recent studies have suggested that fibroblasts could close voids via purse-string contraction, mechanism traditionally ascribed to epithelial cultures. To test their capability to do so, fibroblast recovery on planar wounds of different sizes is analysed. Thereby, in this thesis it is shown that the roles of the formins mDia1 and mDia3 are essential to maintain the structural integrity of the cortex. Upon initial adhesion, fibroblasts fluidise to allow cellular spreading; once stabilised, prestress increases to maintain structural integrity. Formin deletions lead to structural defects in the cortex that are reflected in significantly delayed recovery dynamics upon wound formation. While mDia1 is central in migration and thus recovery kinetics, mDia3 is necessary for re-establishing mechanical integrity of the cortex. However, wound closure can still be initiated in their absence. Finally, based on wound size, two regimes are identified in the closure kinetics; according to fluorescence imaging, the fastest may be provided by purse-string contraction.
Keywords: Fibroblast; Wound healing; Collective dynamics; Purse-string; Actomyosin cortex; Cortical prestress; Formins
